Bostjan Kobe

Structural basis of SARM1 activation, substrate recognition, and inhibition by small molecules

Publisher: Elsevier BV

Date: 05-2022

DOI: 10.1016/J.MOLCEL.2022.03.007

Editorial: Evolution of Innate Immunity in Eukarya: Advances and Implications

Publisher: Frontiers Media SA

Date: 20-04-2022

DOI: 10.3389/FIMMU.2022.879429

A Novel NAD Signaling Mechanism in Axon Degeneration and its Relationship to Innate Immunity

Publisher: Frontiers Media SA

Date: 08-07-2021

DOI: 10.3389/FMOLB.2021.703532

Abstract: Axon degeneration represents a pathological feature of many neurodegenerative diseases, including Alzheimer’s disease and Parkinson’s disease where axons die before the neuronal soma, and axonopathies, such as Charcot-Marie-Tooth disease and hereditary spastic paraplegia. Over the last two decades, it has slowly emerged that a central signaling pathway forms the basis of this process in many circumstances. This is an axonal NAD-related signaling mechanism mainly regulated by the two key proteins with opposing roles: the NAD-synthesizing enzyme NMNAT2, and SARM1, a protein with NADase and related activities. The crosstalk between the axon survival factor NMNAT2 and pro-degenerative factor SARM1 has been extensively characterized and plays an essential role in maintaining the axon integrity. This pathway can be activated in necroptosis and in genetic, toxic or metabolic disorders, physical injury and neuroinflammation, all leading to axon pathology. SARM1 is also known to be involved in regulating innate immunity, potentially linking axon degeneration to the response to pathogens and intercellular signaling. Understanding this NAD-related signaling mechanism enhances our understanding of the process of axon degeneration and enables a path to the development of drugs for a wide range of neurodegenerative diseases.

Substrates of Cyclic Nucleotide-Dependent Protein Kinases

Publisher: Elsevier

Date: 2009

DOI: 10.1016/B978-0-12-374145-5.00182-0

Biochemical characterization of arabidopsis developmentally regulated G-proteins (DRGs)

Publisher: Elsevier BV

Date: 10-2009

DOI: 10.1016/J.PEP.2009.05.009

Abstract: Developmentally regulated G-proteins (DRGs) are a highly conserved family of GTP-binding proteins found in archaea, plants, fungi and animals, indicating important roles in fundamental pathways. Their function is poorly understood, but they have been implicated in cell ision, proliferation, and growth, as well as several medical conditions. In idual subfamilies within the G-protein superfamily possess unique nucleotide binding and hydrolysis rates that are intrinsic to their cellular function, and so characterization of these rates for a particular G-protein may provide insight into its cellular activity. We have produced recombinant active DRG protein using a bacterial expression system and refolding, and performed biochemical characterization of their GTP binding and hydrolysis. We show that recombinant Arabidopsis thaliana atDRG1 and atDRG2a are able to bind GDP and GTP. We also show that DRGs can hydrolyze GTP in vitro without the assistance of GTPase-activating proteins and guanine exchange factors. The atDRG proteins hydrolyze GTP at a relatively slow rate (0.94x10(-3)min(-1) for DRG1 and 1.36x10(-3)min(-1) for DRG2) that is consistent with their nearest characterized relatives, the Obg subfamily. The ability of DRGs to bind nucleotide substrates without assistance, their slow rate of GTP hydrolysis, heat stress activation and domain conservation suggest a possible role as a chaperone in ribosome assembly in response to stress as it has been suggested for the Obg proteins, a different but related G-protein subfamily.

The structure of the caspase recruitment domain of BinCARD reveals that all three cysteines can be oxidized

Publisher: International Union of Crystallography (IUCr)

Date: 11-04-2013

DOI: 10.1107/S0907444913001558

AdcA and AdcAII employ distinct zinc acquisition mechanisms and contribute additively to zinc homeostasis in Streptococcus pneumoniae

Publisher: Wiley

Date: 16-01-2014

DOI: 10.1111/MMI.12504

Abstract: Streptococcus pneumoniae is a globally significant human pathogen responsible for nearly 1 million deaths annually. Central to the ability of S. pneumoniae to colonize and mediate disease in humans is the acquisition of zinc from the host environment. Zinc uptake in S. pneumoniae occurs via the ATP-binding cassette transporter AdcCB, and, unusually, two zinc-binding proteins, AdcA and AdcAII. Studies have suggested that these two proteins are functionally redundant, although AdcA has remained uncharacterized by biochemical methods. Here we show that AdcA is a zinc-specific substrate-binding protein (SBP). By contrast with other zinc-binding SBPs, AdcA has two zinc-binding domains: a canonical amino-terminal cluster A-I zinc-binding domain and a carboxy-terminal zinc-binding domain, which has homology to the zinc-chaperone ZinT from Gram-negative organisms. Intriguingly, this latter feature is absent from AdcAII and suggests that the two zinc-binding SBPs of S. pneumoniae employ different modalities in zinc recruitment. We further show that AdcAII is reliant upon the polyhistidine triad proteins for zinc in vitro and in vivo. Collectively, our studies suggest that, despite the overlapping roles of the two SBPs in zinc acquisition, they may have unique mechanisms in zinc homeostasis and act in a complementary manner during host colonization.

Disruption of de Novo Adenosine Triphosphate (ATP) Biosynthesis Abolishes Virulence in Cryptococcus neoformans

Publisher: American Chemical Society (ACS)

Date: 17-08-2016

DOI: 10.1021/ACSINFECDIS.6B00121

Abstract: Opportunistic fungal pathogens such as Cryptococcus neoformans are a growing cause of morbidity and mortality among immunocompromised populations worldwide. To address the current paucity of antifungal therapeutic agents, further research into fungal-specific drug targets is required. Adenylosuccinate synthetase (AdSS) is a crucial enzyme in the adeosine triphosphate (ATP) biosynthetic pathway, catalyzing the formation of adenylosuccinate from inosine monophosphate and aspartate. We have investigated the potential of this enzyme as an antifungal drug target, finding that loss of function results in adenine auxotrophy in C. neoformans, as well as complete loss of virulence in a murine model. Cryptococcal AdSS was expressed and purified in Escherichia coli and the enzyme's crystal structure determined, the first ex le of a structure of this enzyme from fungi. Together with enzyme kinetic studies, this structural information enabled comparison of the fungal enzyme with the human orthologue and revealed species-specific differences potentially exploitable via rational drug design. These results validate AdSS as a promising antifungal drug target and lay a foundation for future in silico and in vitro screens for novel antifungal compounds.

Structural and biochemical characterization of Acinetobacter baumannii ZnuA.

Publisher: Elsevier BV

Date: 06-2022

DOI: 10.1016/J.JINORGBIO.2022.111787

Abstract: Acinetobacter baumannii is a Gram-negative nosocomial pathogen associated with significant disease. Crucial to the survival and pathogenesis of A. baumannii is the ability to acquire essential micronutrients such as Zn(II). Recruitment of Zn(II) by A. baumannii is mediated, at least in part, by the periplasmic solute-binding protein ZnuA and the ATP-binding cassette transporter ZnuBC. Here, we combined genomic, biochemical, and structural approaches to characterize A. baumannii AB5075_UW ZnuA. Bioinformatic analyses using a erse collection of A. baumannii genomes determined that ZnuA is highly conserved, with the binding site comprised by three strictly conserved histidine residues. The structure of metal-free ZnuA was determined at 2.1 Å resolution, with molecular dynamics analyses revealing loop α2β2, which harbors the putative Zn(II)-coordinating residue His41, to be highly mobile in the metal-free state. The contribution of the putative binding site histidine residues to Zn(II) interaction was further probed by mutagenesis. Analysis of ZnuA mutant variants was performed by quantitative metal binding assays, differential scanning fluorimetry, and affinity measurements, which showed that all three histidine residues contributed to Zn(II)-recruitment, albeit to different extents. Collectively, these analyses provide insight into the mechanism of Zn(II)-binding by A. baumannii ZnuA and expand our understanding of the functional ersity of Zn(II)-recruiting proteins.

Structural basis and prediction of substrate specificity in protein serine/threonine kinases

Publisher: Proceedings of the National Academy of Sciences

Date: 26-12-2002

DOI: 10.1073/PNAS.0134224100

Abstract: The large number of protein kinases makes it impractical to determine their specificities and substrates experimentally. Using the available crystal structures, molecular modeling, and sequence analyses of kinases and substrates, we developed a set of rules governing the binding of a heptapeptide substrate motif (surrounding the phosphorylation site) to the kinase and implemented these rules in a web-interfaced program for automated prediction of optimal substrate peptides, taking only the amino acid sequence of a protein kinase as input. We show the utility of the method by analyzing yeast cell cycle control and DNA damage checkpoint pathways. Our method is the only available predictive method generally applicable for identifying possible substrate proteins for protein serine/threonine kinases and helps in silico construction of signaling pathways. The accuracy of prediction is comparable to the accuracy of data from systematic large-scale experimental approaches.

Turn up the HEAT

Publisher: Elsevier BV

Date: 05-1999

DOI: 10.1016/S0969-2126(99)80060-4

Abstract: The recently determined crystal structure of the PR65/A subunit of protein phosphatase 2A reveals the architecture of proteins containing HEAT repeats. The structural properties of this solenoid protein explain many functional characteristics and account for the involvement of solenoids as scaffold, anchoring and adaptor proteins.

Fibroblast Growth Factor Receptor 2 Phosphorylation on Serine 779 Couples to 14-3-3 and Regulates Cell Survival and Proliferation

Publisher: Informa UK Limited

Date: 05-2008

DOI: 10.1128/MCB.01837-07

A Medium or High Throughput Protein Refolding Assay

Publisher: Humana Press

Date: 2008

DOI: 10.1007/978-1-60327-058-8_17

Abstract: Expression of insoluble protein in E. coli is a major bottleneck of high throughput structural biology projects. Refolding proteins into native conformations from inclusion bodies could significantly increase the number of protein targets that can be taken on to structural studies. This chapter presents a simple assay for screening insoluble protein targets and identifying those that are most amenable to refolding. The assay is based on the observation that when proteins are refolded while bound to metal affinity resin, misfolded proteins are generally not eluted by imidazole. This difference is exploited here to distinguish between folded and misfolded proteins. Two implementations of the assay are described. The assay fits well into a standard high throughput structural biology pipeline, because it begins with the inclusion body preparations that are a byproduct of small-scale, automated expression and purification trials and does not require additional facilities. Two formats of the assay are described, a manual assay that is useful for screening small numbers of targets, and an automated implementation that is useful for large numbers of targets.

The mammalian DUF59 protein Fam96a forms two distinct types of domain-swapped dimer

Publisher: International Union of Crystallography (IUCr)

Date: 17-05-2012

DOI: 10.1107/S0907444912006592

Substrate specificity of protein kinases and computational prediction of substrates

Publisher: Elsevier BV

Date: 12-2005

DOI: 10.1016/J.BBAPAP.2005.07.036

Abstract: To ensure signalling fidelity, kinases must act only on a defined subset of cellular targets. Appreciating the basis for this substrate specificity is essential for understanding the role of an in idual protein kinase in a particular cellular process. The specificity in the cell is determined by a combination of "peptide specificity" of the kinase (the molecular recognition of the sequence surrounding the phosphorylation site), substrate recruitment and phosphatase activity. Peptide specificity plays a crucial role and depends on the complementarity between the kinase and the substrate and therefore on their three-dimensional structures. Methods for experimental identification of kinase substrates and characterization of specificity are expensive and laborious, therefore, computational approaches are being developed to reduce the amount of experimental work required in substrate identification. We discuss the structural basis of substrate specificity of protein kinases and review the experimental and computational methods used to obtain specificity information.

SARM1 is a metabolic sensor activated by an increased NMN/NAD+ ratio to trigger axon degeneration

Publisher: Elsevier BV

Date: 04-2021

DOI: 10.1016/J.NEURON.2021.02.009

Crystallization and preliminary X-ray diffraction analysis of importin-α complexed with NLS peptidomimetics

Publisher: Elsevier BV

Date: 06-2005

DOI: 10.1016/J.BBAPAP.2005.03.014

Abstract: Importin-alpha is the nuclear import receptor that recognizes cargo proteins with nuclear localization sequences (NLSs). The study of NLS peptidomimetics can provide a better understanding of the requirements for the molecular recognition of cargo proteins by importin-alpha, and potentially engender a large number of applications in medicine. Importin-alpha was crystallized with a set of six NLS peptidomimetics, and X-ray diffraction data were collected in the range 2.1-2.5 A resolution. Preliminary electron density calculations show that the ligands are present in the crystals.

Crystal structure determination of the armadillo repeat domain of Drosophila SARM1 using MIRAS phasing

Publisher: International Union of Crystallography (IUCr)

Date: 21-09-2021

DOI: 10.1107/S2053230X21006786

Abstract: The crystal structure determination of the armadillo repeat motif (ARM) domain of Drosophila SARM1 (dSARM1 ARM ) is described, which required the combination of a number of sources of phase information in order to obtain interpretable electron-density maps. SARM1 is a central executioner of programmed axon degeneration, a common feature of the early phase of many neurodegenerative diseases. SARM1 is held in the inactive state in healthy axons by its N-terminal auto-inhibitory ARM domain, and is activated to cleave NAD upon injury, triggering subsequent axon degeneration. To characterize the molecular mechanism of SARM1 activation, it was sought to determine the crystal structure of the SARM1 ARM domain. Here, the recombinant production and crystallization of dSARM1 ARM is described, as well as the unconventional process used for structure determination. Crystals were obtained in the presence of NMN, a precursor of NAD and a potential activator of SARM1, only after in situ proteolysis of the N-terminal 63 residues. After molecular-replacement attempts failed, the crystal structure of dSARM1 ARM was determined at 1.65 Å resolution using the MIRAS phasing technique with autoSHARP , combining data from native, selenomethionine-labelled and bromide-soaked crystals. The structure will further the understanding of SARM1 regulation.

A probabilistic model of nuclear import of proteins

Publisher: Oxford University Press (OUP)

Date: 03-03-2011

DOI: 10.1093/BIOINFORMATICS/BTR121

Abstract: Motivation: Nucleo-cytoplasmic trafficking of proteins is a core regulatory process that sustains the integrity of the nuclear space of eukaryotic cells via an interplay between numerous factors. Despite progress on experimentally characterizing a number of nuclear localization signals, their presence alone remains an unreliable indicator of actual translocation. Results: This article introduces a probabilistic model that explicitly recognizes a variety of nuclear localization signals, and integrates relevant amino acid sequence and interaction data for any candidate nuclear protein. In particular, we develop and incorporate scoring functions based on distinct classes of classical nuclear localization signals. Our empirical results show that the model accurately predicts whether a protein is imported into the nucleus, surpassing the classification accuracy of similar predictors when evaluated on the mouse and yeast proteomes (area under the receiver operator characteristic curve of 0.84 and 0.80, respectively). The model also predicts the sequence position of a nuclear localization signal and whether it interacts with importin-α. Availability: pprowler.itee.uq.edu.au/NucImport Contact: m.boden@uq.edu.au Supplementary information: Supplementary data are available at Bioinformatics online.

Heterogeneous nucleation is required for crystallization of the ZnuA domain of pneumococcal AdcA

Publisher: International Union of Crystallography (IUCr)

Date: 18-11-2015

DOI: 10.1107/S2053230X15021330

Abstract: Zn 2+ is an essential nutrient for all known forms of life. In the major human pathogen Streptococcus pneumoniae , the acquisition of Zn 2+ is facilitated by two Zn 2+ -specific solute-binding proteins: AdcA and AdcAII. To date, there has been a paucity of structural information on AdcA, which has hindered a deeper understanding of the mechanism underlying pneumococcal Zn 2+ acquisition. Native AdcA consists of two domains: an N-terminal ZnuA domain and a C-terminal ZinT domain. In this study, the ZnuA domain of AdcA was crystallized. The initial crystals of the ZnuA-domain protein were obtained using dried seaweed as a heterogeneous nucleating agent. No crystals were obtained in the absence of the heterogeneous nucleating agent. These initial crystals were subsequently used as seeds to produce diffraction-quality crystals. The crystals diffracted to 2.03 Å resolution and had the symmetry of space group P 1. This study demonstrates the utility of heterogeneous nucleation. The solution of the crystal structures will lead to further understanding of Zn 2+ acquisition by S. pneumoniae .

Comparison of three commercial sparse-matrix crystallization screens

Publisher: International Union of Crystallography (IUCr)

Date: 25-03-2003

DOI: 10.1107/S0907444903002919

Abstract: Sparse-matrix s ling using commercially available crystallization screen kits has become the most popular way of determining the preliminary crystallization conditions for macromolecules. In this study, the efficiency of three commercial screening kits, Crystal Screen and Crystal Screen 2 (H ton Research), Wizard Screens I and II (Emerald BioStructures) and Personal Structure Screens 1 and 2 (Molecular Dimensions), has been compared using a set of 19 erse proteins. 18 proteins yielded crystals using at least one crystallization screen. Surprisingly, Crystal Screens and Personal Structure Screens showed dramatically different results, although most of the crystallization formulations are identical as listed by the manufacturers. Higher molecular weight polyethylene glycols and mixed precipitants were found to be the most effective precipitants in this study.

Pathological mutations differentially affect the self-assembly and polymerisation of the innate immune system signalling adaptor molecule MyD88

Publisher: Springer Science and Business Media LLC

Date: 12-2018

DOI: 10.1186/S12915-018-0611-7

Intrasteric Regulation of Calmodulin-Dependent Protein Kinases

Publisher: Elsevier

Date: 1996

DOI: 10.1016/S1054-3589(08)60584-0

Abstract: The accumulation of extracellular amyloid-β (Aβ) and intracellular hyperphosphorylated τ proteins in the brain are the hallmarks of Alzheimer's disease (AD). Much of the research into the pathogenesis of AD has focused on the amyloid or τ hypothesis. These hypotheses propose that Aβ or τ aggregation is the inciting event in AD that leads to downstream neurodegeneration, inflammation, brain atrophy and cognitive impairment. Multiple drugs have been developed and are effective in preventing the accumulation and/or clearing of Aβ or τ proteins. However, clinical trials examining these therapeutic agents have failed to show efficacy in preventing or slowing the progression of the disease. Thus, there is a need for fresh perspectives and the evaluation of alternative therapeutic targets in this field. Epidemiology studies have revealed significant overlap between cardiovascular and cerebrovascular risk factors such as hypertension, diabetes, atherosclerosis and stroke to the development of cognitive impairment. This strong correlation has given birth to a renewed focus on vascular contributions to AD and related dementias. However, few genes and mechanisms have been identified. 20-Hydroxyeicosatetraenoic acid (20-HETE) is a potent vasoconstrictor that plays a complex role in hypertension, autoregulation of cerebral blood flow and blood-brain barrier (BBB) integrity. Multiple human genome-wide association studies have linked mutations in the cytochrome P450 (CYP) 4A (CYP4A) genes that produce 20-HETE to hypertension and stroke. Most recently, genetic variants in the enzymes that produce 20-HETE have also been linked to AD in human population studies. This review examines the emerging role of 20-HETE in AD and related dementias.

Antifungal benzo[b]thiophene 1,1-dioxide IMPDH inhibitors exhibit pan-assay interference (PAINS) profiles

Publisher: Elsevier BV

Date: 11-2018

DOI: 10.1016/J.BMC.2018.09.004

Abstract: Fungi cause serious life-threatening infections in immunocompromised in iduals and current treatments are now complicated by toxicity issues and the emergence of drug resistant strains. Consequently, there is a need for development of new antifungal drugs. Inosine monophosphate dehydrogenase (IMPDH), a key component of the de novo purine biosynthetic pathway, is essential for growth and virulence of fungi and is a potential drug target. In this study, a high-throughput screen of 114,000 drug-like compounds against Cryptococcus neoformans IMPDH was performed. We identified three 3-((5-substituted)-1,3,4-oxadiazol-2-yl)thio benzo[b]thiophene 1,1-dioxides that inhibited Cryptococcus IMPDH and also possessed whole cell antifungal activity. Analogs were synthesized to explore the SAR of these hits. Modification of the fifth substituent on the 1,3,4-oxadiazole ring yielded compounds with nanomolar in vitro activity, but with associated cytotoxicity. In contrast, two analogs generated by substituting the 1,3,4-oxadiazole ring with imidazole and 1,2,4-triazole gave reduced IMPDH inhibition in vitro, but were not cytotoxic. During enzyme kinetic studies in the presence of DTT, nucleophilic attack of a free thiol occurred with the benzo[b]thiophene 1,1-dioxide. Two representative compounds with substitution at the 5 position of the 1,3,4-oxadiazole ring, showed mixed inhibition in the absence of DTT. Incubation of these compounds with Cryptococcus IMPDH followed by mass spectrometry analysis showed non-specific and covalent binding with IMPDH at multiple cysteine residues. These results support recent reports that the benzo[b]thiophene 1,1-dioxides moiety as PAINS (pan-assay interference compounds) contributor.

Use of distance geometry approach for the in vitro antiviral activity evaluation of N-bridgehead C-nucleosides

Publisher: Elsevier BV

Date: 04-1992

DOI: 10.1016/0223-5234(92)90010-X

Structural features of Cryptococcus neoformans bifunctional GAR/AIR synthetase may present novel antifungal drug targets

Publisher: Elsevier BV

Date: 10-2021

DOI: 10.1016/J.JBC.2021.101091

Crystallization and X-ray diffraction analysis of the N-terminal domain of the Toll-like receptor signalling adaptor protein TRIF/TICAM-1

Publisher: International Union of Crystallography (IUCr)

Date: 28-06-2013

DOI: 10.1107/S174430911301419X

Wheat PR ‐1 proteins are targeted by necrotrophic pathogen effector proteins

Publisher: Wiley

Date: 10-08-2016

DOI: 10.1111/TPJ.13228

Abstract: Recent studies have identified that proteinaceous effectors secreted by Parastagonospora nodorum are required to cause disease on wheat. These effectors interact in a gene-for-gene manner with host-dominant susceptibilty loci, resulting in disease. However, whilst the requirement of these effectors for infection is clear, their mechanisms of action remain poorly understood. A yeast-two-hybrid library approach was used to search for wheat proteins that interacted with the necrotrophic effector SnTox3. Using this strategy we indentified an interaction between SnTox3 and the wheat pathogenicity-related protein TaPR-1-1, and confirmed it by in-planta co-immunprecipitation. PR-1 proteins represent a large family (23 in wheat) of proteins that are upregulated early in the defence response however, their function remains ellusive. Interestingly, the P. nodorum effector SnToxA has recently been shown to interact specifically with TaPR-1-5. Our analysis of the SnTox3-TaPR-1 interaction demonstrated that SnTox3 can interact with a broader range of TaPR-1 proteins. Based on these data we utilised homology modeling to predict, and validate, regions on TaPR-1 proteins that are likely to be involved in the SnTox3 interaction. Precipitating from this work, we identified that a PR-1-derived defence signalling peptide from the C-terminus of TaPR-1-1, known as CAPE1, enhanced the infection of wheat by P. nodorum in an SnTox3-dependent manner, but played no role in ToxA-mediated disease. Collectively, our data suggest that P. nodorum has evolved unique effectors that target a common host-protein involved in host defence, albeit with different mechanisms and potentially outcomes.

Structural and Calorimetric Studies Demonstrate that Xeroderma Pigmentosum Type G (XPG) Can Be Imported to the Nucleus by a Classical Nuclear Import Pathway via a Monopartite NLS Sequence

Publisher: Elsevier BV

Date: 05-2016

DOI: 10.1016/J.JMB.2016.01.019

Abstract: Xeroderma pigmentosum type G (XPG) proteins are involved in DNA lesion recognition and promotion of nucleotide excision repair. Specific mutations in these proteins may lead to Cockayne syndrome, in which the patients may display severe developmental retardation and neurological abnormalities. No structural information is available for their spacer region or the C-terminal domain, which are important, respectively, for specific nucleotide excision repair activity and substrate specificity, as well as nuclear translocation. Immunofluorescence studies suggested two specific regions of the XPG C-terminus as potential bipartite nuclear localization sequences, which would be responsible for its translocation to the nucleus by the classical nuclear import pathway mediated by the importin-α (Impα). Thus, in order to test these hypotheses and gain insight into the structural basis for the nuclear import process for the XPG protein, we solved the crystal structures of complexes formed by the Impα and peptides corresponding to both putative nuclear localization signal (NLS) sequences (XPG1 and XPG2) and performed isothermal titration calorimetry assays to determine their binding affinities. Structural experiments confirm the binding of both NLS peptides to Impα but, unexpectedly, they bind to the receptor as monopartite NLSs. The isothermal titration calorimetry assays demonstrated that XPG1 and XPG2 peptides bind to two separate binding sites, but with high affinity to the major NLS-binding site of the Impα, resembling classical monopartite SV40 TAg NLS. The results lead to insights about what distinguishes monopartite and bipartite NLSs, as well as the differential roles of XPG1 and XPG2 NLSs in the nuclear localization of XPG.

Identification of novel target sites and an inhibitor of the dengue virus E protein

Publisher: Springer Science and Business Media LLC

Date: 25-02-2009

DOI: 10.1007/S10822-009-9263-6

Abstract: Dengue and related flaviviruses represent a significant global health threat. The envelope glycoprotein E mediates virus attachment to a host cell and the subsequent fusion of viral and host cell membranes. The fusion process is driven by conformational changes in the E protein and is an essential step in the virus life cycle. In this study, we analyzed the pre-fusion and post-fusion structures of the dengue virus E protein to identify potential novel sites that could bind small molecules, which could interfere with the conformational transitions that mediate the fusion process. We used an in silico virtual screening approach combining three different docking algorithms (DOCK, GOLD and FlexX) to identify compounds that are likely to bind to these sites. Seven structurally erse molecules were selected to test experimentally for inhibition of dengue virus propagation. The best compound showed an IC(50) in the micromolar range against dengue virus type 2.

A General Target Selection Method for Crystallographic Proteomics

Publisher: Humana Press

Date: 2008

DOI: 10.1007/978-1-60327-058-8_2

Abstract: Increasing the success in obtaining structures and maximizing the value of the structures determined are the two major goals of target selection in structural proteomics. This chapter presents an efficient and flexible target selection procedure supplemented with a Web-based resource that is suitable for small- to large-scale structural genomics projects that use crystallography as the major means of structure determination. Based on three criteria, biological significance, structural novelty, and "crystallizability," the approach first removes (filters) targets that do not meet minimal criteria and then ranks the remaining targets based on their "crystallizability" estimates. This novel procedure was designed to maximize selection efficiency, and its prevailing criteria categories make it suitable for a broad range of structural proteomics projects.

Increasing Protein Crystallization Screening Success with Heterogeneous Nucleating Agents

Publisher: Humana Press

Date: 2008

DOI: 10.1007/978-1-60327-058-8_26

Abstract: The crystallization step is considered to be a major bottleneck in the process of structure determination by x-ray crystallography. Successful crystallization requires both the formation of nuclei that are capable of supporting crystal growth, and a subsequent crystal growth. Nucleation can occur spontaneously in a supersaturated solution. However, in the commonly used sparse matrix crystallization screens, protein and precipitant concentrations are not extensively s led, so that suitable supersaturation conditions for nucleation are often missed. This chapter describes a simple method for enhancing nucleation and subsequent crystallization by the addition of heterogeneous nucleating agents.

The many faces of platelet glycoprotein Ibα - Thrombin interaction

Publisher: Bentham Science Publishers Ltd.

Date: 12-2009

DOI: 10.2174/138920309789630570

Abstract: The platelet glycoprotein receptor regulates the adhesion of blood platelets to damaged blood vessel walls and the subsequent platelet aggregation. One of the subunits, platelet glycoprotein Ibalpha (GpIbalpha), binds thrombin, a serine protease with both procoagulant and anticoagulant activities. Two groups reported the crystal structures of the complex between thrombin and the N-terminal extracellular domain (leucine-rich repeat [LRR] domain) of GpIbalpha. In both these structures, GpIbalpha was reported to bind two thrombin molecules, but both the primary and secondary thrombin binding sites differed between them. We performed a detailed comparison of the two structures to look for insights that may explain the differences. Our results show that the 1:1 GpIbalpha-thrombin complex detected in solution between the crystallized proteins is likely the only strong interaction. The anionic sequence (residues 268-284) of GpIbalpha is likely responsible for the initial interaction with thrombin and the interaction with the rest of LRR domain of GpIbalpha occurs subsequently and may alternate between two or more different binding modes. Our modelling suggests the interaction between GpIbalpha and thrombin is highly pH-dependent and a small change in pH is likely to contribute to the formation of alternate binding modes. The differences in the crystal structures reported for the GpIbalpha-thrombin complex suggest a fascinating plasticity in this protein-protein interaction that may be biologically significant.

An optimized SEC-SAXS system enabling high X-ray dose for rapid SAXS assessment with correlated UV measurements for biomolecular structure analysis

Publisher: International Union of Crystallography (IUCr)

Date: 02-2018

DOI: 10.1107/S1600576717017101

Abstract: A new optimized size exclusion chromatography small-angle X-ray scattering (SEC-SAXS) system for biomolecular SAXS at the Australian Synchrotron SAXS/WAXS beamline has been developed. The compact configuration reduces s le dilution to maximize sensitivity. Coflow s le presentation allows an 11-fold increase in flux on s le without capillary fouling, improving throughput and data quality, which are now primarily limited by the full flux available on the beamline. Multi-wavelength fibre optic UV analysis in close proximity to the X-ray beam allows for accurate concentration determination for s les with known UV extinction coefficients and thus estimation of the molecular weight of the scattering particle from the forward X-ray scattering intensity. Fast-flow low-volume SEC columns provide s le throughput competitive with batch concentration series measurements, albeit with a concomitant reduction of potential resolution relative to lower flow rates and larger SEC columns. The performance of the system is demonstrated using a set of model proteins, and its utility to solve various challenges is illustrated with a erse suite of protein s les. These developments increase the quality and rigor of SEC-SAXS analysis and open new avenues for biomolecular solution SEC-SAXS studies that have been challenged by low s le yields, temporal instability, radiation sensitivity and complex mixtures.

Crystallization and preliminary X-ray diffraction analysis of the flax cytokinin oxidase LuCKX1.1

Publisher: International Union of Crystallography (IUCr)

Date: 28-09-2013

DOI: 10.1107/S1744309113023142

Nicotinic acid mononucleotide is an allosteric SARM1 inhibitor promoting axonal protection

Publisher: Elsevier BV

Date: 11-2021

DOI: 10.1016/J.EXPNEUROL.2021.113842

Comment on ‘Protein isoelectric point as a predictor for increased crystallization screening efficiency’

Publisher: Oxford University Press (OUP)

Date: 18-08-2004

DOI: 10.1093/BIOINFORMATICS/BTH452

Autoinhibition by an internal nuclear localization signal revealed by the crystal structure of mammalian importin α

Publisher: Springer Science and Business Media LLC

Date: 04-1999

DOI: 10.1038/7625

Abstract: Importin alpha is the nuclear import receptor that recognizes classical monopartite and bipartite nuclear localization signals (NLSs). The structure of mouse importin alpha has been determined at 2.5 A resolution. The structure shows a large C-terminal domain containing armadillo repeats, and a less structured N-terminal importin beta-binding domain containing an internal NLS bound to the NLS-binding site. The structure explains the regulatory switch between the cytoplasmic, high-affinity form, and the nuclear, low-affinity form for NLS binding of the nuclear import receptor predicted by the current models of nuclear import. Importin beta conceivably converts the low- to high-affinity form by binding to a site overlapping the autoinhibitory sequence. The structure also has implications for understanding NLS recognition, and the structures of armadillo and HEAT repeats.

The Impact of Chromate on Pseudomonas aeruginosa Molybdenum Homeostasis

Publisher: Frontiers Media SA

Date: 24-05-2022

DOI: 10.3389/FMICB.2022.903146

Abstract: Acquisition of the trace-element molybdenum via the high-affinity ATP-binding cassette permease ModABC is essential for Pseudomonas aeruginosa respiration in anaerobic and microaerophilic environments. This study determined the X-ray crystal structures of the molybdenum-recruiting solute-binding protein ModA from P. aeruginosa PAO1 in the metal-free state and bound to the group 6 metal oxyanions molybdate, tungstate, and chromate. Pseudomonas aeruginosa PAO1 ModA has a non-contiguous dual-hinged bilobal structure with a single metal-binding site positioned between the two domains. Metal binding results in a 22° relative rotation of the two lobes with the oxyanions coordinated by four residues, that contribute six hydrogen bonds, distinct from ModA orthologues that feature an additional oxyanion-binding residue. Analysis of 485 Pseudomonas ModA sequences revealed conservation of the metal-binding residues and β-sheet structural elements, highlighting their contribution to protein structure and function. Despite the capacity of ModA to bind chromate, deletion of modA did not affect P. aeruginosa PAO1 sensitivity to chromate toxicity nor impact cellular accumulation of chromate. Exposure to sub-inhibitory concentrations of chromate broadly perturbed P. aeruginosa metal homeostasis and, unexpectedly, was associated with an increase in ModA-mediated molybdenum uptake. Elemental analyses of the proteome from anaerobically grown P. aeruginosa revealed that, despite the increase in cellular molybdenum upon chromate exposure, distribution of the metal within the proteome was substantially perturbed. This suggested that molybdoprotein cofactor acquisition may be disrupted, consistent with the potent toxicity of chromate under anaerobic conditions. Collectively, these data reveal a complex relationship between chromate toxicity, molybdenum homeostasis and anaerobic respiration.

Protein Phosphorylation

Publisher: Springer Berlin Heidelberg

Date: 2013

DOI: 10.1007/978-3-642-16712-6_184

Discovery of Novel Pneumococcal Surface Antigen A (PsaA) Inhibitors Using a Fragment-based Drug Design Approach

Publisher: American Chemical Society (ACS)

Date: 30-03-2015

DOI: 10.1021/CB501032X

Abstract: Streptococcus pneumoniae is a leading cause of life-threatening bacterial infections, especially in young children in developing countries. Pneumococcal infections can be treated with β-lactam antibiotics, but rapid emergence of multidrug-resistant strains of S. pneumoniae over the past two decades has emphasized the need to identify novel drug targets. Pneumococcal surface antigen A (PsaA) is one such target, found on the cell surface of S. pneumoniae. It functions as a high-affinity substrate-binding protein, facilitating acquisition of Mn(2+), which has an important role in protecting S. pneumoniae from reactive oxygen species and, hence, oxidative stress. Consequently, PsaA is essential for bacterial survival and an important virulence factor, which makes it a promising target for antibiotic drug development. To design novel PsaA inhibitors, we used a combination of de novo fragment-based drug discovery and in silico virtual screening methods. We profiled a collection of low molecular weight compounds that were selected based on their structural ersity and ability to bind to apo-PsaA in a virtual docking experiment. The screening resulted in two initial hits that were further optimized by structural variation to improve their potency while maintaining their ligand efficiency and favorable physicochemical properties. The optimized hits were validated using a cell-based assay and molecular dynamics simulations. We found that virtual screening substantially augmented fragment-based drug design approaches, leading to the identification of novel pneumococcal PsaA inhibitors.

Crystallization and preliminary X-ray analysis of ferric enterobactin receptor FepA, an integral membrane protein fromEscherichia coli

Publisher: International Union of Crystallography (IUCr)

Date: 19-10-0077

DOI: 10.1107/S0907444997020222

Abstract: Diffraction-quality crystals have been obtained of the integral membrane protein ferric enterobactin receptor (FepA) from the outer membrane of Escherichia coli. Crystals were grown using the zwitterionic detergent lauryldimethylamine oxide (LDAO), the precipitants polyethylene glycol (PEG) 1000 and sodium chloride, and the additive heptane-1,2,3-triol they have the symmetry of the orthorhomic space group C2221 with a = 112.2, b = 137.2 and c = 135. 4 A and diffract to 2.5 A resolution. The crystals were flash-cooled and a preliminary data set was collected at 103 K. The crystals are suitable for three-dimensional structure analysis.

Functional Implications of the Human T-Lymphotropic Virus Type 1 Transmembrane Glycoprotein Helical Hairpin Structure

Publisher: American Society for Microbiology

Date: 15-07-2000

DOI: 10.1128/JVI.74.14.6614-6621.2000

Abstract: Retrovirus entry into cells follows receptor binding by the surface-exposed envelope glycoprotein (Env) subunit (SU), which triggers the membrane fusion activity of the transmembrane (TM) protein. TM protein fragments expressed in the absence of SU adopt helical hairpin structures comprising a central coiled coil, a region of chain reversal containing a disulfide-bonded loop, and a C-terminal segment that packs onto the exterior of the coiled coil in an antiparallel manner. Here we used in vitro mutagenesis to test the functional role of structural elements observed in a model helical hairpin, gp21 of human T-lymphotropic virus type 1. Membrane fusion activity requires the stabilization of the N and C termini of the central coiled coil by a hydrophobic N cap and a small hydrophobic core, respectively. A conserved Gly-Gly hinge motif preceding the disulfide-bonded loop, a salt bridge that stabilizes the chain reversal region, and interactions between the C-terminal segment and the coiled coil are also critical for fusion activity. Our data support a model whereby the chain reversal region transmits a conformational signal from receptor-bound SU to induce the fusion-activated helical hairpin conformation of the TM protein.

Expression and biochemical analysis of the entire HIV‐2 gp41 ectodomain: determinants of stability map to N‐ and C‐terminal sequences outside the 6‐helix bundle core

Publisher: Wiley

Date: 03-05-2004

DOI: 10.1016/J.FEBSLET.2004.04.054

A Molecular Mechanism for Bacterial Susceptibility to Zinc

Publisher: Public Library of Science (PLoS)

Date: 03-11-2011

DOI: 10.1371/JOURNAL.PPAT.1002357

Assessment of the ability to model proteins with leucine‐rich repeats in light of the latest structural information

Publisher: Wiley

Date: 05-2002

DOI: 10.1110/PS.4010102

Abstract: The three-dimensional structures of leucine-rich repeat (LRR)-containing proteins from five different families were previously predicted based on the crystal structure of the ribonuclease inhibitor, using an approach that combined homology-based modeling, structure-based sequence alignment of LRRs, and several rational assumptions. The structural models have been produced based on very limited sequence similarity, which, in general, cannot yield trustworthy predictions. Recently, the protein structures from three of these five families have been determined. In this report we estimate the quality of the modeling approach by comparing the models with the experimentally determined structures. The comparison suggests that the general architecture, curvature, "interior/exterior" orientations of side chains, and backbone conformation of the LRR structures can be predicted correctly. On the other hand, the analysis revealed that, in some cases, it is difficult to predict correctly the twist of the overall super-helical structure. Taking into consideration the conclusions from these comparisons, we identified a new family of bacterial LRR proteins and present its structural model. The reliability of the LRR protein modeling suggests that it would be informative to apply similar modeling approaches to other classes of solenoid proteins.

Overview of the pipeline for structural and functional characterization of macrophage proteins at the university of queensland.

Publisher: Humana Press

Date: 2008

DOI: 10.1007/978-1-60327-058-8_38

Abstract: This chapter describes the methodology adopted in a project aimed at structural and functional characterization of proteins that potentially play an important role in mammalian macrophages. The methodology that underpins this project is applicable to both small research groups and larger structural genomics consortia. Gene products with putative roles in macrophage function are identified using gene expression information obtained via DNA microarray technology. Specific targets for structural and functional characterization are then selected based on a set of criteria aimed at maximizing insight into function. The target proteins are cloned using a modification of Gateway cloning technology, expressed with hexa-histidine tags in E. coli, and purified to homogeneity using a combination of affinity and size exclusion chromatography. Purified proteins are finally subjected to crystallization trials and/or NMR-based screening to identify candidates for structure determination. Where crystallography and NMR approaches are unsuccessful, chemical cross-linking is employed to obtain structural information. This resulting structural information is used to guide cell biology experiments to further investigate the cellular and molecular function of the targets in macrophage biology. Jointly, the data sheds light on the molecular and cellular functions of macrophage proteins.

Crystal structure of the Toll/interleukin‐1 receptor (TIR) domain of IL‐1R10 provides structural insights into TIR domain signalling

Publisher: Wiley

Date: 02-2022

DOI: 10.1002/1873-3468.14288

Abstract: The Toll/interleukin‐1 receptor (TIR) domains are key innate immune signalling modules. Here, we present the crystal structure of the TIR domain of human interleukin‐1 receptor 10 (IL‐1R10), also called interleukin 1 receptor accessory protein like 2. It is similar to that of IL‐1R9 (IL‐1RAPL1) but shows significant structural differences to those from Toll‐like receptors (TLRs) and the adaptor proteins MyD88 adaptor‐like protein (MAL) and MyD88. Interactions of TIR domains in their respective crystals and the higher‐order assemblies (MAL and MyD88) reveal the presence of a common ‘BCD surface’, suggesting its functional significance. We also show that the TIR domains of IL‐1R10 and IL‐1R9 lack NADase activity, consistent with their structures. Our study provides a foundation for unravelling the functions of IL‐1R9 and IL‐1R10.

Crystallization of the C-terminal domain of the mouse brain cytosolic long-chain acyl-CoA thioesterase

Publisher: International Union of Crystallography (IUCr)

Date: 27-01-2006

DOI: 10.1107/S1744309106000030

Protein structure determination using a combination of cross-linking, mass spectrometry, and molecular modeling.

Publisher: Humana Press

Date: 2008

DOI: 10.1007/978-1-60327-058-8_31

Abstract: Cross-linking in combination with mass spectrometry can be used as a tool for structural modeling of protein complexes and multidomain proteins. Although cross-links represent only weak structural constraints, the combination of a limited set of experimental cross-links with molecular docking/modeling is often sufficient to determine the structure of a protein complex or multidomain protein at low resolution.

Cyclic ADP ribose isomers: Production, chemical structures, and immune signaling

Publisher: American Association for the Advancement of Science (AAAS)

Date: 30-09-2022

DOI: 10.1126/SCIENCE.ADC8969

Abstract: Cyclic adenosine diphosphate (ADP)–ribose (cADPR) isomers are signaling molecules produced by bacterial and plant Toll/interleukin-1 receptor (TIR) domains via nicotinamide adenine dinucleotide (oxidized form) (NAD + ) hydrolysis. We show that v-cADPR (2′cADPR) and v2-cADPR (3′cADPR) isomers are cyclized by O-glycosidic bond formation between the ribose moieties in ADPR. Structures of 2′cADPR-producing TIR domains reveal conformational changes that lead to an active assembly that resembles those of Toll-like receptor adaptor TIR domains. Mutagenesis reveals a conserved tryptophan that is essential for cyclization. We show that 3′cADPR is an activator of ThsA effector proteins from the bacterial antiphage defense system termed Thoeris and a suppressor of plant immunity when produced by the effector HopAM1. Collectively, our results reveal the molecular basis of cADPR isomer production and establish 3′cADPR in bacteria as an antiviral and plant immunity–suppressing signaling molecule.

Nicotinic acid mononucleotide is an allosteric SARM1 inhibitor promoting axonal protection

Publisher: Cold Spring Harbor Laboratory

Date: 16-07-2021

DOI: 10.1101/2021.07.15.452560

Abstract: SARM1 is an inducible NAD + hydrolase that is the central executioner of pathological axon loss. Recently, we elucidated the molecular mechanism of SARM1 activation, demonstrating that SARM1 is a metabolic sensor regulated by the levels of NAD + and its precursor, nicotinamide mononucleotide (NMN), via their competitive binding to an allosteric site within the SARM1 N-terminal ARM domain. In healthy neurons with abundant NAD + , binding of NAD + blocks access of NMN to this allosteric site. However, with injury or disease the levels of the NAD + biosynthetic enzyme NMNAT2 drop, increasing the NMN/ NAD + ratio and thereby promoting NMN binding to the SARM1 allosteric site, which in turn induces a conformational change activating the SARM1 NAD + hydrolase. Hence, NAD + metabolites both regulate the activation of SARM1 and, in turn, are regulated by the SARM1 NAD + hydrolase. This dual upstream and downstream role for NAD + metabolites in SARM1 function has hindered mechanistic understanding of axoprotective mechanisms that manipulate the NAD + metabolome. Here we reevaluate two methods that potently block axon degeneration via modulation of NAD + related metabolites, 1) the administration of the NMN biosynthesis inhibitor FK866 in conjunction with the NAD + precursor nicotinic acid riboside (NaR) and 2) the neuronal expression of the bacterial enzyme NMN deamidase. We find that these approaches not only lead to a decrease in the levels of the SARM1 activator NMN, but also an increase in the levels of the NAD + precursor nicotinic acid mononucleotide (NaMN). We show that NaMN inhibits SARM1 activation, and demonstrate that this NaMN-mediated inhibition is important for the long-term axon protection induced by these treatments. Analysis of the NaMN-ARM domain co-crystal structure shows that NaMN competes with NMN for binding to the SARM1 allosteric site and promotes the open, autoinhibited configuration of SARM1 ARM domain. Together, these results demonstrate that the SARM1 allosteric pocket can bind a erse set of metabolites including NMN, NAD + , and NaMN to monitor cellular NAD + homeostasis and regulate SARM1 NAD + hydrolase activity. The relative promiscuity of the allosteric site may enable the development of potent pharmacological inhibitors of SARM1 activation for the treatment of neurodegenerative disorders. NaMN binds to SARM1 N-terminal allosteric site and inhibits SARM1 NAD+ hydrolase NaMN inhibits SARM1 activation by stabilizing its open, inactive structure NMN deamidase promotes strong axonal protection by reducing NMN and increasing NaMN

A structural basis of the interactions between leucine-rich repeats and protein ligands

Publisher: Springer Science and Business Media LLC

Date: 03-1995

DOI: 10.1038/374183A0

Abstract: The leucine-rich repeat is a recently characterized structural motif used in molecular recognition processes as erse as signal transduction, cell adhesion, cell development, DNA repair and RNA processing. We present here the crystal structure at 2.5 A resolution of the complex between ribonuclease A and ribonuclease inhibitor, a protein built entirely of leucine-rich repeats. The unusual non-globular structure of ribonuclease inhibitor, its solvent-exposed parallel beta-sheet and the conformational flexibility of the structure are used in the interaction they appear to be the principal reasons for the effectiveness of leucine-rich repeats as protein-binding motifs. The structure can serve as a model for the interactions of other proteins containing leucine-rich repeats with their ligands.

United we stand: combining structural methods

Publisher: Elsevier BV

Date: 10-2008

DOI: 10.1016/J.SBI.2008.07.004

Abstract: High-resolution techniques are the mainstay of structural biologists however, to address challenging biological systems many are now turning to hybrid approaches that use complementary structural data. In this review we outline the types of structural problems that benefit from combining results of many methods, we summarise the types of data that can be generated by complementary approaches, and we highlight the application of combined methods in structural biology with recent structural studies of membrane proteins, mega-complexes and inherently flexible proteins.

Structural Proteomics

Publisher: Humana Press

Date: 2008

DOI: 10.1007/978-1-60327-058-8

Solution structure of the TLR adaptor MAL/TIRAP reveals an intact BB loop and supports MAL Cys91 glutathionylation for signaling

Publisher: Proceedings of the National Academy of Sciences

Date: 24-07-2017

DOI: 10.1073/PNAS.1701868114

Abstract: Toll-like receptor (TLR) signaling pathways are targeted to limit inflammation in immune cells. TLRs use adaptor proteins to drive inflammatory signaling platforms for effective microbial clearance. Here we show that MyD88 adaptor-like (MAL), an adaptor protein in TLR signaling, undergoes glutathionylation in response to LPS, driving macrophage responses to proinflammatory stimuli. We also determined the solution structure of MAL in the reduced form without disulfides, revealing a typical BB loop observed in adaptor proteins, in contrast to previously reported crystal structures. This alternate solution structure reveals the inherent flexibility of MAL, supporting the hypothesis that glutathionylation may reposition the MAL BB loop for MyD88 interaction to drive inflammation. This discovery could lead to novel approaches to target MAL glutathionylation in dysregulated TLR signaling, limiting inflammation.

Distinctive Conformation of Minor Site-Specific Nuclear Localization Signals Bound to Importin-alpha

Publisher: Wiley

Date: 08-2013

DOI: 10.1111/TRA.12098

Abstract: Nuclear localization signals (NLSs) contain one or two clusters of basic residues and are recognized by the import receptor importin-α. There are two NLS-binding sites (major and minor) on importin-α and the major NLS-binding site is considered to be the primary binding site. Here, we used crystallographic and biochemical methods to investigate the binding between importin-α and predicted 'minor site-specific' NLSs: four peptide library-derived peptides, and the NLS from mouse RNA helicase II/Guα. The crystal structures reveal that these atypical NLSs indeed preferentially bind to the minor NLS-binding site. Unlike previously characterized NLSs, the C-terminal residues of these NLSs form an α-helical turn, stabilized by internal H-bond and cation-π interactions between the aromatic residues from the NLSs and the positively charged residues from importin-α. This helical turn sterically hinders binding at the major NLS-binding site, explaining the minor-site preference. Our data suggest the sequence RXXKR[K/X][F/Y/W]XXAF as the optimal minor NLS-binding site-specific motif, which may help identify novel proteins with atypical NLSs.

The crystal structure of SnTox3 from the necrotrophic fungus Parastagonospora nodorum reveals a unique effector fold and insights into Kex2 protease processing of fungal effectors

Publisher: Cold Spring Harbor Laboratory

Date: 27-05-2020

DOI: 10.1101/2020.05.27.120113

Abstract: Plant pathogens cause disease through secreted effector proteins, which act to modulate host physiology and promote infection. Typically, the sequences of effectors provide little functional information and further targeted experimentation is required. Here, we utilised a structure/function approach to study SnTox3, an effector from the necrotrophic fungal pathogen Parastagonospora nodorum , which causes cell death in wheat-lines carrying the sensitivity gene Snn3 . We developed a workflow for the production of SnTox3 in a heterologous host that enabled crystal structure determination. We show this approach can be successfully applied to effectors from other pathogenic fungi. Complementing this, an in-silico study uncovered the prevalence of an expanded subclass of effectors from fungi. The β-barrel fold of SnTox3 is a novel fold among fungal effectors. We demonstrate that SnTox3 is a pre-pro-protein and that the protease Kex2 removes the pro-domain. Our in-silico studies suggest that Kex2-processed pro-domain (designated here as K2PP) effectors are common in fungi, and we demonstrate this experimentally for effectors from Fusarium oxysporum f sp. lycopersici . We propose that K2PP effectors are highly prevalent among fungal effectors. The identification and classification of K2PP effectors has broad implications for the approaches used to study their function in fungal virulence.

Crystal structure of Mycobacterium tuberculosis ketol‐acid reductoisomerase at 1.0 Å resolution – a potential target for anti‐tuberculosis drug discovery

Publisher: Wiley

Date: 18-02-2016

DOI: 10.1111/FEBS.13672

Abstract: The biosynthetic pathway for the branched-chain amino acids is present in plants, fungi and bacteria, but not in animals, making it an attractive target for herbicidal and antimicrobial drug discovery. Ketol-acid reductoisomerase (KARI EC 1.1.1.86) is the second enzyme in this pathway, converting in a Mg(2+) - and NADPH-dependent reaction either 2-acetolactate or 2-aceto-2-hydroxybutyrate to their corresponding 2,3-dihydroxy-3-alkylbutyrate products. Here, we have determined the crystal structure of Mycobacterium tuberculosis (Mt) KARI, a class I KARI, with two magnesium ions bound in the active site. X-ray data were obtained to 1.0 Å resolution and the final model has an Rfree of 0.163. The structure shows that the active site is solvent-accessible with the two metal ions separated by 4.7 Å. A comparison of this structure with that of Mg(2+) -free Pseudomonas aeruginosa KARI suggests that upon magnesium binding no movement of the N domain relative to the C domain occurs. However, upon formation of the Michaelis complex, as illustrated in the structure of Slackia exigua KARI in complex with NADH.Mg(2+) . N-hydroxy-N-isopropyloxamate (IpOHA, a transition state analog), domain movements and reduction of the metal-metal distance to 3.5 Å are observed. This inherent flexibility therefore appears to be critical for initiation of the KARI-catalyzed reaction. This study provides new insights into the complex structural rearrangements required for activity of KARIs, particularly those belonging to class I, and provides the framework for the rational design of Mt KARI inhibitors that can be tested as novel antituberculosis agents. Coordinates and structure factors for the Mt KARI.Mg(2+) complex are available in the Protein Data Bank under accession number 4YPO.

Principles of Kinase Regulation

Publisher: Elsevier

Date: 2003

DOI: 10.1016/B978-012124546-7/50450-2

The use of Co²⁺for crystallization and structure determination, using a conventional monochromatic X-ray source, of flax rust avirulence protein

Publisher: International Union of Crystallography (IUCr)

Date: 23-02-2007

DOI: 10.1107/S1744309107004599

PREDIVAC: CD4+ T-cell epitope prediction for vaccine design that covers 95% of HLA class II DR protein diversity

Publisher: Springer Science and Business Media LLC

Date: 14-02-2013

DOI: 10.1186/1471-2105-14-52

The Single Nucleotide Polymorphism Mal-D96N Mice Provide New Insights into Functionality of Mal in TLR Immune Responses

Publisher: The American Association of Immunologists

Date: 15-04-2019

DOI: 10.4049/JIMMUNOL.1800501

Abstract: MyD88 adaptor-like (Mal) protein is the most polymorphic of the four key adaptor proteins involved in TLR signaling. TLRs play a critical role in the recognition and immune response to pathogens through activation of the prototypic inflammatory transcription factor NF-κB. The study of single nucleotide polymorphisms in TLRs, adaptors, and signaling mediators has provided key insights into the function of the corresponding genes but also into the susceptibility to infectious diseases in humans. In this study, we have analyzed the immune response of mice carrying the human Mal-D96N genetic variation that has previously been proposed to confer protection against septic shock. We have found that Mal-D96N macrophages display reduced cytokine expression in response to TLR4 and TLR2 ligand challenge. Mal-D96N macrophages also display reduced MAPK activation, NF-κB transactivation, and delayed NF-κB nuclear translocation, presumably via delayed kinetics of Mal interaction with MyD88 following LPS stimulation. Importantly, Mal-D96N genetic variation confers a physiological protective phenotype to in vivo models of LPS-, Escherichia coli–, and influenza A virus–induced hyperinflammatory disease in a gene dosage-dependent manner. Together, these results highlight the critical role Mal plays in regulating optimal TLR-induced inflammatory signaling pathways and suggest the potential therapeutic advantages of targeting the Mal D96 signaling nexus.

Purification of the M flax‐rust resistance protein expressed in Pichia pastoris

Publisher: Wiley

Date: 23-05-2007

DOI: 10.1111/J.1365-313X.2007.03104.X

The transmembrane adapter SCIMP recruits tyrosine kinase Syk to phosphorylate Toll-like receptors to mediate selective inflammatory outputs

Publisher: Elsevier BV

Date: 05-2022

DOI: 10.1016/J.JBC.2022.101857

A computational analysis of substrate binding strength by phosphorylase kinase and protein kinase A

Publisher: Wiley

Date: 2002

DOI: 10.1002/JMR.563

Abstract: Protein kinases exhibit various degrees of substrate specificity. The large number of different protein kinases in the eukaryotic proteomes makes it impractical to determine the specificity of each enzyme experimentally. To test if it were possible to discriminate potential substrates from non-substrates by simple computational techniques, we analysed the binding enthalpies of modelled enzyme-substrate complexes and attempted to correlate it with experimental enzyme kinetics measurements. The crystal structures of phosphorylase kinase and cAMP-dependent protein kinase were used to generate models of the enzyme with a series of known peptide substrates and non-substrates, and the approximate enthalpy of binding assessed following energy minimization. We show that the computed enthalpies do not correlate closely with kinetic measurements, but the method can distinguish good substrates from weak substrates and non-substrates.

Comparative Analysis of the Flax Immune Receptors L6 and L7 Suggests an Equilibrium-Based Switch Activation Model

Publisher: Oxford University Press (OUP)

Date: 2016

DOI: 10.1105/TPC.15.00303

Author response: Neurotoxin-mediated potent activation of the axon degeneration regulator SARM1

Publisher: eLife Sciences Publications, Ltd

Date: 30-11-2021

DOI: 10.7554/ELIFE.72823.SA2

Mildew RALPHs up in arms with cereals

Publisher: Proceedings of the National Academy of Sciences

Date: 23-08-2023

DOI: 10.1073/PNAS.2311817120

Structural mix-n-match reveals molecular secrets of platelets

Publisher: American Society of Hematology

Date: 10-11-2011

DOI: 10.1182/BLOOD-2011-09-379859

Structural characterization of the N‐terminal autoregulatory sequence of phenylalanine hydroxylase

Publisher: Wiley

Date: 08-2002

DOI: 10.1110/PS.4560102

Identification and predicted structure of a leucine-rich repeat motif shared by Leishmania major proteophosphoglycan and Parasite Surface Antigen 2

Publisher: Elsevier BV

Date: 15-04-2000

DOI: 10.1016/S0166-6851(00)00186-9

The role of histidine residues in low-pH-mediated viral membrane fusion

Publisher: Elsevier BV

Date: 10-2006

DOI: 10.1016/J.STR.2006.07.011

Abstract: A central event in the invasion of a host cell by an enveloped virus is the fusion of viral and cell membranes. For many viruses, membrane fusion is driven by specific viral surface proteins that undergo large-scale conformational rearrangements, triggered by exposure to low pH in the endosome upon internalization. Here, we present evidence suggesting that in both class I (helical hairpin proteins) and class II (beta-structure-rich proteins) pH-dependent fusion proteins the protonation of specific histidine residues triggers fusion via an analogous molecular mechanism. These histidines are located in the vicinity of positively charged residues in the prefusion conformation, and they subsequently form salt bridges with negatively charged residues in the postfusion conformation. The molecular surfaces involved in the corresponding structural rearrangements leading to fusion are highly conserved and thus might provide a suitable common target for the design of antivirals, which could be active against a erse range of pathogenic viruses.

Peptide Substrates of Cyclic Nucleotide-Dependent Protein Kinases

Publisher: Elsevier

Date: 2003

DOI: 10.1016/B978-012124546-7/50563-5

Interaction between plate make and protein in protein crystallisation screening

Publisher: Public Library of Science (PLoS)

Date: 16-11-2009

DOI: 10.1371/JOURNAL.PONE.0007851

Structural basis of TIR-domain-assembly formation in MAL- and MyD88-dependent TLR4 signaling

Publisher: Springer Science and Business Media LLC

Date: 31-07-2017

DOI: 10.1038/NSMB.3444

Conformational behavior of Escherichia coli OmpA signal peptides in membrane mimetic environments

Publisher: American Chemical Society (ACS)

Date: 11-05-1993

DOI: 10.1021/BI00069A025

Abstract: Nuclear magnetic resonance and circular dichroism (CD) studies of isolated peptides corresponding to WT and mutant OmpA signal sequences are reported all of the peptides adopt substantial amounts of alpha-helical structure both in 1:1 (v/v) trifluoroethanol (TFE)/water and in sodium dodecyl sulfate (SDS) micelles. In TFE/water, the helix begins after the positively charged N-terminal residues and is most stable in the hydrophobic core, which correlates with results obtained previously for other signal sequences. The helix is weaker between the hydrophobic core and the C-terminus such a break in the helix appears to be common to other signal peptides studied previously and could be of functional importance. No clear correlation could be established between the helicity of the peptides in TFE/water and their in vivo activities. All the peptides have a higher alpha-helix content in SDS than in TFE/water, and there is a good correlation between helix content in SDS and in vivo activity. Helicity in SDS for the functional peptides increases both at the N-terminus and in the hydrophobic core, and is driven by a strong association of the core with the hydrophobic chains of the detergent. The extension of the helix toward the N-terminus may be a result of neutralization of the N-terminal positive charges by the headgroups of the micelles, which removes unfavorable electrostatic interactions with the helix dipole. All these comparisons were facilitated by the use of upfield shifts of H alpha protons in helical regions relative to random coil chemical shifts, which also yielded estimates of helical content that correlated well with the CD results.

Giant protein kinases: domain interactions and structural basis of autoregulation.

Publisher: Wiley

Date: 12-1996

DOI: 10.1002/J.1460-2075.1996.TB01072.X

Mapping the stabilome: A novel computational method for classifying metabolic protein stability

Publisher: Springer Science and Business Media LLC

Date: 08-06-2012

DOI: 10.1186/1752-0509-6-60

Abstract: The half-life of a protein is regulated by a range of system properties, including the abundance of components of the degradative machinery and protein modifiers. It is also influenced by protein-specific properties, such as a protein’s structural make-up and interaction partners. New experimental techniques coupled with powerful data integration methods now enable us to not only investigate what features govern protein stability in general, but also to build models that identify what properties determine each protein’s metabolic stability. In this work we present five groups of features useful for predicting protein stability: (1) post-translational modifications, (2) domain types, (3) structural disorder, (4) the identity of a protein’s N-terminal residue and (5) amino acid sequence. We incorporate these features into a predictive model with promising accuracy. At a 20% false positive rate, the model exhibits an 80% true positive rate, outperforming the only previously proposed stability predictor. We also investigate the impact of N-terminal protein tagging as used to generate the data set, in particular the impact it may have on the measurements for secreted and transmembrane proteins we train and test our model on a subset of the data with those proteins removed, and show that the model sustains high accuracy. Finally, we estimate system-wide metabolic stability by surveying the whole human proteome. We describe a variety of protein features that are significantly over- or under-represented in stable and unstable proteins, including phosphorylation, acetylation and destabilizing N-terminal residues. Bayesian networks are ideal for combining these features into a predictive model with superior accuracy and transparency compared to the only other proposed stability predictor. Furthermore, our stability predictions of the human proteome will find application in the analysis of functionally related proteins, shedding new light on regulation by protein synthesis and degradation.

Proteins with leucine-rich repeats

Publisher: Elsevier BV

Date: 06-1995

DOI: 10.1016/0959-440X(95)80105-7

Abstract: Leucine-rich repeats are short sequence motifs present in over sixty proteins, all of which appear to be involved in protein-protein interactions. The crystal structure of ribonuclease inhibitor demonstrated that the repeats correspond to beta-alpha structural units. The recently determined crystal structure of the ribonuclease A-ribonuclease inhibitor complex suggests the basis for the protein-binding function of leucine-rich repeats.

PR1‐mediated defence via C‐terminal peptide release is targeted by a fungal pathogen effector

Publisher: Wiley

Date: 03-01-2021

DOI: 10.1111/NPH.17128

Crystal structure of the Melampsora lini effector AvrP reveals insights into a possible nuclear function and recognition by the flax disease resistance protein P

Publisher: Wiley

Date: 16-11-2017

DOI: 10.1111/MPP.12597

Structure and Metal Binding Properties of Chlamydia trachomatis YtgA.

Publisher: American Society for Microbiology

Date: 06-12-2019

DOI: 10.1128/JB.00580-19

Abstract: Chlamydia trachomatis is the most common bacterial sexually transmitted infection in developed countries, with an estimated global prevalence of 4.2% in the 15- to 49-year age group. Although infection is asymptomatic in more than 80% of infected women, about 10% of cases result in serious disease. Infection by C. trachomatis is dependent on the ability to acquire essential nutrients, such as the transition metal iron, from host cells. In this study, we show that iron is the most abundant transition metal in C. trachomatis and report the structural and biochemical properties of the iron-recruiting protein YtgA. Knowledge of the high-resolution structure of YtgA will provide a platform for future structure-based antimicrobial design approaches.

Functional and structural properties of mammalian acyl-coenzyme A thioesterases

Publisher: Elsevier BV

Date: 10-2010

DOI: 10.1016/J.PLIPRES.2010.04.001

Abstract: Acyl-coenzyme A thioesterases (Acots) play important cellular roles in mammalian fatty acid metabolism through modulation of cellular concentrations of activated fatty acyl-CoAs. Acots catalyse the hydrolysis of the thioester bond present within acyl-CoA ester molecules to yield coenzyme A (CoASH) and the corresponding non-esterified fatty acid. Acyl-CoA thioesterases are expressed ubiquitously in both prokaryotes and eukaryotes and, in higher order organisms, the enzymes are expressed and localised in a tissue-dependent manner within the cytosol, mitochondria, peroxisomes and endoplasmic reticulum. Recent studies have led to advances in the functional and structural characterization of many mammalian Acot family members. These include the structure determination of both type-I and type-II Acot family members, structural elucidation of the START domain of ACOT11, identification of roles in arachidonic acid and inflammatory prostaglandin production by Acot7, and inclusion of a 13th Acot family member. Here, we review and analyse the current literature on mammalian Acots with respect to their characterization and summarize the current knowledge on the structure, function and regulation of this enzyme family.

Histidine protonation and the activation of viral fusion proteins

Publisher: Portland Press Ltd.

Date: 22-01-2008

DOI: 10.1042/BST0360043

Abstract: Many viral fusion proteins only become activated under mildly acidic condition (pH 4.5–6.5) close to the pKa of histidine side-chain protonation. Analysis of the sequences and structures of influenza HA (haemagglutinin) and flaviviral envelope glycoproteins has led to the identification of a number of histidine residues that are not only fully conserved themselves but have local environments that are also highly conserved [K mann, Mueller, Mark, Young and Kobe (2006) Structure 14, 1481–1487]. Here, we summarize studies aimed at determining the role, if any, that protonation of these potential switch histidine residues plays in the low-pH-dependent conformational changes associated with fusion activation of a flaviviral envelope protein. Specifically, we report on MD (Molecular Dynamics) simulations of the DEN2 (dengue virus type 2) envelope protein ectodomain sE (soluble E) performed under varied pH conditions designed to test the histidine switch hypothesis of K mann et al. (2006).

Fusion-protein-assisted protein crystallization

Publisher: International Union of Crystallography (IUCr)

Date: 27-06-2015

DOI: 10.1107/S2053230X15011061

Abstract: Fusion proteins can be used directly in protein crystallization to assist crystallization in at least two different ways. In one approach, the `heterologous fusion-protein approach', the fusion partner can provide additional surface area to promote crystal contact formation. In another approach, the `fusion of interacting proteins approach', protein assemblies can be stabilized by covalently linking the interacting partners. The linker connecting the proteins plays different roles in the two applications: in the first approach a rigid linker is required to reduce conformational heterogeneity in the second, conversely, a flexible linker is required that allows the native interaction between the fused proteins. The two approaches can also be combined. The recent applications of fusion-protein technology in protein crystallization from the work of our own and other laboratories are briefly reviewed.

The TLR signaling adaptor TRAM interacts with TRAF6 to mediate activation of the inflammatory response by TLR4

Publisher: Oxford University Press (OUP)

Date: 08-05-2014

DOI: 10.1189/JLB.2A0913-487R

Abstract: TLRs act as sentinels in professional immune cells to detect and initiate the innate immune response to pathogen challenge. TLR4 is a widely expressed TLR, responsible for initiating potent immune responses to LPS. TRAM acts to bridge TLR4 with TRIF, orchestrating the inflammatory response to pathogen challenge. We have identified a putative TRAF6-binding motif in TRAM that could mediate a novel signaling function for TRAM in TLR4 signaling. TRAM and TRAF6 association was confirmed by immunoprecipitation of endogenous, ectopically expressed and recombinant proteins, which was ablated upon mutation of a key Glu residue in TRAM (TRAM E183A). TRAF6 and TRAM were observed colocalizing using confocal microscopy following ectopic expression in cells and the ability of TRAM and TRAM E183A to activate luciferase-linked reporter assays was determined in HEK293 and TRAF6-deficient cells. Importantly, TRAM-deficient macrophages reconstituted with TRAM E183A display significantly reduced inflammatory TNF-α, IL-6, and RANTES protein production compared with WT TRAM. These results demonstrate a novel role for TRAM in TLR4-mediated signaling in regulating inflammatory responses via its interaction with TRAF6, distinct from its role as a bridging adaptor between TLR4 and TRIF.

Role of flanking sequences and phosphorylation in the recognition of the simian-virus-40 large T-antigen nuclear localization sequences by importin-

Publisher: Portland Press Ltd.

Date: 15-10-2003

DOI: 10.1042/BJ20030510

Abstract: The nuclear import of simian-virus-40 large T-antigen (tumour antigen) is enhanced via phosphorylation by the protein kinase CK2 at Ser112 in the vicinity of the NLS (nuclear localization sequence). To determine the structural basis of the effect of the sequences flanking the basic cluster KKKRK, and the effect of phosphorylation on the recognition of the NLS by the nuclear import factor importin-α (Impα), we co-crystallized non-autoinhibited Impα with peptides corresponding to the phosphorylated and non-phosphorylated forms of the NLS, and determined the crystal structures of the complexes. The structures show that the amino acids N-terminally flanking the basic cluster make specific contacts with the receptor that are distinct from the interactions between bipartite NLSs and Impα. We confirm the important role of flanking sequences using binding assays. Unexpectedly, the regions of the peptides containing the phosphorylation site do not make specific contacts with the receptor. Binding assays confirm that phosphorylation does not increase the affinity of the T-antigen NLS to Impα. We conclude that the sequences flanking the basic clusters in NLSs play a crucial role in nuclear import by modulating the recognition of the NLS by Impα, whereas phosphorylation of the T-antigen enhances nuclear import by a mechanism that does not involve a direct interaction of the phosphorylated residue with Impα.

Crystallization and X-ray diffraction analysis of the C-terminal domain of the flax rust effector protein AvrM

Publisher: International Union of Crystallography (IUCr)

Date: 26-11-2011

DOI: 10.1107/S1744309111037675

Abstract: The flax rust effector AvrM is a secreted protein of unknown fold that is recognized by the M resistance protein in flax. In order to investigate the structural basis of the AvrM–M interaction and possible virulence-associated functions of AvrM, the C-terminal domains of two different AvrM variants (AvrM-A and avrM) were crystallized. Crystals of native AvrM-A were obtained using pentaerythritol ethoxylate (15/4 EO/OH) as a precipitant and diffracted X-rays to 2.9 Å resolution. Selenomethionine-derivative crystals of similar quality were obtained using PEG 1500 as a precipitant. Both the native and selenomethionine-labelled AvrM-A crystals had symmetry of space group C 222 1 with eight molecules in the asymmetric unit. Crystals of avrM had symmetry of space group P 2 1 2 1 2 1 and diffracted X-rays to 2.7 Å resolution. Initial AvrM-A phases were calculated using the single-wavelength anomalous dispersion (SAD) method and a partial model was built. Phases for avrM were obtained by molecular replacement using the partial AvrM-A model.

Multiple Domain Associations within the Arabidopsis Immune Receptor RPP1 Regulate the Activation of Programmed Cell Death

Publisher: Public Library of Science (PLoS)

Date: 18-07-2016

DOI: 10.1371/JOURNAL.PPAT.1005769

Defining the structural basis of human plasminogen binding by streptococcal surface enolase

Publisher: Elsevier BV

Date: 06-2009

DOI: 10.1074/JBC.M109.004317

Regulation of signaling by cooperative assembly formation in mammalian innate immunity signalosomes by molecular mimics

Publisher: Elsevier BV

Date: 03-2020

DOI: 10.1016/J.SEMCDB.2018.05.002

Abstract: Innate immunity pathways constitute the first line of defense against infections and cellular damage. An emerging concept in these pathways is that signaling involves the formation of finite (e.g. rings in NLRs) or open-ended higher-order assemblies (e.g. filamentous assemblies by members of the death-fold family and TIR domains). This signaling by cooperative assembly formation (SCAF) mechanism allows rapid and strongly lified responses to minute amounts of stimulus. While the characterization of the molecular mechanisms of SCAF has seen rapid progress, little is known about its regulation. One emerging theme involves proteins produced both in host cells and by pathogens that appear to mimic the signaling components. Recently characterized ex les involve the capping of the filamentous assemblies formed by caspase-1 CARDs by the CARD-only protein INCA, and those formed by caspase-8 by the DED-containing protein MC159. By contrast, the CARD-only protein ICEBERG and the DED-containing protein cFLIP incorporate into signaling filaments and presumably interfere with proximity based activation of caspases. We review selected ex les of SCAF in innate immunity pathways and focus on the current knowledge on signaling component mimics produced by mammalian and pathogen cells and what is known about their mechanisms of action.

Towards the structure of the TIR-domain signalosome

Publisher: Elsevier BV

Date: 04-2017

DOI: 10.1016/J.SBI.2016.12.014

Abstract: TIR (Toll/interleukin-1 receptor/resistance protein) domains feature in animal, plant and bacterial proteins involved in innate immunity pathways and associated processes. They function through protein:protein interactions, in particular self-association and homotypic association with other TIR domains. Structures of TIR domains from all phyla have been determined, but common association modes have only emerged for plant and bacterial TIR domains, and not for mammalian TIR domains. Numerous attempts involving hybrid approaches, which have combined structural, computational, mutagenesis and biophysical data, have failed to converge onto common models of how these domains associate and function. We propose that the available data can be reconciled in the context of higher-order assembly formation, and that TIR domains function through signaling by cooperative assembly formation (SCAF).

Structural Biology and Regulation of Protein Import into the Nucleus

Publisher: Elsevier BV

Date: 05-2016

DOI: 10.1016/J.JMB.2015.10.023

Abstract: Proteins are translated in the cytoplasm, but many need to access the nucleus to perform their functions. Understanding how these nuclear proteins are transported through the nuclear envelope and how the import processes are regulated is therefore an important aspect of understanding cell function. Structural biology has played a key role in understanding the molecular events during the transport processes and their regulation, including the recognition of nuclear targeting signals by the corresponding receptors. Here, we review the structural basis of the principal nuclear import pathways and the molecular basis of their regulation. The pathways involve transport factors that are members of the β-karyopherin family, which can bind cargo directly (e.g., importin-β, transportin-1, transportin-3, importin-13) or through adaptor proteins (e.g., importin-α, snurportin-1, symportin-1), as well as unrelated transport factors such as Hikeshi, involved in the transport of heat-shock proteins, and NTF2, involved in the transport of RanGDP. Solenoid proteins feature prominently in these pathways. Nuclear transport factors recognize nuclear targeting signals on the cargo proteins, including the classical nuclear localization signals, recognized by the adaptor importin-α, and the PY nuclear localization signals, recognized by transportin-1. Post-translational modifications, particularly phosphorylation, constitute key regulatory mechanisms operating in these pathways.

Exon shuffling potentiates a diverse repertoire of brown algal NB‐ARC‐TPR candidate immune receptor proteins via alternative splicing

Publisher: Wiley

Date: 22-02-2023

DOI: 10.1111/TPJ.16131

Abstract: Like other organisms, brown algae are subject to diseases caused by bacteria, fungi, and viruses. Brown algal immunity mechanisms are not well characterized however, there is evidence suggesting that pathogen receptors exist in brown algae. One key protein family likely associated with brown algal innate immunity possesses an NB‐ARC domain analogous to innate immune proteins in plants and animals. In this study, we conducted an extensive survey of NB‐ARC genes in brown algae and obtained insights into the domain organization and evolutionary history of the encoded proteins. Our data show that brown algae possess an ancient NB‐ARC‐tetratricopeptide repeat (NB‐TPR) domain architecture. We identified an N‐terminal effector domain, the four‐helix bundle, which was not previously found associated with NB‐ARC domains. The phylogenetic tree including NB‐ARC domains from all kingdoms of life suggests the three clades of brown algal NB‐TPRs are likely monophyletic, whereas their TPRs seem to have distinct origins. One group of TPRs exhibit intense exon shuffling, with various alternative splicing and ersifying selection acting on them, suggesting exon shuffling is an important mechanism for evolving ligand‐binding specificities. The reconciliation of gene duplication and loss events of the NB‐ARC genes reveals that more independent gene gains than losses have occurred during brown algal evolution, and that tandem duplication has played a major role in the expansion of NB‐ARC genes. Our results substantially enhance our understanding of the evolutionary history and exon shuffling mechanisms of the candidate innate immune repertoire of brown algae.

Neurotoxin-mediated potent activation of the axon degeneration regulator SARM1

Publisher: eLife Sciences Publications, Ltd

Date: 06-12-2021

DOI: 10.7554/ELIFE.72823

Abstract: Axon loss underlies symptom onset and progression in many neurodegenerative disorders. Axon degeneration in injury and disease is promoted by activation of the NAD-consuming enzyme SARM1. Here, we report a novel activator of SARM1, a metabolite of the pesticide and neurotoxin vacor. Removal of SARM1 completely rescues mouse neurons from vacor-induced neuron and axon death in vitro and in vivo. We present the crystal structure of the Drosophila SARM1 regulatory domain complexed with this activator, the vacor metabolite VMN, which as the most potent activator yet known is likely to support drug development for human SARM1 and NMNAT2 disorders. This study indicates the mechanism of neurotoxicity and pesticide action by vacor, raises important questions about other pyridines in wider use today, provides important new tools for drug discovery, and demonstrates that removing SARM1 can robustly block programmed axon death induced by toxicity as well as genetic mutation.

Structural Basis of Interaction of Bipartite Nuclear Localization Signal from Agrobacterium VirD2 with Rice Importin-α

Publisher: Elsevier BV

Date: 06-2014

DOI: 10.1093/MP/SSU014

Blood group antigen recognition via the group a streptococcal m protein mediates host colonization

Publisher: American Society for Microbiology

Date: 08-03-2017

DOI: 10.1128/MBIO.02237-16

Abstract: Streptococcus pyogenes (group A streptococcus [GAS]) is responsible for over 500,000 deaths worldwide each year. The highly virulent M1T1 GAS clone is one of the most frequently isolated serotypes from streptococcal pharyngitis and invasive disease. The oral epithelial tract is a niche highly abundant in glycosylated structures, particularly those of the ABO(H) blood group antigen family. Using a high-throughput approach, we determined that a strain representative of the globally disseminated M1T1 GAS clone 5448 interacts with numerous, structurally erse glycans. Preeminent among GAS virulence factors is the surface-expressed M protein. M1 protein showed high affinity for several terminal galactose blood group antigen structures. Deletion mutagenesis shows that M1 protein mediates glycan binding via its B repeat domains. Association of M1T1 GAS with oral epithelial cells varied significantly as a result of phenotypic differences in blood group antigen expression, with significantly higher adherence to those cells expressing H antigen structures compared to cells expressing A, B, or AB antigen structures. These data suggest a novel mechanism for GAS attachment to host cells and propose a link between host blood group antigen expression and M1T1 GAS colonization. IMPORTANCE There has been a resurgence in group A streptococcal (GAS) invasive disease, which has been paralleled by the emergence of the highly virulent M1T1 GAS clone. Intensive research has focused on mechanisms that contribute to the invasive nature of this serotype, while the mechanisms that contribute to host susceptibility to disease and bacterial colonization and persistence are still poorly understood. The M1T1 GAS clone is frequently isolated from the throat, an environment highly abundant in blood group antigen structures. This work examined the interaction of the M1 protein, the preeminent GAS surface protein, against a wide range of host-expressed glycan structures. Our data suggest that susceptibility to infection by GAS in the oral tract may correlate with phenotypic differences in host blood group antigen expression. Thus, variations in host blood group antigen expression may serve as a selective pressure contributing to the dissemination and overrepresentation of M1T1 GAS.

Structural Evolution of TIR-Domain Signalosomes

Publisher: Frontiers Media SA

Date: 17-11-2021

DOI: 10.3389/FIMMU.2021.784484

Abstract: TIR (Toll/interleukin-1 receptor/resistance protein) domains are cytoplasmic domains widely found in animals and plants, where they are essential components of the innate immune system. A key feature of TIR-domain function in signaling is weak and transient self-association and association with other TIR domains. An additional new role of TIR domains as catalytic enzymes has been established with the recent discovery of NAD + -nucleosidase activity by several TIR domains, mostly involved in cell-death pathways. Although self-association of TIR domains is necessary in both cases, the functional specificity of TIR domains is related in part to the nature of the TIR : TIR interactions in the respective signalosomes. Here, we review the well-studied TIR domain-containing proteins involved in eukaryotic immunity, focusing on the structures, interactions and their corresponding functional roles. Structurally, the signalosomes fall into two separate groups, the scaffold and enzyme TIR-domain assemblies, both of which feature open-ended complexes with two strands of TIR domains, but differ in the orientation of the two strands. We compare and contrast how TIR domains assemble and signal through distinct scaffolding and enzymatic roles, ultimately leading to distinct cellular innate-immunity and cell-death outcomes.

Predikin and PredikinDB: A computational framework for the prediction of protein kinase peptide specificity and an associated database of phosphorylation sites

Publisher: Springer Science and Business Media LLC

Date: 26-05-2008

DOI: 10.1186/1471-2105-9-245

An Autoactive Mutant of the M Flax Rust Resistance Protein Has a Preference for Binding ATP, Whereas Wild-Type M Protein Binds ADP

Publisher: Scientific Societies

Date: 08-2011

DOI: 10.1094/MPMI-03-11-0052

Abstract: Resistance (R) proteins are key regulators of the plant innate immune system and are capable of pathogen detection and activation of the hypersensitive cell death immune response. To understand the molecular mechanism of R protein activation, we undertook a phenotypic and biochemical study of the flax nucleotide binding (NB)-ARC leucine-rich repeat protein, M. Using Agrobacterium-mediated transient expression in flax cotyledons, site-directed mutations of key residues within the P-loop, kinase 2, and MHD motifs within the NB-ARC domain of M were shown to affect R protein function. When purified using a yeast expression system and assayed for ATP and ADP, these mutated proteins exhibited marked differences in the quantity and identity of the bound nucleotide. ADP was bound to recombinant wild-type M protein, while the nonfunctional P-loop mutant did not have any nucleotides bound. In contrast, ATP was bound to an autoactive M protein mutated in the highly conserved MHD motif. These data provide direct evidence supporting a model of R protein function in which the “off” R protein binds ADP and activation of R protein defense signaling involves the exchange of ADP for ATP.

Cortactin adopts a globular conformation and bundles actin into sheets

Publisher: Elsevier BV

Date: 06-2008

DOI: 10.1074/JBC.M708917200

Structural basis of nuclear import of flap endonuclease 1 (FEN1)

Publisher: International Union of Crystallography (IUCr)

Date: 15-06-2012

DOI: 10.1107/S0907444912010281

International year of crystallography

Publisher: CSIRO Publishing

Date: 2014

DOI: 10.1071/CH14557

NAD ⁺ cleavage activity by animal and plant TIR domains in cell death pathways

Publisher: American Association for the Advancement of Science (AAAS)

Date: 23-08-2019

DOI: 10.1126/SCIENCE.AAX1911

Abstract: One way that plants respond to pathogen infection is by sacrificing the infected cells. The nucleotide-binding leucine-rich repeat immune receptors responsible for this hypersensitive response carry Toll/interleukin-1 receptor (TIR) domains. In two papers, Horsefield et al. and Wan et al. report that these TIR domains cleave the metabolic cofactor nicotinamide adenine dinucleotide (NAD + ) as part of their cell-death signaling in response to pathogens. Similar signaling links mammalian TIR-containing proteins to NAD + depletion during Wallerian degeneration of neurons. Science , this issue p. 793 , p. 799

De novo GTP Biosynthesis Is Critical for Virulence of the Fungal Pathogen Cryptococcus neoformans

Publisher: Public Library of Science (PLoS)

Date: 11-10-2012

DOI: 10.1371/JOURNAL.PPAT.1002957

Structure and Function of the TIR Domain from the Grape NLR Protein RPV1

Publisher: Frontiers Media SA

Date: 08-12-2016

DOI: 10.3389/FPLS.2016.01850

Recombinant production of functional full-length and truncated human TRAM/TICAM-2 adaptor protein involved in Toll-like receptor and interferon signaling

Publisher: Elsevier BV

Date: 02-2015

DOI: 10.1016/J.PEP.2014.09.019

Abstract: TRAM/TICAM-2 is used by Toll-like receptor 4 (TLR4) as a bridging adaptor during the mammalian innate immune response. It recruits TRIF, another TIR domain-containing adaptor protein, to TLR4 via TIR domain interactions, which leads to the activation of transcription factors responsible for the production of type-1 interferon and cytokines. The molecular mechanisms of these dual interactions mediated by the TRAM TIR domain are not clear. To understand the molecular basis of TIR:TIR domain interactions, structural and biochemical studies of TRAM TIR domain are necessary, and require a functional soluble protein. In this paper, we report a successful purification and characterization of full-length TRAM. Because full-length TRAM likely contains unstructured regions that may be disadvantageous for structural studies, we also carried out a systematic construct design to determine the boundaries of the TRAM TIR domain. The truncated TRAM constructs were designed based on secondary structure predictions and screened by small-scale expression. Selected constructs were subjected to biophysical analyses. We show that the expressed TRAM TIR domain is functional using in vitro GST pull-down assays that demonstrate a physical interaction with the TLR4 TIR domain. We further show, by site-directed mutagenesis, that the "BB loop" regions of both the TRAM TIR domain and the TLR4 TIR domain are crucial for this physical interaction.

Enhancing the Thermal and Kinetic Stability of Ketol-Acid Reductoisomerase, a Central Catalyst of a Cell-Free Enzyme Cascade for the Manufacture of Platform Chemicals

Publisher: MDPI AG

Date: 10-08-2022

DOI: 10.3390/APPLBIOSCI1020011

Abstract: The branched-chain amino acids (BCAAs) leucine, isoleucine and valine are synthesized via a common biosynthetic pathway. Ketol-acid reductoisomerase (KARI) is the second enzyme in this pathway. In addition to its role in BCAA biosynthesis, KARI catalyzes two rate-limiting steps that are key components of a cell-free biofuel biosynthesis route. For industrial applications, reaction temperature and enzyme stability are key factors that affect process robustness and product yield. Here, we have solved the cryo-EM structure (2.94 Å resolution) of a homododecameric Class I KARI (from C ylobacter jejuni) and demonstrated how a triad of amino acid side chains plays a crucial role in promoting the oligomerization of this enzyme. Importantly, both its thermal and solvent stability are greatly enhanced in the dodecameric state when compared to its dimeric counterpart (apparent melting temperatures (Tm) of 83.1 °C and 51.5 °C, respectively). We also employed protein design (PROSS) for a tetrameric Class II KARI (from Escherichia coli) to generate a variant with improved thermal and solvent stabilities. In total, 34 mutations were introduced, which did not affect the oligomeric state of this enzyme but resulted in a fully functional catalyst with a significantly elevated Tm (58.5 °C vs. 47.9 °C for the native version).

MyD88 TIR domain higher-order assembly interactions revealed by microcrystal electron diffraction and serial femtosecond crystallography

Publisher: Springer Science and Business Media LLC

Date: 10-05-2021

DOI: 10.1038/S41467-021-22590-6

Abstract: MyD88 and MAL are Toll-like receptor (TLR) adaptors that signal to induce pro-inflammatory cytokine production. We previously observed that the TIR domain of MAL (MAL TIR ) forms filaments in vitro and induces formation of crystalline higher-order assemblies of the MyD88 TIR domain (MyD88 TIR ). These crystals are too small for conventional X-ray crystallography, but are ideally suited to structure determination by microcrystal electron diffraction (MicroED) and serial femtosecond crystallography (SFX). Here, we present MicroED and SFX structures of the MyD88 TIR assembly, which reveal a two-stranded higher-order assembly arrangement of TIR domains analogous to that seen previously for MAL TIR . We demonstrate via mutagenesis that the MyD88 TIR assembly interfaces are critical for TLR4 signaling in vivo, and we show that MAL promotes unidirectional assembly of MyD88 TIR . Collectively, our studies provide structural and mechanistic insight into TLR signal transduction and allow a direct comparison of the MicroED and SFX techniques.

Structural basis of autoregulation of phenylalanine hydroxylase.

Publisher: Springer Science and Business Media LLC

Date: 05-1999

DOI: 10.1038/8247

Abstract: Phenylalanine hydroxylase converts phenylalanine to tyrosine, a rate-limiting step in phenylalanine catabolism and protein and neurotransmitter biosynthesis. It is tightly regulated by the substrates phenylalanine and tetrahydrobiopterin and by phosphorylation. We present the crystal structures of dephosphorylated and phosphorylated forms of a dimeric enzyme with catalytic and regulatory properties of the wild-type protein. The structures reveal a catalytic domain flexibly linked to a regulatory domain. The latter consists of an N-terminal autoregulatory sequence (containing Ser 16, which is the site of phosphorylation) that extends over the active site pocket, and an alpha-beta sandwich core that is, unexpectedly, structurally related to both pterin dehydratase and the regulatory domains of metabolic enzymes. Phosphorylation has no major structural effects in the absence of phenylalanine, suggesting that phenylalanine and phosphorylation act in concert to activate the enzyme through a combination of intrasteric and possibly allosteric mechanisms.

Intramolecular interaction influences binding of the Flax L5 and L6 resistance proteins to their AvrL567 ligands.

Publisher: Public Library of Science (PLoS)

Date: 29-11-2012

DOI: 10.1371/JOURNAL.PPAT.1003004

Improved Success of Sparse Matrix Protein Crystallization Screening with Heterogeneous Nucleating Agents

Publisher: Public Library of Science (PLoS)

Date: 31-10-2007

DOI: 10.1371/JOURNAL.PONE.0001091

Crystallization of a trimeric human T cell leukemia virus type 1 gp21 ectodomain fragment as a chimera with maltose‐binding protein

Publisher: Wiley

Date: 07-1998

DOI: 10.1002/PRO.5560070715

Abstract: We present a novel protein crystallization strategy, applied to the crystallization of human T cell leukemia virus type 1 (HTLV-1) transmembrane protein gp21 lacking the fusion peptide and the transmembrane domain, as a chimera with the Escherichia coli maltose binding protein (MBP). Crystals could not be obtained with a MBP/gp21 fusion protein in which fusion partners were separated by a flexible linker, but were obtained after connecting the MBP C-terminal alpha-helix to the predicted N-terminal alpha-helical sequence of gp21 via three alanine residues. The gp21 sequences conferred a trimeric structure to the soluble fusion proteins as assessed by sedimentation equilibrium and X-ray diffraction, consistent with the trimeric structures of other retroviral transmembrane proteins. The envelope protein precursor, gp62, is likewise trimeric when expressed in mammalian cells. Our results suggest that MBP may have a general application for the crystallization of proteins containing N-terminal alpha-helical sequences.

The AvrM Effector from Flax Rust Has a Structured C-Terminal Domain and Interacts Directly with the M Resistance Protein

Publisher: Scientific Societies

Date: 2010

DOI: 10.1094/MPMI-23-1-0049

Abstract: In plant immunity, recognition of pathogen effectors by plant resistance proteins leads to the activation of plant defenses and a localized cell death response. The AvrM effector from flax rust is a small secreted protein that is recognized by the M resistance protein in flax. Here, we investigate the mechanism of M–AvrM recognition and show that these two proteins directly interact in a yeast two-hybrid assay, and that this interaction correlates with the recognition specificity observed for each of the different AvrM variants. We further characterize this interaction by demonstrating that the C-terminal domain of AvrM is required for M-dependent cell death, and show that this domain also interacts with the M protein in yeast. We investigate the role of C-terminal differences among the different AvrM proteins for their involvement in this interaction and establish that M recognition is hindered by an additional 34 amino acids present at the C terminus of several AvrM variants. Structural characterization of recombinant AvrM-A protein revealed a globular C-terminal domain that dimerizes.

Structural interpretation of mutations in phenylalanine hydroxylase protein aids in identifying genotype–phenotype correlations in phenylketonuria

Publisher: Springer Science and Business Media LLC

Date: 31-08-2000

DOI: 10.1038/SJ.EJHG.5200518

Abstract: Phenylalanine hydroxylase (PAH) is the enzyme that converts phenylalanine to tyrosine as a rate-limiting step in phenylalanine catabolism and protein and neurotransmitter biosynthesis. Over 300 mutations have been identified in the gene encoding PAH that result in a deficient enzyme activity and lead to the disorders hyperphenylalaninaemia and phenylketonuria. The determination of the crystal structure of PAH now allows the determination of the structural basis of mutations resulting in PAH deficiency. We present an analysis of the structural basis of 120 mutations with a 'classified' biochemical phenotype and/or available in vitro expression data. We find that the mutations can be grouped into five structural categories, based on the distinct expected structural and functional effects of the mutations in each category. Missense mutations and small amino acid deletions are found in three categories: 'active site mutations', 'dimer interface mutations', and 'domain structure mutations'. Nonsense mutations and splicing mutations form the category of 'proteins with truncations and large deletions'. The final category, 'fusion proteins', is caused by frameshift mutations. We show that the structural information helps formulate some rules that will help predict the likely effects of unclassified and newly discovered mutations: proteins with truncations and large deletions, fusion proteins and active site mutations generally cause severe phenotypes domain structure mutations and dimer interface mutations spread over a range of phenotypes, but domain structure mutations in the catalytic domain are more likely to be severe than domain structure mutations in the regulatory domain or dimer interface mutations.

A bioinformatics tool for epitope-based vaccine design that accounts for human ethnic diversity: Application to emerging infectious diseases

Publisher: Elsevier BV

Date: 03-2015

DOI: 10.1016/J.VACCINE.2015.01.040

Abstract: Peptide vaccination based on multiple T-cell epitopes can be used to target well-defined ethnic populations. Because the response to T-cell epitopes is restricted by HLA proteins, the HLA specificity of T-cell epitopes becomes a major consideration for epitope-based vaccine design. We have previously shown that CD4+ T-cell epitopes restricted by 95% of human MHC class II proteins can be predicted with high-specificity. We describe here the integration of epitope prediction with population coverage and epitope selection algorithms. The population coverage assessment makes use of the Allele Frequency Net Database. We present the computational platform Pre ac-2.0 for HLA class II-restricted epitope-based vaccine design, which accounts comprehensively for human genetic ersity. We validated the performance of the tool on the identification of promiscuous and immunodominant CD4+ T-cell epitopes from the human immunodeficiency virus (HIV) protein Gag. We further describe an application for epitope-based vaccine design in the context of emerging infectious diseases associated with Lassa, Nipah and Hendra viruses. Putative CD4+ T-cell epitopes were mapped on the surface glycoproteins of these pathogens and are good candidates to be experimentally tested, as they hold potential to provide cognate help in vaccination settings in their respective target populations. Pre ac-2.0 is a novel approach in epitope-based vaccine design, particularly suited to be applied to virus-related emerging infectious diseases, because the geographic distributions of the viruses are well defined and ethnic populations in need of vaccination can be determined ("ethnicity-oriented approach"). Pre ac-2.0 is accessible through the website pre ac.biosci.uq.edu.au/.

Verprolin Cytokinesis Function Mediated by the Hof One Trap Domain

Publisher: Wiley

Date: 21-05-2005

DOI: 10.1111/J.1600-0854.2005.00300.X

Abstract: In budding yeast, partitioning of the cytoplasm during cytokinesis can proceed via a pathway dependent on the contractile actomyosin ring, as in other eukaryotes, or alternatively via a septum deposition pathway dependent on an SH3 domain protein, Hof1/Cyk2 (the yeast PSTPIP1 ortholog). In iding yeast cells, Hof1 forms a ring at the bud neck distinct from the actomyosin ring, and this zone is active in septum deposition. We previously showed the yeast Wiskott-Aldrich syndrome protein (WASP)-interacting protein (WIP) ortholog, verprolin/Vrp1/End5, interacts with Hof1 and facilitates Hof1 recruitment to the bud neck. A Vrp1 fragment unable to interact with yeast WASP (Las17/Bee1), localize to the actin cytoskeleton or function in polarization of the cortical actin cytoskeleton nevertheless retains function in Hof1 recruitment and cytokinesis. Here, we show the ability of this Vrp1 fragment to bind the Hof1 SH3 domain via its Hof one trap (HOT) domain is critical for cytokinesis. The Vrp1 HOT domain consists of three tandem proline-rich motifs flanked by serines. Unexpectedly, the Hof1 SH3 domain itself is not required for cytokinesis and indeed appears to negatively regulate cytokinesis. The Vrp1 HOT domain promotes cytokinesis by binding to the Hof1 SH3 domain and counteracting its inhibitory effect.

Autoimmunity and effector recognition in Arabidopsis thaliana can be uncoupled by mutations in the RRS1-R immune receptor.

Publisher: Wiley

Date: 03-02-2019

DOI: 10.1111/NPH.15617

Abstract: Plant nucleotide-binding leucine-rich repeat (NLR) disease resistance proteins recognize specific pathogen effectors and activate a cellular defense program. In Arabidopsis thaliana (Arabidopsis), Resistance to Ralstonia solanacearum 1 (RRS1-R) and Resistance to Pseudomonas syringae 4 (RPS4) function together to recognize the unrelated bacterial effectors PopP2 and AvrRps4. In the plant cell nucleus, the RRS1-R/RPS4 complex binds to and signals the presence of AvrRps4 or PopP2. The exact mechanism underlying NLR signaling and immunity activation remains to be elucidated. Using genetic and biochemical approaches, we characterized the intragenic suppressors of sensitive to low humidity 1 (slh1), a temperature-sensitive autoimmune allele of RRS1-R. Our analyses identified five amino acid residues that contribute to RRS1-R

The TLR signalling adaptor TRIF/TICAM-1 has an N-terminal helical domain with structural similarity to IFIT proteins

Publisher: International Union of Crystallography (IUCr)

Date: 19-11-2013

DOI: 10.1107/S0907444913022385

Abstract: TRIF/TICAM-1 (TIR domain-containing adaptor inducing interferon-β/TIR domain-containing adaptor molecule 1) is the adaptor protein in the Toll-like receptor (TLR) 3 and 4 signalling pathway that leads to the production of type 1 interferons and cytokines. The signalling involves TIR (Toll/interleukin-1 receptor) domain-dependent TRIF oligomerization. A protease-resistant N-terminal region is believed to be involved in self-regulation of TRIF by interacting with its TIR domain. Here, the structural and functional characterization of the N-terminal domain of TRIF (TRIF-NTD) comprising residues 1–153 is reported. The 2.22 Å resolution crystal structure was solved by single-wavelength anomalous diffraction (SAD) using selenomethionine-labelled crystals of TRIF-NTD containing two additional introduced Met residues (TRIF-NTD A66M/L113M ). The structure consists of eight antiparallel helices that can be ided into two subdomains, and the overall fold shares similarity to the interferon-induced protein with tetratricopeptide repeats (IFIT) family of proteins, which are involved in both the recognition of viral RNA and modulation of innate immune signalling. Analysis of TRIF-NTD surface features and the mapping of sequence conservation onto the structure suggest several possible binding sites involved in either TRIF auto-regulation or interaction with other signalling molecules or ligands. TRIF-NTD suppresses TRIF-mediated activation of the interferon-β promoter, as well as NF-κB-dependent reporter-gene activity. These findings thus identify opportunities for the selective targeting of TLR3- and TLR4-mediated inflammation.

Prokaryotic Substrate-Binding Proteins as Targets for Antimicrobial Therapies

Publisher: Bentham Science Publishers Ltd.

Date: 08-2012

DOI: 10.2174/138945012803530170

Abstract: The rapid emergence of multidrug-resistant bacteria over the last two decades has catalyzed a shift away from traditional antibiotic development strategies and encouraged the search for unconventional drug targets. Prokaryotic substrate- binding proteins (SBPs), together with their cognate ATP-binding cassette (ABC) transporters, facilitate the unidirectional, transbilayer movement of specific extracytosolic cargoes against a concentration gradient, powered by ATP hydrolysis. In Gram-negative bacteria, SBPs are found in the periplasmic space, whereas in Gram-positive organisms these proteins are anchored to the outer cell wall by a lipid moiety. SBPs are vital components of the substrate-translocation machinery, as they determine cargo specificity and are involved in coupling the cargo uptake process with ABC transporter- mediated ATP hydrolysis. In this review, we focus on "Cluster A-1" alent metal-binding proteins from within the SBP family. Acquisition of transition row metal ions is essential for bacterial colonization and virulence and Cluster A-1 SBPs play an integral role in this process. Cluster A-1 SBPs lack homologs in humans, bypass the need to deliver compounds into the bacterial cell, and are therefore potential drug targets against Gram-positive bacteria. Here we discuss the role SBPs play in the prokaryotic substrate-translocation machinery with emphasis in the substrate-binding mechanism of Cluster A-1 SBPs, the role of these proteins in virulence and their potential use as drug targets.

Importin-β is a GDP-to-GTP exchange factor of Ran. Implications for the mechanism of nuclear import

Publisher: Elsevier BV

Date: 08-2009

DOI: 10.1074/JBC.M109.019935

Phosphorylation adjacent to the nuclear localization signal of human dUTPase abolishes nuclear import: structural and mechanistic insights

Publisher: International Union of Crystallography (IUCr)

Date: 19-11-2013

DOI: 10.1107/S0907444913023354

Abstract: Phosphorylation adjacent to nuclear localization signals (NLSs) is involved in the regulation of nucleocytoplasmic transport. The nuclear isoform of human dUTPase, an enzyme that is essential for genomic integrity, has been shown to be phosphorylated on a serine residue (Ser11) in the vicinity of its nuclear localization signal however, the effect of this phosphorylation is not yet known. To investigate this issue, an integrated set of structural, molecular and cell biological methods were employed. It is shown that NLS-adjacent phosphorylation of dUTPase occurs during the M phase of the cell cycle. Comparison of the cellular distribution of wild-type dUTPase with those of hyperphosphorylation- and hypophosphorylation-mimicking mutants suggests that phosphorylation at Ser11 leads to the exclusion of dUTPase from the nucleus. Isothermal titration microcalorimetry and additional independent biophysical techniques show that the interaction between dUTPase and importin-α, the karyopherin molecule responsible for `classical' NLS binding, is weakened significantly in the case of the S11E hyperphosphorylation-mimicking mutant. The structures of the importin-α–wild-type and the importin-α–hyperphosphorylation-mimicking dUTPase NLS complexes provide structural insights into the molecular details of this regulation. The data indicate that the post-translational modification of dUTPase during the cell cycle may modulate the nuclear availability of this enzyme.

DNA mismatch repair proteins MLH1 and PMS2 can be imported to the nucleus by a classical nuclear import pathway

Publisher: Elsevier BV

Date: 03-2018

DOI: 10.1016/J.BIOCHI.2017.11.013

Abstract: MLH1 and PMS2 proteins form the MutLα heterodimer, which plays a major role in DNA mismatch repair (MMR) in humans. Mutations in MMR-related proteins are associated with cancer, especially with colon cancer. The N-terminal region of MutLα comprises the N-termini of PMS2 and MLH1 and, similarly, the C-terminal region of MutLα is composed by the C-termini of PMS2 and MLH1, and the two are connected by linker region. The nuclear localization sequences (NLSs) necessary for the nuclear transport of the two proteins are found in this linker region. However, the exact NLS sequences have been controversial, with different sequences reported, particularly for MLH1. The in idual components are not imported efficiently, presumably due to their C-termini masking their NLSs. In order to gain insights into the nuclear transport of these proteins, we solved the crystal structures of importin-α bound to peptides corresponding to the supposed NLSs of MLH1 and PMS2 and performed isothermal titration calorimetry to study their binding affinities. Both putative MLH1 and PMS2 NLSs can bind to importin-α as monopartite NLSs, which is in agreement with some previous studies. However, MLH1-NLS has the highest affinity measured by a natural NLS peptide, suggesting a major role of MLH1 protein in nuclear import compared to PMS2. Finally, the role of MLH1 and PMS2 in the nuclear transport of the MutLα heterodimer is discussed.

Functional Analysis, Using in Vitro Mutagenesis, of Amino Acids Located in the Phenylalanine Hydroxylase Active Site

Publisher: Elsevier BV

Date: 12-2000

DOI: 10.1006/ABBI.2000.2111

Abstract: The 3-dimensional structure determination of rat phenylalanine hydroxylase (PAH) has identified potentially important amino acids lining the active site cleft with the majority of these having hydrophobic side-chains including several with aromatic side chains. Here we have analyzed the effect on rat PAH enzyme kinetics of in vitro mutagenesis of a number of these amino acids lining the PAH active site. Mutation of F299, Y324, F331, and Y343 caused a significant decrease in enzyme activity but no change in the Km for substrate or cofactor. We conclude that these aromatic residues are essential for activity but are not significantly involved in binding of the substrate or cofactor. In contrast the PAH mutant, S349T, showed an 18-fold increase in Km for phenylalanine, showing the first functional evidence that this residue was binding at or near the phenylalanine binding site. This confirms the recently published model for the binding of phenylalanine to the PAH active site that postulated S349 interacts with the amino group on the main chain of the phenylalanine molecule. This result differs with that found for the equivalent mutation (S395T), in the closely related tyrosine hydroxylase, which had no effect on substrate Km, showing that while the architecture of the two active sites are very similar the amino acids that bind to the respective substrates are different.

Frequent somatic mutations in MAP3K5 and MAP3K9 in metastatic melanoma identified by exome sequencing

Publisher: Springer Science and Business Media LLC

Date: 25-12-2012

DOI: 10.1038/NG.1041

Structural Basis of Importin-α-Mediated Nuclear Transport for Ku70 and Ku80

Publisher: Elsevier BV

Date: 09-2011

DOI: 10.1016/J.JMB.2011.07.038

Abstract: Ku70 and Ku80 form a heterodimeric complex involved in multiple nuclear processes. This complex plays a key role in DNA repair due to its ability to bind DNA double-strand breaks and facilitate repair by the nonhomologous end-joining pathway. Ku70 and Ku80 have been proposed to contain bipartite and monopartite nuclear localization sequences (NLSs), respectively, that allow them to be translocated to the nucleus independently of each other via the classical importin-α (Impα)/importin-β-mediated nuclear import pathway. To determine the structural basis of the recognition of Ku70 and Ku80 proteins by Impα, we solved the crystal structures of the complexes of Impα with the peptides corresponding to the Ku70 and Ku80 NLSs. Our structural studies confirm the binding of the Ku80 NLS as a classical monopartite NLS but reveal an unexpected binding mode for Ku70 NLS with only one basic cluster bound to the receptor. Both Ku70 and Ku80 therefore contain monopartite NLSs, and sequences outside the basic cluster make favorable interactions with Impα, suggesting that this may be a general feature in monopartite NLSs. We show that the Ku70 NLS has a higher affinity for Impα than the Ku80 NLS, consistent with more extensive interactions in its N-terminal region. The prospect of nuclear import of Ku70 and Ku80 independently of each other provides a powerful regulatory mechanism for the function of the Ku70/Ku80 heterodimer and independent functions of the two proteins.

A linker strategy for the production and crystallization of Toll/interleukin-1 receptor/resistance protein domain complexes

Publisher: Oxford University Press (OUP)

Date: 15-03-2015

DOI: 10.1093/PROTEIN/GZV013

Abstract: Structural characterization of protein-protein complexes is required to fully understand biological processes. However, such studies can be difficult, particularly when the interactions are transient. In some cases, the covalent linking of weakly interacting binding partners has been shown to facilitate structural studies. Here, we used this approach to investigate, by X-ray crystallography, the interactions between TIR (Toll/interleukin-1 receptor/resistance protein) domains from proteins involved in plant and animal innate immunity. Combinations of TIR domains known to interact were covalently attached using short glycine- and serine-rich linkers. This approach enabled the production of a number of TIR-TIR domain complexes in soluble form, facilitating crystallization studies. Crystallization of two of the tested combinations was achieved. Furthermore, production in soluble form was achieved for another two combinations, where this was not possible for in idual proteins. Our results demonstrate that the linker strategy can aid in the structural studies of TIR domains. Similarly, this approach has potential for improving protein production and facilitating structural studies of other protein-protein interaction domains.

Structural basis of recognition of monopartite and bipartite nuclear localization sequences by mammalian importin-α11Edited by K. Nagai

Publisher: Elsevier BV

Date: 04-2000

DOI: 10.1006/JMBI.2000.3642

Molybdenum Enzymes and How They Support Virulence in Pathogenic Bacteria.

Publisher: Frontiers Media SA

Date: 11-12-2020

DOI: 10.3389/FMICB.2020.615860

Abstract: Mononuclear molybdoenzymes are highly versatile catalysts that occur in organisms in all domains of life, where they mediate essential cellular functions such as energy generation and detoxification reactions. Molybdoenzymes are particularly abundant in bacteria, where over 50 distinct types of enzymes have been identified to date. In bacterial pathogens, all aspects of molybdoenzyme biology such as molybdate uptake, cofactor biosynthesis, and function of the enzymes themselves, have been shown to affect fitness in the host as well as virulence. Although current studies are mostly focused on a few key pathogens such as Escherichia coli, Salmonella enterica, C ylobacter jejuni , and Mycobacterium tuberculosis , some common themes for the function and adaptation of the molybdoenzymes to pathogen environmental niches are emerging. Firstly, for many of these enzymes, their role is in supporting bacterial energy generation and the corresponding pathogen fitness and virulence defects appear to arise from a suboptimally poised metabolic network. Secondly, all substrates converted by virulence-relevant bacterial Mo enzymes belong to classes known to be generated in the host either during inflammation or as part of the host signaling network, with some enzyme groups showing adaptation to the increased conversion of such substrates. Lastly, a specific adaptation to bacterial in-host survival is an emerging link between the regulation of molybdoenzyme expression in bacterial pathogens and the presence of immune system-generated reactive oxygen species. The prevalence of molybdoenzymes in key bacterial pathogens including ESKAPE pathogens, paired with the mounting evidence of their central roles in bacterial fitness during infection, suggest that they could be important future drug targets.

The Plant Resistosome: Structural Insights into Immune Signaling

Publisher: Elsevier BV

Date: 08-2019

DOI: 10.1016/J.CHOM.2019.07.020

Abstract: Plant innate immunity is triggered via direct or indirect recognition of pathogen effectors by the NLR family immune receptors. Mechanistic understanding of plant NLR function has relied on structural information from in idual NLR domains and inferences from studies on animal NLRs. Recent reports of the cryo-EM structures of the Arabidopsis plant immune receptor ZAR1 in monomeric inactive and transition states, as well as the active oligomeric state or the "resistosome," have afforded a quantum leap in our understanding of how plant NLRs function. In this Review, we outline the recent structural findings and examine their implications for the activation of plant immune receptors more broadly. We also discuss how NLR signaling in plants, as illustrated by the ZAR1 structure, is analogous to innate immune receptor signaling mechanisms across kingdoms, drawing particular attention to the concept of signaling by cooperative assembly formation.

Structural basis of NLR activation and innate immune signalling in plants

Publisher: Springer Science and Business Media LLC

Date: 04-01-2022

DOI: 10.1007/S00251-021-01242-5

Abstract: Animals and plants have NLRs (nucleotide-binding leucine-rich repeat receptors) that recognize the presence of pathogens and initiate innate immune responses. In plants, there are three types of NLRs distinguished by their N-terminal domain: the CC (coiled-coil) domain NLRs, the TIR (Toll/interleukin-1 receptor) domain NLRs and the RPW8 (resistance to powdery mildew 8)-like coiled-coil domain NLRs. CC-NLRs (CNLs) and TIR-NLRs (TNLs) generally act as sensors of effectors secreted by pathogens, while RPW8-NLRs (RNLs) signal downstream of many sensor NLRs and are called helper NLRs. Recent studies have revealed three dimensional structures of a CNL (ZAR1) including its inactive, intermediate and active oligomeric state, as well as TNLs (RPP1 and ROQ1) in their active oligomeric states. Furthermore, accumulating evidence suggests that members of the family of lipase-like EDS1 (enhanced disease susceptibility 1) proteins, which are uniquely found in seed plants, play a key role in providing a link between sensor NLRs and helper NLRs during innate immune responses. Here, we summarize the implications of the plant NLR structures that provide insights into distinct mechanisms of action by the different sensor NLRs and discuss plant NLR-mediated innate immune signalling pathways involving the EDS1 family proteins and RNLs.

Open up review system

Publisher: Springer Science and Business Media LLC

Date: 12-1998

DOI: 10.1038/24969

An automatable screen for the rapid identification of proteins amenable to refolding

Publisher: Wiley

Date: 03-2006

DOI: 10.1002/PMIC.200500056

Abstract: Insoluble expression of heterologous proteins in Escherichia coli is a major bottleneck of many structural genomics and high-throughput protein biochemistry projects. Many of these proteins may be amenable to refolding, but their identification is h ered by a lack of high-throughput methods. We have developed a matrix-assisted refolding approach in which correctly folded proteins are distinguished from misfolded proteins by their elution from affinity resin under non-denaturing conditions. Misfolded proteins remain adhered to the resin, presumably via hydrophobic interactions. The assay can be applied to insoluble proteins on an in idual basis but is particularly well suited for high-throughput applications because it is rapid, automatable and has no rigorous s le preparation requirements. The efficacy of the screen is demonstrated on small-scale expression s les for 15 proteins. Refolding is then validated by large-scale expressions using SEC and circular dichroism.

Mechanism of Bacterial Interference with TLR4 Signaling by Brucella Toll/Interleukin-1 Receptor Domain-containing Protein TcpB

Publisher: Elsevier BV

Date: 2000

DOI: 10.1074/JBC.M113.523274

Incorporating a TEV cleavage site reduces the solubility of nine recombinant mouse proteins

Publisher: Elsevier BV

Date: 11-2006

DOI: 10.1016/J.PEP.2006.05.006

Abstract: Failure to express soluble proteins in bacteria is mainly attributed to the properties of the target protein itself, as well as the choice of the vector, the purification tag and the linker between the tag and protein, and codon usage. The expression of proteins with fusion tags to facilitate subsequent purification steps is a widely used procedure in the production of recombinant proteins. However, the additional residues can affect the properties of the protein therefore, it is often desirable to remove the tag after purification. This is usually done by engineering a cleavage site between the tag and the encoded protein that is recognised by a site-specific protease, such as the one from tobacco etch virus (TEV). In this study, we investigated the effect of four different tags on the bacterial expression and solubility of nine mouse proteins. Two of the four engineered constructs contained hexahistidine tags with either a long or short linker. The other two constructs contained a TEV cleavage site engineered into the linker region. Our data show that inclusion of the TEV recognition site directly downstream of the recombination site of the Invitrogen Gateway vector resulted in a loss of solubility of the nine mouse proteins. Our work suggests that one needs to be very careful when making modifications to expression vectors and combining different affinity and fusion tags and cleavage sites.

Structural characterisation of the nuclear import receptor importin alpha in complex with the bipartite NLS of Prp20

Publisher: Public Library of Science (PLoS)

Date: 10-12-2013

DOI: 10.1371/JOURNAL.PONE.0082038

Chemical structures of cyclic ADP ribose (cADPR) isomers and the molecular basis of their production and signaling

Publisher: Cold Spring Harbor Laboratory

Date: 08-05-2022

DOI: 10.1101/2022.05.07.491051

Abstract: Cyclic ADP ribose (cADPR) isomers are important signaling molecules produced by bacterial and plant Toll/interleukin-1 receptor (TIR) domains via NAD + hydrolysis, yet their chemical structures are unknown. We show that v-cADPR (2’cADPR) and v2-cADPR (3’cADPR) isomers are cyclized by O -glycosidic bond formation between the ribose moieties in ADPR. Structures of v-cADPR (2’cADPR)-producing TIR domains reveal that conformational changes are required for the formation of the active assembly that resembles those of Toll-like receptor adaptor TIR domains, and mutagenesis data demonstrate that a conserved tryptophan is essential for cyclization. We show that v2-cADPR (3’cADPR) is a potent activator of ThsA effector proteins from Thoeris anti-phage defence systems and is responsible for suppression of plant immunity by the effector HopAM1. Collectively, our results define new enzymatic activities of TIR domains, reveal the molecular basis of cADPR isomer production, and establish v2-cADPR (3’cADPR) as an antiviral signaling molecule and an effector-mediated signaling molecule for plant immunity suppression. The chemical structures of two O -glycosidic bond-containing cyclic ADP ribose isomers, the molecular basis of their production, and their function in antiviral and plant immunity suppression by bacteria are reported.

Crystal structure of rice importin-α and structural basis of its interaction with plant-specific nuclear localization signals

Publisher: Oxford University Press (OUP)

Date: 12-2013

DOI: 10.1105/TPC.112.104422

Abstract: In the classical nucleocytoplasmic import pathway, nuclear localization signals (NLSs) in cargo proteins are recognized by the import receptor importin-α. Importin-α has two separate NLS binding sites (the major and the minor site), both of which recognize positively charged amino acid clusters in NLSs. Little is known about the molecular basis of the unique features of the classical nuclear import pathway in plants. We determined the crystal structure of rice (Oryza sativa) importin-α1a at 2-Å resolution. The structure reveals that the autoinhibitory mechanism mediated by the importin-β binding domain of importin-α operates in plants, with NLS-mimicking sequences binding to both minor and major NLS binding sites. Consistent with yeast and mammalian proteins, rice importin-α binds the prototypical NLS from simian virus 40 large T-antigen preferentially at the major NLS binding site. We show that two NLSs, previously described as plant specific, bind to and are functional with plant, mammalian, and yeast importin-α proteins but interact with rice importin-α more strongly. The crystal structures of their complexes with rice importin-α show that they bind to the minor NLS binding site. By contrast, the crystal structures of their complexes with mouse (Mus musculus) importin-α show preferential binding to the major NLS binding site. Our results reveal the molecular basis of a number of features of the classical nuclear transport pathway specific to plants.

Structural and functional analysis of a plant resistance protein TIR domain reveals interfaces for self-association, signaling, and autoregulation.

Publisher: Elsevier BV

Date: 03-2011

DOI: 10.1016/J.CHOM.2011.02.009

PhosphoPICK-SNP: Quantifying the effect of amino acid variants on protein phosphorylation

Publisher: Oxford University Press (OUP)

Date: 10-02-2017

DOI: 10.1093/BIOINFORMATICS/BTX072

Abstract: Genome-wide association studies are identifying single nucleotide variants (SNVs) linked to various diseases, however the functional effect caused by these variants is often unknown. One potential functional effect, the loss or gain of protein phosphorylation sites, can be induced through variations in key amino acids that disrupt or introduce valid kinase binding patterns. Current methods for predicting the effect of SNVs on phosphorylation operate on the sequence content of reference and variant proteins. However, consideration of the amino acid sequence alone is insufficient for predicting phosphorylation change, as context factors determine kinase-substrate selection. We present here a method for quantifying the effect of SNVs on protein phosphorylation through an integrated system of motif analysis and context-based assessment of kinase targets. By predicting the effect that known variants across the proteome have on phosphorylation, we are able to use this background of proteome-wide variant effects to quantify the significance of novel variants for modifying phosphorylation. We validate our method on a manually curated set of phosphorylation change-causing variants from the primary literature, showing that the method predicts known ex les of phosphorylation change at high levels of specificity. We apply our approach to data-sets of variants in phosphorylation site regions, showing that variants causing predicted phosphorylation loss are over-represented among disease-associated variants. The method is freely available as a web-service at the website bioinf.scmb.uq.edu.au hosphopick/snp Supplementary data are available at Bioinformatics online.

Multiple functional self-association interfaces in plant TIR domains

Publisher: Proceedings of the National Academy of Sciences

Date: 03-02-2017

DOI: 10.1073/PNAS.1621248114

Abstract: Toll/interleukin-1 receptor/resistance protein (TIR) domains are present in plant and animal innate immunity receptors and appear to play a scaffold function in defense signaling. In both systems, self-association of TIR domains is crucial for their function. In plants, the TIR domain is associated with intracellular immunity receptors, known as nucleotide-binding oligomerization domain-like receptors (NLRs). Previous studies from several plant NLRs have identified two distinct interfaces that are required for TIR:TIR dimerization in different NLRs. We show that the two interfaces previously identified are both important for self-association and defense signaling of multiple TIR–NLR proteins. Collectively, this work suggests that there is a common mechanism of TIR domain self-association in signaling across the TIR–NLR class of receptor proteins.

Predicting Protein Kinase Specificity: Predikin Update and Performance in the DREAM4 Challenge

Publisher: Public Library of Science (PLoS)

Date: 28-07-2011

DOI: 10.1371/JOURNAL.PONE.0021169

Ca2+ /S100 regulation of giant protein kinases

Publisher: Springer Science and Business Media LLC

Date: 04-1996

DOI: 10.1038/380636A0

Abstract: Protein phosphorylation by protein kinases plays a central regulatory role in cellular processes and these kinases are themselves tightly regulated. One common mechanism of regulation involves Ca2+-binding proteins (CaBP) such as calmodulin (CaM). Here we report a Ca2+-effector mechanism for protein kinase activation by demonstrating the specific and >1,000-fold activation of the myosin-associated giant protein kinase twitchin by Ca2+/S100A1(2). S100A1(2) is a member of a large CaBP family that is implicated in various cellular processes, including cell growth, differentiation and motility, but whose molecular actions are largely unknown. The S100A1(2)-binding site is a part of the autoregulatory sequence positioned in the active site that is responsible for intrasteric autoinhibition of twitchin kinase the mechanism of autoinhibition based on the crystal structures of two twitchin kinase fragments is described elsewhere. Ca2+/S100 represents a likely physiological activator for the entire family of giant protein kinases involved in muscle contractions and cytoskeletal structure.

TRIF-dependent TLR signaling, its functions in host defense and inflammation, and its potential as a therapeutic target

Publisher: Oxford University Press (OUP)

Date: 09-05-2016

DOI: 10.1189/JLB.2RI1115-531R

Abstract: Toll/IL-1R domain-containing adaptor-inducing IFN-β (TRIF)-dependent signaling is required for TLR-mediated production of type-I IFN and several other proinflammatory mediators. Various pathogens target the signaling molecules and transcriptional regulators acting in the TRIF pathway, thus demonstrating the importance of this pathway in host defense. Indeed, the TRIF pathway contributes to control of both viral and bacterial pathogens through promotion of inflammatory mediators and activation of antimicrobial responses. TRIF signaling also has both protective and pathologic roles in several chronic inflammatory disease conditions, as well as an essential function in wound-repair processes. Here, we review our current understanding of the regulatory mechanisms that control TRIF-dependent TLR signaling, the role of the TRIF pathway in different infectious and noninfectious pathologic states, and the potential for manipulating TRIF-dependent TLR signaling for therapeutic benefit.

Vaccinia Virus Immunomodulator A46: Destructive Interactions with MAL and MyD88 Shown by Negative-Stain EM

Publisher: Elsevier BV

Date: 2020

DOI: 10.2139/SSRN.3600564

Characterizing the conformational dynamics of metal-free PsaA using molecular dynamics simulations and electron paramagnetic resonance spectroscopy

Publisher: Elsevier BV

Date: 12-2015

DOI: 10.1016/J.BPC.2015.08.004

Abstract: Prokaryotic metal-ion receptor proteins, or solute-binding proteins, facilitate the acquisition of metal ions from the extracellular environment. Pneumococcal surface antigen A (PsaA) is the primary Mn(2+)-recruiting protein of the human pathogen Streptococcus pneumoniae and is essential for its in vivo colonization and virulence. The recently reported high-resolution structures of metal-free and metal-bound PsaA have provided the first insights into the mechanism of PsaA-facilitated metal binding. However, the conformational dynamics of metal-free PsaA in solution remain unknown. Here, we use continuous wave electron paramagnetic resonance (EPR) spectroscopy and molecular dynamics (MD) simulations to study the relative flexibility of the structural domains in metal-free PsaA and its distribution of conformations in solution. The results show that the crystal structure of metal-free PsaA is a good representation of the dominant conformation in solution, but the protein also s les structurally distinct conformations that are not captured by the crystal structure. Further, these results suggest that the metal binding site is both larger and more solvent exposed than indicated by the metal-free crystal structure. Collectively, this study provides atomic-resolution insight into the conformational dynamics of PsaA prior to metal binding and lays the groundwork for future EPR and MD based studies of PsaA in solution.

Adaptors in toll-like receptor signaling and their potential as therapeutic targets

Publisher: Bentham Science Publishers Ltd.

Date: 08-2012

DOI: 10.2174/138945012803530260

Abstract: To initiate the innate immune response, Toll-like receptors (TLRs) associate with cytoplasmic adaptor proteins through TIR (Toll/interleukin-1 receptor) domain interactions. The four principal signaling adaptor proteins include MyD88, MAL, TRIF and TRAM, and the fifth protein SARM, involved in negative regulation of TLR pathways, is usually considered a part of the TIR domain-containing adaptor protein group. Other TIR domain-containing proteins have also been shown to regulate these signaling pathways, including ST2 and SIGIRR, as well as several bacterial and viral TIR domain-containing proteins that modulate these pathways as virulence factors. TLR pathways and the adaptor proteins are associated with a number of diseases, including infection, sepsis, inflammatory, allergic and autoimmune diseases and cancer. We review our current understanding of the structure and function of adaptor proteins and their regulatory proteins, their association with disease and their potential as therapeutic targets in human disease.

3 Intrasteric regulation of protein kinases

Publisher: Elsevier

Date: 1997

DOI: 10.1016/S1040-7952(97)80006-7

Abstract: Crosstalk between the gut microbiota and bile acid plays an important role in the pathogenesis of gastrointestinal disorders. We investigated the relationship between microbial structure and bile acid metabolism in the ileal mucosa of Crohn's disease (CD). Twelve non-CD controls and 38 CD patients in clinical remission were enrolled. S les were collected from the distal ileum under balloon-assisted enteroscopy. Bile acid composition was analyzed by liquid chromatography-mass spectrometry. The gut microbiota was analyzed by 16S rRNA gene sequencing. The Shannon evenness index was significantly lower in endoscopically active lesions than in non-CD controls. β-Diversity, evaluated by the UniFrac metric, revealed a significant difference between the active lesions and non-CD controls (P=0.039). The relative abundance of Escherichia was significantly higher and that of Faecalibacterium and Roseburia was significantly lower in CD s les than in non-CD controls. The increased abundance of Escherichia was more prominent in active lesions than in inactive lesions. The proportion of conjugated bile acids was significantly higher in CD patients than in non-CD controls, but there was no difference in the proportion of primary or secondary bile acids. The genera Escherichia and Lactobacillus were positively correlated with the proportion of conjugated bile acids. On the other hand, Roseburia, Intestinibacter, and Faecalibacterium were negatively correlated with the proportion of conjugated bile acids. Mucosa-associated dysbiosis and the alteration of bile acid composition were identified in the ileum of CD patients. These may play a role in the pathophysiology of ileal lesions in CD patients.

Discovering Sequence Motifs with Arbitrary Insertions and Deletions

Publisher: Public Library of Science (PLoS)

Date: 09-05-2008

DOI: 10.1371/JOURNAL.PCBI.1000071

Computer‐aided design of T‐cell epitope‐based vaccines: addressing population coverage

Publisher: Wiley

Date: 24-07-2015

DOI: 10.1111/IJI.12214

Abstract: Epitope-based vaccines (EVs) make use of short antigen-derived peptides corresponding to immune epitopes, which are administered to trigger a protective humoral and/or cellular immune response. EVs potentially allow for precise control over the immune response activation by focusing on the most relevant - immunogenic and conserved - antigen regions. Experimental screening of large sets of peptides is time-consuming and costly therefore, in silico methods that facilitate T-cell epitope mapping of protein antigens are paramount for EV development. The prediction of T-cell epitopes focuses on the peptide presentation process by proteins encoded by the major histocompatibility complex (MHC). Because different MHCs have different specificities and T-cell epitope repertoires, in iduals are likely to respond to a different set of peptides from a given pathogen in genetically heterogeneous human populations. In addition, protective immune responses are only expected if T-cell epitopes are restricted by MHC proteins expressed at high frequencies in the target population. Therefore, without careful consideration of the specificity and prevalence of the MHC proteins, EVs could fail to adequately cover the target population. This article reviews state-of-the-art algorithms and computational tools to guide EV design through all the stages of the process: epitope prediction, epitope selection and vaccine assembly, while optimizing vaccine immunogenicity and coping with genetic variation in humans and pathogens.

Structural and Functional Characterization of a CrosssReactive Dengue Virus Neutralizing Antibody That Recognizes a Cryptic Epitope

Publisher: Elsevier BV

Date: 2018

DOI: 10.2139/SSRN.3155754

Mechanism of Ribonuclease Inhibition by Ribonuclease Inhibitor Protein Based on the Crystal Structure of its Complex with Ribonuclease A

Publisher: Elsevier BV

Date: 12-1996

DOI: 10.1006/JMBI.1996.0694

Abstract: We describe the mechanism of ribonuclease inhibition by ribonuclease inhibitor, a protein built of leucine-rich repeats, based on the crystal structure of the complex between the inhibitor and ribonuclease A. The structure was determined by molecular replacement and refined to an Rcryst of 19.4% at 2.5 A resolution. Ribonuclease A binds to the concave region of the inhibitor protein comprising its parallel beta-sheet and loops. The inhibitor covers the ribonuclease active site and directly contacts several active-site residues. The inhibitor only partially mimics the RNase-nucleotide interaction and does not utilize the p1 phosphate-binding pocket of ribonuclease A, where a sulfate ion remains bound. The 2550 A2 of accessible surface area buried upon complex formation may be one of the major contributors to the extremely tight association (Ki = 5.9 x 10(-14) M). The interaction is predominantly electrostatic there is a high chemical complementarity with 18 putative hydrogen bonds and salt links, but the shape complementarity is lower than in most other protein-protein complexes. Ribonuclease inhibitor changes its conformation upon complex formation the conformational change is unusual in that it is a plastic reorganization of the entire structure without any obvious hinge and reflects the conformational flexibility of the structure of the inhibitor. There is a good agreement between the crystal structure and other biochemical studies of the interaction. The structure suggests that the conformational flexibility of RI and an unusually large contact area that compensates for a lower degree of complementarity may be the principal reasons for the ability of RI to potently inhibit erse ribonucleases. However, the inhibition is lost with hibian ribonucleases that have substituted most residues corresponding to inhibitor-binding residues in RNase A, and with bovine seminal ribonuclease that prevents inhibitor binding by forming a dimer.

Protein crystal screening and characterization for serial femtosecond nanocrystallography

Publisher: Springer Science and Business Media LLC

Date: 03-05-2016

DOI: 10.1038/SREP25345

Abstract: The recent development of X-ray free electron lasers (XFELs) has spurred the development of serial femtosecond nanocrystallography (SFX) which, for the first time, is enabling structure retrieval from sub-micron protein crystals. Although there are already a growing number of structures published using SFX, the technology is still very new and presents a number of unique challenges as well as opportunities for structural biologists. One of the biggest barriers to the success of SFX experiments is the preparation and selection of suitable protein crystal s les. Here we outline a protocol for preparing and screening for suitable XFEL targets.

Conformation of the Solute-Binding Protein AdcAII Influences Zinc Uptake in Streptococcus pneumoniae

Publisher: Frontiers Media SA

Date: 19-08-2021

DOI: 10.3389/FCIMB.2021.729981

Abstract: Streptococcus pneumoniae scavenges essential zinc ions from the host during colonization and infection. This is achieved by the ATP-binding cassette transporter, AdcCB, and two solute-binding proteins (SBPs), AdcA and AdcAII. It has been established that AdcAII serves a greater role during initial infection, but the molecular details of how the protein selectively acquires Zn(II) remain poorly understood. This can be attributed to the refractory nature of metal-free AdcAII to high-resolution structural determination techniques. Here, we overcome this issue by separately mutating the Zn(II)-coordinating residues and performing a combination of structural and biochemical analyses on the variant proteins. Structural analyses of Zn(II)-bound AdcAII variants revealed that specific regions within the protein underwent conformational changes via direct coupling to each of the metal-binding residues. Quantitative in vitro metal-binding assays combined with affinity determination and phenotypic growth assays revealed that each of the four Zn(II)-coordinating residues contributes to metal binding by AdcAII. Intriguingly, the phenotypic growth impact of the mutant adcAII alleles was, in general, independent of affinity, suggesting that the Zn(II)-bound conformation of the SBP is crucial for efficacious metal uptake. Collectively, these data highlight the intimate coupling of ligand affinity with protein conformational change in ligand-receptor proteins and provide a putative mechanism for AdcAII. These findings provide further mechanistic insight into the structural and functional ersity of SBPs that is broadly applicable to other prokaryotes.

Structural Basis for Assembly and Function of a Heterodimeric Plant Immune Receptor

Publisher: American Association for the Advancement of Science (AAAS)

Date: 18-04-2014

DOI: 10.1126/SCIENCE.1247357

Abstract: Certain pathogen effectors are detected in plants by cytoplasmic receptors. First solving the crystal structures of Arabidopsis receptors, Williams et al. (p. 299 see the Perspective by Nishimura and Dangl ) discovered that in the resting state, the structures form a heterodimer that readies the complex for effector binding and keeps the signaling domains from firing too early. Once the pathogen effector binds, the structure of the complex shifts such that the signaling domains can form a homodimer to initiate downstream signaling. Similarities between these plant-pathogen receptors and Toll-like receptors in animals suggest the molecular mechanisms may translate broadly.

Protein kinases associated with the yeast phosphoproteome

Publisher: Springer Science and Business Media LLC

Date: 31-01-2006

DOI: 10.1186/1471-2105-7-47

Abstract: Protein phosphorylation is an extremely important mechanism of cellular regulation. A large-scale study of phosphoproteins in a whole-cell lysate of Saccharomyces cerevisiae has previously identified 383 phosphorylation sites in 216 peptide sequences. However, the protein kinases responsible for the phosphorylation of the identified proteins have not previously been assigned. We used Predikin in combination with other bioinformatic tools, to predict which of 116 unique protein kinases in yeast phosphorylates each experimentally determined site in the phosphoproteome. The prediction was based on the match between the phosphorylated 7-residue sequence and the predicted substrate specificity of each kinase, with the highest weight applied to the residues or positions that contribute most to the substrate specificity. We estimated the reliability of the predictions by performing a parallel prediction on phosphopeptides for which the kinase has been experimentally determined. The results reveal that the functions of the protein kinases and their predicted phosphoprotein substrates are often correlated, for ex le in endocytosis, cytokinesis, transcription, replication, carbohydrate metabolism and stress response. The predictions link phosphoproteins of unknown function with protein kinases with known functions and vice versa, suggesting functions for the uncharacterized proteins. The study indicates that the phosphoproteins and the associated protein kinases represented in our dataset have housekeeping cellular roles certain kinases are not represented because they may only be activated during specific cellular responses. Our results demonstrate the utility of our previously reported protein kinase substrate prediction approach (Predikin) as a tool for establishing links between kinases and phosphoproteins that can subsequently be tested experimentally.

Direct protein interaction underlies gene-for-gene specificity and coevolution of the flax resistance genes and flax rust avirulence genes

Publisher: Proceedings of the National Academy of Sciences

Date: 06-06-2006

DOI: 10.1073/PNAS.0602577103

Abstract: Plant resistance proteins (R proteins) recognize corresponding pathogen avirulence (Avr) proteins either indirectly through detection of changes in their host protein targets or through direct R–Avr protein interaction. Although indirect recognition imposes selection against Avr effector function, pathogen effector molecules recognized through direct interaction may overcome resistance through sequence ersification rather than loss of function. Here we show that the flax rust fungus AvrL567 genes, whose products are recognized by the L5, L6, and L7 R proteins of flax, are highly erse, with 12 sequence variants identified from six rust strains. Seven AvrL567 variants derived from Avr alleles induce necrotic responses when expressed in flax plants containing corresponding resistance genes ( R genes), whereas five variants from avr alleles do not. Differences in recognition specificity between AvrL567 variants and evidence for ersifying selection acting on these genes suggest they have been involved in a gene-specific arms race with the corresponding flax R genes. Yeast two-hybrid assays indicate that recognition is based on direct R–Avr protein interaction and recapitulate the interaction specificity observed in planta . Biochemical analysis of Escherichia coli -produced AvrL567 proteins shows that variants that escape recognition nevertheless maintain a conserved structure and stability, suggesting that the amino acid sequence differences directly affect the R–Avr protein interaction. We suggest that direct recognition associated with high genetic ersity at corresponding R and Avr gene loci represents an alternative outcome of plant–pathogen coevolution to indirect recognition associated with simple balanced polymorphisms for functional and nonfunctional R and Avr genes.

Quantitative Structural Analysis of Importin-beta Flexibility: Paradigm for Solenoid Protein Structures

Publisher: Elsevier BV

Date: 09-2010

DOI: 10.1016/J.STR.2010.06.015

Abstract: The structure of solenoid proteins facilitates a higher degree of flexibility than most folded proteins. In importin-β, a nuclear import factor built from 19 tandem HEAT repeats, flexibility plays a crucial role in allowing interactions with a range of different partners. We present a comprehensive analysis of importin-β flexibility based on a number of different approaches. We determined the crystal structure of unliganded Saccharomyces cerevisiae importin-β (Kap95) to allow a quantitative comparison with importin-β bound to different partners. Complementary mutagenesis, small angle X-ray scattering and molecular dynamics studies suggest that the protein s les several conformations in solution. The analyses suggest the flexibility of the solenoid is generated by cumulative small movements along its length. Importin-β illustrates how solenoid proteins can orchestrate protein interactions in many cellular pathways.

Plant and prokaryotic TIR domains generate distinct cyclic ADPR NADase products

Publisher: American Association for the Advancement of Science (AAAS)

Date: 15-03-2023

DOI: 10.1126/SCIADV.ADE8487

Abstract: Toll/interleukin-1 receptor (TIR) domain proteins function in cell death and immunity. In plants and bacteria, TIR domains are often enzymes that produce isomers of cyclic adenosine 5′-diphosphate–ribose (cADPR) as putative immune signaling molecules. The identity and functional conservation of cADPR isomer signals is unclear. A previous report found that a plant TIR could cross-activate the prokaryotic Thoeris TIR–immune system, suggesting the conservation of plant and prokaryotic TIR-immune signals. Here, we generate autoactive Thoeris TIRs and test the converse hypothesis: Do prokaryotic Thoeris TIRs also cross-activate plant TIR immunity? Using in planta and in vitro assays, we find that Thoeris and plant TIRs generate overlapping sets of cADPR isomers and further clarify how plant and Thoeris TIRs activate the Thoeris system via producing 3′cADPR. This study demonstrates that the TIR signaling requirements for plant and prokaryotic immune systems are distinct and that TIRs across kingdoms generate a ersity of small-molecule products.

Distinctive Conformation of Minor Site-Specific Nuclear Localization Signals Bound to Importin-α

Publisher: Wiley

Date: 13-05-2016

DOI: 10.1111/TRA.12395

Ucl fimbriae regulation and glycan receptor specificity contribute to gut colonisation by extra-intestinal pathogenic Escherichia coli

Publisher: Public Library of Science (PLoS)

Date: 14-06-2022

DOI: 10.1371/JOURNAL.PPAT.1010582

Abstract: Extra-intestinal pathogenic Escherichia coli (ExPEC) belong to a critical priority group of antibiotic resistant pathogens. ExPEC establish gut reservoirs that seed infection of the urinary tract and bloodstream, but the mechanisms of gut colonisation remain to be properly understood. Ucl fimbriae are attachment organelles that facilitate ExPEC adherence. Here, we investigated cellular receptors for Ucl fimbriae and Ucl expression to define molecular mechanisms of Ucl-mediated ExPEC colonisation of the gut. We demonstrate differential expression of Ucl fimbriae in ExPEC sequence types associated with disseminated infection. Genome editing of strains from two common sequence types, F11 (ST127) and UTI89 (ST95), identified a single nucleotide polymorphism in the ucl promoter that changes fimbriae expression via activation by the global stress-response regulator OxyR, leading to altered gut colonisation. Structure-function analysis of the Ucl fimbriae tip-adhesin (UclD) identified high-affinity glycan receptor targets, with highest affinity for sialyllacto-N-fucopentose VI, a structure likely to be expressed on the gut epithelium. Comparison of the UclD adhesin to the homologous UcaD tip-adhesin from Proteus mirabilis revealed that although they possess a similar tertiary structure, apart from lacto-N-fucopentose VI that bound to both adhesins at low-micromolar affinity, they recognize different fucose- and glucose-containing oligosaccharides. Competitive surface plasmon resonance analysis together with co-structural investigation of UcaD in complex with monosaccharides revealed a broad-specificity glycan binding pocket shared between UcaD and UclD that could accommodate these interactions. Overall, our study describes a mechanism of adaptation that augments establishment of an ExPEC gut reservoir to seed disseminated infections, providing a pathway for the development of targeted anti-adhesion therapeutics.

Imperfect coordination chemistry facilitates metal ion release in the Psa permease

Publisher: Springer Science and Business Media LLC

Date: 10-11-2014

DOI: 10.1038/NCHEMBIO.1382

Abstract: The relative stability of alent first-row transition metal ion complexes, as defined by the Irving-Williams series, poses a fundamental chemical challenge for selectivity in bacterial metal ion acquisition. Here we show that although the substrate-binding protein of Streptococcus pneumoniae, PsaA, is finely attuned to bind its physiological substrate manganese, it can also bind a broad range of other alent transition metal cations. By combining high-resolution structural data, metal-binding assays and mutational analyses, we show that the inability of open-state PsaA to satisfy the preferred coordination chemistry of manganese enables the protein to undergo the conformational changes required for cargo release to the Psa permease. This is specific for manganese ions, whereas zinc ions remain bound to PsaA. Collectively, these findings suggest a new ligand binding and release mechanism for PsaA and related substrate-binding proteins that facilitate specificity for alent cations during competition from zinc ions, which are more abundant in biological systems.

Plant and prokaryotic TIR domains generate distinct cyclic ADPR NADase products

Publisher: Cold Spring Harbor Laboratory

Date: 19-09-2022

DOI: 10.1101/2022.09.19.508568

Abstract: Toll/interleukin-1 receptor (TIR) domain proteins function in cell death and immunity. In plants and bacteria, TIR domains are enzymes that produce isomers of cyclic ADPR (cADPR) as putative immune signaling molecules. The identity and functional conservation of cADPR isomer signals is unclear. A previous report found that a plant TIR could cross-activate the prokaryotic Thoeris TIR-immune system, suggesting the conservation of plant and prokaryotic TIR-immune signals. Here, we generate auto-active Thoeris TIRs and test the converse hypothesis: do prokaryotic Thoeris TIRs also cross-activate plant TIR-immunity? Using in planta and in vitro assays, we find that Thoeris and plant TIRs generate overlapping sets of cADPR isomers, and further clarify how plant and Thoeris TIRs activate the Thoeris system via producing 3’cADPR. This study demonstrates that the TIR-signaling requirements for plant and prokaryotic immune systems are distinct and that TIRs across kingdoms generate a ersity of small molecule products.

Soybean Nodule Autoregulation Receptor Kinase Phosphorylates Two Kinase-associated Protein Phosphatases in Vitro

Publisher: Elsevier BV

Date: 09-2008

DOI: 10.1074/JBC.M800400200

Crystal structure of human T cell leukemia virus type 1 gp21 ectodomain crystallized as a maltose-binding protein chimera reveals structural evolution of retroviral transmembrane proteins

Publisher: Proceedings of the National Academy of Sciences

Date: 13-04-1999

DOI: 10.1073/PNAS.96.8.4319

Abstract: Retroviral entry into cells depends on envelope glycoproteins, whereby receptor binding to the surface-exposed subunit triggers membrane fusion by the transmembrane protein (TM) subunit. We determined the crystal structure at 2.5-Å resolution of the ectodomain of gp21, the TM from human T cell leukemia virus type 1. The gp21 fragment was crystallized as a maltose-binding protein chimera, and the maltose-binding protein domain was used to solve the initial phases by the method of molecular replacement. The structure of gp21 comprises an N-terminal trimeric coiled coil, an adjacent disulfide-bonded loop that stabilizes a chain reversal, and a C-terminal sequence structurally distinct from HIV type 1/simian immunodeficiency virus gp41 that packs against the coil in an extended antiparallel fashion. Comparison of the gp21 structure with the structures of other retroviral TMs contrasts the conserved nature of the coiled coil-forming region and adjacent disulfide-bonded loop with the variable nature of the C-terminal ectodomain segment. The structure points to these features having evolved to enable the dual roles of retroviral TMs: conserved fusion function and an ability to anchor erse surface-exposed subunit structures to the virion envelope and infected cell surface. The structure of gp21 implies that the N-terminal fusion peptide is in close proximity to the C-terminal transmembrane domain and likely represents a postfusion conformation.

Wzy-Dependent Bacterial Capsules as Potential Drug Targets

Publisher: Bentham Science Publishers Ltd.

Date: 08-2012

DOI: 10.2174/138945012803530279

Abstract: The bacterial capsule is a recognized virulence factor in pathogenic bacteria. It likely works as an antiphagocytic barrier by minimizing complement deposition on the bacterial surface. With the continual rise of bacterial pathogens resistant to multiple antibiotics, there is an increasing need for novel drugs. In the Wzy-dependent pathway, the biosynthesis of capsular polysaccharide (CPS) is regulated by a phosphoregulatory system, whose main components consist of bacterial-tyrosine kinases (BY-kinases) and their cognate phosphatases. The ability to regulate capsule biosynthesis has been shown to be vital for pathogenicity, because different stages of infection require a shift in capsule thickness, making the phosphoregulatory proteins suitable as drug targets. Here, we review the role of regulatory proteins focusing on Streptococcus pneumoniae, Staphylococcus aureus, and Escherichia coli and discuss their suitability as targets in structure-based drug design.

Evolutionary Conservation of the Membrane Fusion Machine

Publisher: Informa UK Limited

Date: 08-1999

DOI: 10.1080/152165499307152

Comprehensive analysis of IncC plasmid conjugation identifies a crucial role for the transcriptional regulator AcaB

Publisher: Springer Science and Business Media LLC

Date: 17-08-2020

DOI: 10.1038/S41564-020-0775-0

Abstract: The IncC family of broad-host-range plasmids enables the spread of antibiotic resistance genes among human enteric pathogens

Proteomic and electron microscopy survey of large assemblies in macrophage cytoplasm

Publisher: Elsevier BV

Date: 06-2011

DOI: 10.1074/MCP.M111.008763

Prediction of kinase-specific phosphorylation sites through an integrative model of protein context and sequence

Publisher: Cold Spring Harbor Laboratory

Date: 15-03-2016

DOI: 10.1101/043679

Abstract: The identification of kinase substrates and the specific phosphorylation sites they regulate is an important factor in understanding protein function regulation and signalling pathways. Computational prediction of kinase targets – assigning kinases to putative substrates, and selecting from protein sequence the sites that kinases can phosphorylate – requires the consideration of both the cellular context that kinases operate in, as well as their binding affinity. This consideration enables investigation of how phosphorylation influences a range of biological processes. We report here a novel probabilistic model for the classification of kinase-specific phosphorylation sites from sequence across three model organisms: human, mouse and yeast. The model incorporates position-specific amino acid frequencies, and counts of co-occurring amino acids from kinase binding sites in a kinase‐ and family-specific manner. We show how this model can be seamlessly integrated with protein interactions and cell-cycle abundance profiles. When evaluating the prediction accuracy of our method, PhosphoPICK, on an independent hold-out set of kinase-specific phosphorylation sites, we found it achieved an average specificity of 97% while correctly predicting 32% of true positives. We also compared PhosphoPICK’s ability, through cross-validation, to predict kinase-specific phosphorylation sites with alternative methods, and found that at high levels of specificity PhosphoPICK outperforms alternative methods for most comparisons made. We investigated the relationship between experimentally confirmed phosphorylation sites and predicted nuclear localisation signals by predicting the most likely kinases to be regulating the phosphorylated residues immediately upstream or downstream from the localisation signal. We show that kinases PKA, Akt1 and AurB have an over-representation of predicted binding sites at particular positions downstream from predicted nuclear localisation signals, demonstrating an important role for these kinases in regulating the nuclear import of proteins. PhosphoPICK is freely available online as a web-service at bioinf.scmb.uq.edu.au hosphopick .

Structures of the flax-rust effector AvrM reveal insights into the molecular basis of plant-cell entry and effector-triggered immunity

Publisher: Proceedings of the National Academy of Sciences

Date: 07-10-2013

DOI: 10.1073/PNAS.1307614110

Abstract: Fungal and oomycete pathogens cause devastating diseases in crop plants and facilitate infection by delivering effector molecules into the plant cell. The secreted effector protein AvrM from flax rust, a fungal pathogen that infects flax plants, internalizes into host cells in the absence of the pathogen, binds to phosphoinositides, and is recognized directly by the resistance protein M in flax to initiate effector-triggered immunity. We describe the crystal structure of AvrM and identify functionally important surface regions in the protein, which advances our understanding of the molecular mechanisms underlying how effectors enter host cells and how they are detected by the plant immune system.

Leucines on a roll

Publisher: Springer Science and Business Media LLC

Date: 12-1996

DOI: 10.1038/NSB1296-977

Abstract: Epithelial cells that line tissues such as the intestine serve as the primary barrier to the outside world. Epithelia provide selective permeability in the presence of a large constellation of microbes, termed the microbiota. Recent studies have revealed that the symbiotic relationship between the healthy host and the microbiota includes the regulation of cell-cell interactions at the level of epithelial tight junctions. The most recent findings have identified multiple microbial-derived metabolites that influence intracellular signaling pathways which elicit activities at the epithelial apical junction complex. Here, we review recent findings that place microbiota-derived metabolites as primary regulators of epithelial cell-cell interactions and ultimately mucosal permeability in health and disease.

Dysregulation of transition metal ion homeostasis is the molecular basis for cadmium toxicity in Streptococcus pneumoniae

Publisher: Springer Science and Business Media LLC

Date: 03-03-2015

DOI: 10.1038/NCOMMS7418

Abstract: Cadmium is a transition metal ion that is highly toxic in biological systems. Although relatively rare in the Earth’s crust, anthropogenic release of cadmium since industrialization has increased biogeochemical cycling and the abundance of the ion in the biosphere. Despite this, the molecular basis of its toxicity remains unclear. Here we combine metal-accumulation assays, high-resolution structural data and biochemical analyses to show that cadmium toxicity, in Streptococcus pneumoniae , occurs via perturbation of first row transition metal ion homeostasis. We show that cadmium uptake reduces the millimolar cellular accumulation of manganese and zinc, and thereby increases sensitivity to oxidative stress. Despite this, high cellular concentrations of cadmium (~17 mM) are tolerated, with negligible impact on growth or sensitivity to oxidative stress, when manganese and glutathione are abundant. Collectively, this work provides insight into the molecular basis of cadmium toxicity in prokaryotes, and the connection between cadmium accumulation and oxidative stress.

Crystal structure of porcine ribonuclease inhibitor, a protein with leucine-rich repeats

Publisher: Springer Science and Business Media LLC

Date: 12-1993

DOI: 10.1038/366751A0

Abstract: Ribonuclease inhibitor is a cytoplasmic protein that tightly binds and inhibits ribonucleases of the pancreatic ribonuclease superfamily. The primary sequence of this inhibitor contains leucine-rich repeats (LRRs) these motifs are present in many proteins that participate in protein-protein interactions and have different functions and cellular locations. In vivo, ribonuclease inhibitor may have a role in the regulation of RNA turnover in mammalian cells and in angiogenesis. To define the structural features of LRR proteins and to understand better the nature of the tight interaction of ribonuclease inhibitor with ribonucleases, we have determined the crystal structure of the porcine inhibitor. To our knowledge, this is the first three-dimensional structure of a protein containing LRRs and represents a new class of alpha/beta protein fold. In idual repeats constitute beta-alpha structural units that probably also occur in other proteins containing LRRs. The non-globular shape of the structure and the exposed face of the parallel beta-sheet may explain why LRRs are used to achieve strong protein-protein interactions. A possible ribonuclease-binding region incorporates the surface formed by the parallel beta-sheet and the beta alpha loops.

Ligand-induced conformational changes within a hexameric acyl-CoA thioesterase

Publisher: Elsevier BV

Date: 10-2011

DOI: 10.1074/JBC.M111.225953

Kap95p Binding Induces the Switch Loops of RanGDP to Adopt the GTP-Bound Conformation: Implications for Nuclear Import Complex Assembly Dynamics

Publisher: Elsevier BV

Date: 11-2008

DOI: 10.1016/J.JMB.2008.07.090

Abstract: The asymmetric distribution of the nucleotide-bound state of Ran across the nuclear envelope is crucial for determining the directionality of nuclear transport. In the nucleus, Ran is primarily in the guanosine 5'-triphosphate (GTP)-bound state, whereas in the cytoplasm, Ran is primarily guanosine 5'-diphosphate (GDP)-bound. Conformational changes within the Ran switch I and switch II loops are thought to modulate its affinity for importin-beta. Here, we show that RanGDP and importin-beta form a stable complex with a micromolar dissociation constant. This complex can be dissociated by importin-beta binding partners such as importin-alpha. Surprisingly, the crystal structure of the Kap95p-RanGDP complex shows that Kap95p induces the switch I and II regions of RanGDP to adopt a conformation that resembles that of the GTP-bound form. The structure of the complex provides insights into the structural basis for the gradation of affinities regulating nuclear protein transport.

Evolutionary model of protein secondary structure capable of revealing new biological relationships

Publisher: Cold Spring Harbor Laboratory

Date: 28-02-2019

DOI: 10.1101/563452

Abstract: Ancestral sequence reconstruction has had recent success in decoding the origins and the determinants of complex protein functions. However, phylo­genetic analyses of remote homologues must handle extreme amino-acid se­quence ersity resulting from extended periods of evolutionary change. We exploited the wealth of protein structures to develop an evolutionary model based on protein secondary structure. The approach follows the differences between discrete secondary structure states observed in modern proteins and those hypothesised in their immediate ancestors. We implemented maximum likelihood-based phylogenetic inference to reconstruct ancestral secondary structure. The predictive accuracy from the use of the evolutionary model surpasses that of comparative modelling and sequence-based prediction the reconstruction extracts information not available from modern structures or the ancestral sequences alone. Based on a phylogenetic analysis of multiple protein families, we showed that the model can highlight relationships that are evolutionarily rooted in structure and not evident in amino acid-based analysis.

Evolution of Innate Immunity in Eukarya: Advances and Implications

Publisher: Frontiers Media SA

Date: 2022

DOI: 10.3389/978-2-88976-133-3

Erratum: Leucines on a roll

Publisher: Springer Science and Business Media LLC

Date: 04-1997

DOI: 10.1038/NSB0497-327

A non-uniform sampling approach enables studies of dilute and unstable proteins

Publisher: Springer Science and Business Media LLC

Date: 10-02-2017

DOI: 10.1007/S10858-017-0091-Z

Abstract: NMR spectroscopy is a powerful method in structural and functional analysis of macromolecules and has become particularly prevalent in studies of protein structure, function and dynamics. Unique to NMR spectroscopy is the relatively low constraints on s le preparation and the high level of control of s le conditions. Proteins can be studied in a wide range of buffer conditions, e.g. different pHs and variable temperatures, allowing studies of proteins under conditions that are closer to their native environment compared to other structural methods such as X-ray crystallography and electron microscopy. The key disadvantage of NMR is the relatively low sensitivity of the method, requiring either concentrated s les or very lengthy data-acquisition times. Thus, proteins that are unstable or can only be studied in dilute solutions are often considered practically unfeasible for NMR studies. Here, we describe a general method, where non-uniform s ling (NUS) allows for signal averaging to be monitored in an iterative manner, enabling efficient use of spectrometer time, ultimately leading to savings in costs associated with instrument and isotope-labelled protein use. The method requires preparation of multiple aliquots of the protein s le that are flash-frozen and thawed just before acquisition of a short NMR experiments carried out while the protein is stable (12 h in the presented case). Non-uniform s ling enables sufficient resolution to be acquired for each short experiment. Identical NMR datasets are acquired and sensitivity is monitored after each co-added spectrum is reconstructed. The procedure is repeated until sufficient signal-to-noise is obtained. We discuss how maximum entropy reconstruction is used to process the data, and propose a variation on the previously described method of automated parameter selection. We conclude that combining NUS with iterative co-addition is a general approach, and particularly powerful when applied to unstable proteins.

The leucine-rich repeat as a protein recognition motif

Publisher: Elsevier BV

Date: 12-2001

DOI: 10.1016/S0959-440X(01)00266-4

Abstract: Leucine-rich repeats (LRRs) are 20-29-residue sequence motifs present in a number of proteins with erse functions. The primary function of these motifs appears to be to provide a versatile structural framework for the formation of protein-protein interactions. The past two years have seen an explosion of new structural information on proteins with LRRs. The new structures represent different LRR subfamilies and proteins with erse functions, including GTPase-activating protein rna1p from the ribonuclease-inhibitor-like subfamily spliceosomal protein U2A', Rab geranylgeranyltransferase, internalin B, dynein light chain 1 and nuclear export protein TAP from the SDS22-like subfamily Skp2 from the cysteine-containing subfamily and YopM from the bacterial subfamily. The new structural information has increased our understanding of the structural determinants of LRR proteins and our ability to model such proteins with unknown structures, and has shed new light on how these proteins participate in protein-protein interactions.

The myosin-I-binding protein Acan125 binds the SH3 domain and belongs to the superfamily of leucine-rich repeat proteins

Publisher: Proceedings of the National Academy of Sciences

Date: 15-04-1997

DOI: 10.1073/PNAS.94.8.3685

Abstract: The SH3 domains of src and other nonreceptor tyrosine kinases have been shown to associate with the motif PXXP, where P and X stand for proline and an unspecified amino acid, but a motif that binds to the SH3 domain of myosin has thus far not been characterized. We previously showed that the SH3 domain of Acanthamoeba myosin-IC interacts with the protein Acan125. We now report that the Acan125 protein sequence contains two tandem consensus PXXP motifs near the C terminus. To test for binding, we expressed a polypeptide, AD3p, which includes 344 residues of native C-terminal sequence and a mutant polypeptide, AD3Δ977–994p, which lacks the sequence RPKPVPPPRGAKPAPPPR containing both PXXP motifs. The SH3 domain of Acanthamoeba myosin-IC bound AD3p and not AD3Δ977–994p, showing that the PXXP motifs are required for SH3 binding. The sequence of Acan125 is related overall to a protein of unknown function coded by Caenorhabditis elegans gene K07G5.1. The K07G5.1 gene product contains a proline-rich segment similar to the SH3 binding motif found in Acan125. The aligned sequences show considerable conservation of leucines and other hydrophobic residues, including the spacing of these residues, which matches a motif for leucine-rich repeats (LRRs). LRR domains have been demonstrated to be sites for ligand binding. Having an LRR domain and an SH3-binding domain, Acan125 and the C. elegans homologue define a novel family of bifunctional binding proteins.

Emerging Insights into the Functions of Pathogenesis-Related Protein 1

Publisher: Elsevier BV

Date: 10-2017

DOI: 10.1016/J.TPLANTS.2017.06.013

Abstract: The members of the pathogenesis-related protein 1 (PR-1) family are among the most abundantly produced proteins in plants on pathogen attack, and PR-1 gene expression has long been used as a marker for salicylic acid-mediated disease resistance. However, despite considerable interest over several decades, their requirement and role in plant defence remains poorly understood. Recent reports have emerged demonstrating that PR-1 proteins possess sterol-binding activity, harbour an embedded defence signalling peptide, and are targeted by plant pathogens during host infection. These studies have re-energised the field and provided long-awaited insights into a possible PR-1 function. Here we review the current status of PR-1 proteins and discuss how these recent advances shed light on putative roles for these enigmatic proteins.

Handling of nutrient copper in the bacterial envelope

Publisher: Oxford University Press (OUP)

Date: 2019

DOI: 10.1039/C8MT00218E

Abstract: The insertion of copper into bacterial cuproenzymes in vivo does not always require a copper-binding metallochaperone – why?

Structure and Regulation of Phenylalanine Hydroxylase, and Implications for Related Enzymes

Publisher: Springer US

Date: 2002

DOI: 10.1007/978-1-4615-0945-5_13

Crystallization and Preliminary X-ray Analysis of Porcine Ribonuclease Inhibitor, a Protein with Leucine-rich Repeats

Publisher: Elsevier BV

Date: 05-1993

DOI: 10.1006/JMBI.1993.1263

Abstract: Ribonuclease inhibitor was purified from pig liver and crystallized at 21 degrees C from solutions containing dithiothreitol as an additive and ammonium sulfate, lithium sulfate or combinations of both as precipitants. Crystals have the symmetry of the tetragonal space group I4 with a = 134.9 A and c = 83.6 A, and diffract to better than 3 A resolution. Self rotation functions and packing density of the crystals are consistent with two molecules in the asymmetric unit.

The CC domain structure from the wheat stem rust resistance protein Sr33 challenges paradigms for dimerization in plant NLR proteins

Publisher: Proceedings of the National Academy of Sciences

Date: 17-10-2016

DOI: 10.1073/PNAS.1609922113

Abstract: Plants and animals use intracellular immunity receptors, known as nucleotide-binding oligomerization domain-like receptors (NLRs), to defend themselves against invading microbes. In this study, we report the solution structure of the N-terminal coiled-coil (CC) domain from the wheat stem rust resistance protein Sr33. Remarkably, this structure differs substantially from the published crystal structure of the equivalent region from the orthologous barley powdery mildew resistance protein MLA10. Using a structural, biophysical, and functional approach, we compare the Sr33 CC domain with other structurally defined NLR CC domains. Collectively, this work redefines our current understanding of the structure and function of plant NLR CC domains, which has significant implications for future studies into this important class of defense receptors.

Structural basis for the specificity of bipartite nuclear localisation sequence binding by importin-

Publisher: Elsevier BV

Date: 12-2009

DOI: 10.1074/JBC.M303275200

Crystallization and preliminary X-ray diffraction studies of FHA domains of Dun1 and Rad53 protein kinases

Publisher: International Union of Crystallography (IUCr)

Date: 03-2001

DOI: 10.1107/S0907444901002116

Abstract: Forkhead-associated (FHA) domains are modular protein-protein interaction domains of approximately 130 amino acids present in numerous signalling proteins. FHA-domain-dependent protein interactions are regulated by phosphorylation of target proteins and FHA domains may be multifunctional phosphopeptide-recognition modules. FHA domains of the budding yeast cell-cycle checkpoint protein kinases Dun1p and Rad53p have been crystallized. Crystals of the Dun1-FHA domain exhibit the symmetry of the space group P6(1)22 or P6(5)22, with unit-cell parameters a = b = 127.3, c = 386.3 A diffraction data have been collected to 3.1 A resolution on a synchrotron source. Crystals of the N-terminal FHA domain (FHA1) of Rad53p diffract to 4.0 A resolution on a laboratory X-ray source and have Laue-group symmetry 4/mmm, with unit-cell parameters a = b = 61.7, c = 104.3 A.

Structure and function of Toll/interleukin-1 receptor/resistance protein (TIR) domains

Publisher: Springer Science and Business Media LLC

Date: 02-12-2014

DOI: 10.1007/S10495-014-1064-2

Abstract: The Toll/interleukin-1 receptor/resistance protein (TIR) domain is a protein-protein interaction domain consisting of 125-200 residues, widely distributed in animals, plants and bacteria but absent from fungi, archea and viruses. In plants and animals, these domains are found in proteins with functions in innate immune pathways, while in bacteria, some TIR domain-containing proteins interfere with the innate immune pathways in the host. TIR domains function as protein scaffolds, mostly involving self-association and homotypic interactions with other TIR domains. In the last 15 years, the three-dimensional structures of TIR domains from several mammalian, plant and bacterial proteins have been reported. These structures, jointly with functional data including the identification of interacting proteins, have started to provide insight into the molecular basis of the assembly of animal and plant immune signaling complexes, and for host immunosuppression by bacterial pathogens. This review focuses on the current knowledge of the structures of the TIR domains and how the structure relates to function.

Production of small cysteine‐rich effector proteins in Escherichia coli for structural and functional studies

Publisher: Wiley

Date: 12-04-2016

DOI: 10.1111/MPP.12385

UpaH Is a Newly Identified Autotransporter Protein That Contributes to Biofilm Formation and Bladder Colonization by Uropathogenic Escherichia coli CFT073

Publisher: American Society for Microbiology

Date: 04-2010

DOI: 10.1128/IAI.01010-09

Abstract: Escherichia coli is the primary cause of urinary tract infection (UTI) in the developed world. The major factors associated with virulence of uropathogenic E. coli (UPEC) are fimbrial adhesins, which mediate specific attachment to host receptors and trigger innate host responses. Another group of adhesins is represented by the autotransporter (AT) subgroup of proteins. In this study, we identified a new AT-encoding gene, termed upaH , present in a 6.5-kb unannotated intergenic region in the genome of the prototypic UPEC strain CFT073. Cloning and sequencing of the upaH gene from CFT073 revealed an intact 8.535-kb coding region, contrary to the published genome sequence. The upaH gene was widely distributed among a large collection of UPEC isolates as well as the E. coli Reference (ECOR) strain collection. Bioinformatic analyses suggest β-helix as the predominant structure in the large N-terminal passenger (α) domain and a 12-strand β-barrel for the C-terminal β-domain of UpaH. We demonstrated that UpaH is expressed at the cell surface of CFT073 and promotes biofilm formation. In the mouse UTI model, deletion of the upaH gene in CFT073 and in two other UPEC strains did not significantly affect colonization of the bladder in single-challenge experiments. However, in competitive colonization experiments, CFT073 significantly outcompeted its upaH isogenic mutant strain in urine and the bladder.

Structural Basis of High-Affinity Nuclear Localization Signal Interactions with Importin-α

Publisher: Wiley

Date: 07-02-2012

DOI: 10.1111/J.1600-0854.2012.01329.X

Abstract: Classical nuclear localization signals (cNLSs), comprising one (monopartite cNLSs) or two clusters of basic residues connected by a 10-12 residue linker (bipartite cNLSs), are recognized by the nuclear import factor importin-α. The cNLSs bind along a concave groove on importin-α however, specificity determinants of cNLSs remain poorly understood. We present a structural and interaction analysis study of importin-α binding to both designed and naturally occurring high-affinity cNLS-like sequences the peptide inhibitors Bimax1 and Bimax2, and cNLS peptides of cap-binding protein 80. Our data suggest that cNLSs and cNLS-like sequences can achieve high affinity through maximizing interactions at the importin-α minor site, and by taking advantage of multiple linker region interactions. Our study defines an extended set of binding cavities on the importin-α surface, and also expands on recent observations that longer linker sequences are allowed, and that long-range electrostatic complementarity can contribute to cNLS-binding affinity. Altogether, our study explains the molecular and structural basis of the results of a number of recent studies, including systematic mutagenesis and peptide library approaches, and provides an improved level of understanding on the specificity determinants of a cNLS. Our results have implications for identifying cNLSs in novel proteins.

Crystal structures of fusion proteins with large‐affinity tags

Publisher: Wiley

Date: 07-2003

DOI: 10.1110/PS.0243403

Abstract: The fusion of a protein of interest to a large-affinity tag, such as the maltose-binding protein (MBP), thioredoxin (TRX), or glutathione-S-transferase (GST), can be advantageous in terms of increased expression, enhanced solubility, protection from proteolysis, improved folding, and protein purification via affinity chromatography. Unfortunately, crystal growth is hindered by the conformational heterogeneity induced by the fusion tag, requiring that the tag is removed by a potentially problematic cleavage step. The first three crystal structures of fusion proteins with large-affinity tags have been reported recently. All three structures used a novel strategy to rigidly fuse the protein of interest to MBP via a short three- to five-amino acid spacer. This strategy has the potential to aid structure determination of proteins that present particular experimental challenges and are not conducive to more conventional crystallization strategies (e.g., membrane proteins). Structural genomics initiatives may also benefit from this approach as a way to crystallize problematic proteins of significant interest.

Megahertz pulse trains enable multi-hit serial femtosecond crystallography experiments at X-ray free electron lasers

Publisher: Springer Science and Business Media LLC

Date: 11-08-2022

DOI: 10.1038/S41467-022-32434-6

Abstract: The European X-ray Free Electron Laser (XFEL) and Linac Coherent Light Source (LCLS) II are extremely intense sources of X-rays capable of generating Serial Femtosecond Crystallography (SFX) data at megahertz (MHz) repetition rates. Previous work has shown that it is possible to use consecutive X-ray pulses to collect diffraction patterns from in idual crystals. Here, we exploit the MHz pulse structure of the European XFEL to obtain two complete datasets from the same lysozyme crystal, first hit and the second hit, before it exits the beam. The two datasets, separated by µs, yield up to 2.1 Å resolution structures. Comparisons between the two structures reveal no indications of radiation damage or significant changes within the active site, consistent with the calculated dose estimates. This demonstrates MHz SFX can be used as a tool for tracking sub-microsecond structural changes in in idual single crystals, a technique we refer to as multi-hit SFX.

Crystallization of the flexible nuclear import receptor importin-β in the unliganded state

Publisher: International Union of Crystallography (IUCr)

Date: 22-05-2009

DOI: 10.1107/S1744309109016820

Computational modelling of linear motif-mediated protein interactions

Publisher: Bentham Science Publishers Ltd.

Date: 08-2012

DOI: 10.2174/156802612802652439

Abstract: Chemical information can be used to inform biology through being employed to develop bioinformatic tools. One area where bioinformatic tools are valuable is the study of linear motif-mediated protein interactions. Linear motifs are short sequences found mostly in disordered regions of proteins that function in cellular signaling and regulation, by binding to protein interaction domains or by being the target of post-translational modifications. Linear motifs pose difficulty not only to experimental study, but also computational methods they are difficult to identify due to their small size and their binding specificity is affected by several factors acting in concert. We discuss the different ways linear motifs can be represented computationally, and how computational approaches can integrate the different specificity-determining factors. We illustrate these issues on our own work focusing on the use of three-dimensional structural information in predicting protein phosphorylation sites, and the integration of erse types of data in predicting nuclear localization. Computational approaches will play an increasing role in the future, allowing new relationships and system-wide understanding to be unearthed from the large datasets becoming available through high-throughput studies.

Flow cytometric reporter assays provide robust functional analysis of signaling complexes

Publisher: Elsevier BV

Date: 12-2022

DOI: 10.1016/J.JBC.2022.102666

Abstract: Conventional assays to probe signaling protein interactions and function involve measurement of luciferase reporter expression within the bulk cell population, with lack of control over target-protein expression level. To address this issue, we have developed a rapid and robust flow cytometric assay for analysis of signaling protein function. A fluorescent reporter and fluorescent tagging of the target protein enables simultaneous assessment of protein expression and signaling within in idual cells. We have applied our technique to the analysis of variants of the lipopolysaccharide (LPS) receptor Toll-like receptor 4 (TLR4) and its adapter protein MyD88, using a NF-кB-responsive promoter driving mScarlet-I expression. The assay enables exclusion of non-transfected cells and overexpressing cells that signal spontaneously. Additionally, our assay allows for the identification of protein variants that fail to express. We found that the assays were highly sensitive, with cells expressing an appropriate level of GFP-MyD88 showing approximately 200-fold induction of mScarlet-I by LPS, and we can detect subtle protein concentration-dependent effects of mutations. Importantly, the assay is adaptable to various signaling pathways.

Crystal structure of importin‐α bound to a peptide bearing the nuclear localisation signal from chloride intracellular channel protein 4

Publisher: Wiley

Date: 30-03-2011

DOI: 10.1111/J.1742-4658.2011.08086.X

Abstract: It has been reported that a human chloride intracellular channel (CLIC) protein, CLIC4, translocates to the nucleus in response to cellular stress, facilitated by a putative CLIC4 nuclear localization signal (NLS). The CLIC4 NLS adopts an α-helical structure in the native CLIC4 fold. It is proposed that CLIC4 is transported to the nucleus via the classical nuclear import pathway after binding the import receptor, importin-α. In this study, we have determined the X-ray crystal structure of a truncated form of importin-α lacking the importin-β binding domain, bound to a CLIC4 NLS peptide. The NLS peptide binds to the major binding site in an extended conformation similar to that observed for the classical simian virus 40 large T-antigen NLS. A Tyr residue within the CLIC4 NLS makes surprisingly favourable interactions by forming side-chain hydrogen bonds to the importin-α backbone. This structural evidence supports the hypothesis that CLIC4 translocation to the nucleus is governed by the importin-α nuclear import pathway, provided that CLIC4 can undergo a conformational rearrangement that exposes the NLS in an extended conformation.

A Trap-Door Mechanism for Zinc Acquisition by Streptococcus pneumoniae AdcA

Publisher: American Society for Microbiology

Date: 23-02-2021

DOI: 10.1128/MBIO.01958-20

Abstract: Zinc is an essential nutrient for the virulence of bacterial pathogens such as Streptococcus pneumoniae . Many Gram-positive bacteria use a two-domain lipoprotein for zinc acquisition, but how this class of metal-recruiting proteins acquire zinc and interact with the uptake machinery has remained poorly defined.

Uses for JNK: the Many and Varied Substrates of the c-Jun N-Terminal Kinases

Publisher: American Society for Microbiology

Date: 12-2006

DOI: 10.1128/MMBR.00025-06

Abstract: The c-Jun N-terminal kinases (JNKs) are members of a larger group of serine/threonine (Ser/Thr) protein kinases from the mitogen-activated protein kinase family. JNKs were originally identified as stress-activated protein kinases in the livers of cycloheximide-challenged rats. Their subsequent purification, cloning, and naming as JNKs have emphasized their ability to phosphorylate and activate the transcription factor c-Jun. Studies of c-Jun and related transcription factor substrates have provided clues about both the preferred substrate phosphorylation sequences and additional docking domains recognized by JNK. There are now more than 50 proteins shown to be substrates for JNK. These include a range of nuclear substrates, including transcription factors and nuclear hormone receptors, heterogeneous nuclear ribonucleoprotein K, and the Pol I-specific transcription factor TIF-IA, which regulates ribosome synthesis. Many nonnuclear substrates have also been characterized, and these are involved in protein degradation (e.g., the E3 ligase Itch), signal transduction (e.g., adaptor and scaffold proteins and protein kinases), apoptotic cell death (e.g., mitochondrial Bcl2 family members), and cell movement (e.g., paxillin, DCX, microtubule-associated proteins, the stathmin family member SCG10, and the intermediate filament protein keratin 8). The range of JNK actions in the cell is therefore likely to be complex. Further characterization of the substrates of JNK should provide clearer explanations of the intracellular actions of the JNKs and may allow new avenues for targeting the JNK pathways with therapeutic agents downstream of JNK itself.

Death, TIR, and RHIM: Self-assembling domains involved in innate immunity and cell-death signaling

Publisher: Oxford University Press (OUP)

Date: 05-12-2019

DOI: 10.1002/JLB.MR0318-123R

Abstract: The innate immune system consists of pattern recognition receptors (PRRs) that detect pathogen- and endogenous danger-associated molecular patterns (PAMPs and DAMPs), initiating signaling pathways that lead to the induction of cytokine expression, processing of pro-inflammatory cytokines, and induction of cell-death responses. An emerging concept in these pathways and associated processes is signaling by cooperative assembly formation (SCAF), which involves formation of higher order oligomeric complexes, and enables rapid and strongly lified signaling responses to minute amounts of stimulus. Many of these signalosomes assemble through homotypic interactions of members of the death-fold (DF) superfamily, Toll/IL-1 receptor (TIR) domains, or the RIP homotypic interaction motifs (RHIM). We review the current understanding of the structure and function of these domains and their molecular interactions with a particular focus on higher order assemblies. We review the structure and function of domains involved in signaling by cooperative assembly formation (SCAF) in innate immunity and cell-death pathways.

Structural and Functional Characterization of a Cross-Reactive Dengue Virus Neutralizing Antibody that Recognizes a Cryptic Epitope

Publisher: Elsevier BV

Date: 2018

DOI: 10.1016/J.STR.2017.11.017

Abstract: Understanding the molecular basis of the neutralizing antibody response to dengue virus (DENV) is an essential component in the design and development of effective vaccines and immunotherapeutics. Here we present the structure of a cross-reactive, neutralizing antibody, 3E31, in complex with domain III (DIII) of the DENV envelope (E) protein and reveal a conserved, temperature-sensitive, cryptic epitope on DIII that is not available in any of the known conformations of E on the dengue virion. We observed that 3E31 inhibits E-mediated membrane fusion, suggesting that the antibody is able to neutralize virus through binding an as-yet uncharacterized intermediate conformation of DENV E and sterically block trimer formation. Finally, we show that, unlike cross-reactive fusion peptide-specific antibodies, 3E31 does not promote antibody-dependent enhancement of infection at sub-neutralizing concentrations. Our results highlight the 3E31 epitope on the E protein DIII as a promising target for immunotherapeutics or vaccine design.

Structural basis of thiol-based regulation of formaldehyde detoxification inH. influenzaeby a MerR regulator with no sensor region

Publisher: Oxford University Press (OUP)

Date: 15-06-2016

DOI: 10.1093/NAR/GKW543

The leucine-rich repeat: a versatile binding motif

Publisher: Elsevier BV

Date: 10-1994

DOI: 10.1016/0968-0004(94)90090-6

Abstract: Leucine-rich repeats are short sequence motifs present in a number of proteins with erse functions and cellular locations. All proteins containing these repeats are thought to be involved in protein-protein interactions. The crystal structure of ribonuclease inhibitor protein has revealed that leucine-rich repeats correspond to beta-alpha structural units. These units are arranged so that they form a parallel beta-sheet with one surface exposed to solvent, so that the protein acquires an unusual, nonglobular shape. These two features may be responsible for the protein-binding functions of proteins containing leucine-rich repeats.

Engineering indel and substitution variants of diverse and ancient enzymes using Graphical Representation of Ancestral Sequence Predictions (GRASP)

Publisher: Public Library of Science (PLoS)

Date: 24-10-2022

DOI: 10.1371/JOURNAL.PCBI.1010633

Abstract: Ancestral sequence reconstruction is a technique that is gaining widespread use in molecular evolution studies and protein engineering. Accurate reconstruction requires the ability to handle appropriately large numbers of sequences, as well as insertion and deletion (indel) events, but available approaches exhibit limitations. To address these limitations, we developed Graphical Representation of Ancestral Sequence Predictions (GRASP), which efficiently implements maximum likelihood methods to enable the inference of ancestors of families with more than 10,000 members. GRASP implements partial order graphs (POGs) to represent and infer insertion and deletion events across ancestors, enabling the identification of building blocks for protein engineering. To validate the capacity to engineer novel proteins from realistic data, we predicted ancestor sequences across three distinct enzyme families: glucose-methanol-choline (GMC) oxidoreductases, cytochromes P450, and dihydroxy/sugar acid dehydratases (DHAD). All tested ancestors demonstrated enzymatic activity. Our study demonstrates the ability of GRASP (1) to support large data sets over 10,000 sequences and (2) to employ insertions and deletions to identify building blocks for engineering biologically active ancestors, by exploring variation over evolutionary time.

Recombinant and epitope-based vaccines on the road to the market and implications for vaccine design and production

Publisher: Informa UK Limited

Date: 02-10-2015

DOI: 10.1080/21645515.2015.1094595

Crystal structure and insights into the oligomeric state of UDP-glucose pyrophosphorylase from sugarcane

Publisher: Public Library of Science (PLoS)

Date: 03-2018

DOI: 10.1371/JOURNAL.PONE.0193667

Focusing in on structural genomics: The University of Queensland structural biology pipeline

Publisher: Elsevier BV

Date: 12-2006

DOI: 10.1016/J.BIOENG.2006.09.002

Abstract: The flood of new genomic sequence information together with technological innovations in protein structure determination have led to worldwide structural genomics (SG) initiatives. The goals of SG initiatives are to accelerate the process of protein structure determination, to fill in protein fold space and to provide information about the function of uncharacterized proteins. In the long-term, these outcomes are likely to impact on medical biotechnology and drug discovery, leading to a better understanding of disease as well as the development of new therapeutics. Here we describe the high throughput pipeline established at the University of Queensland in Australia. In this focused pipeline, the targets for structure determination are proteins that are expressed in mouse macrophage cells and that are inferred to have a role in innate immunity. The aim is to characterize the molecular structure and the biochemical and cellular function of these targets by using a parallel processing pipeline. The pipeline is designed to work with tens to hundreds of target gene products and comprises target selection, cloning, expression, purification, crystallization and structure determination. The structures from this pipeline will provide insights into the function of previously uncharacterized macrophage proteins and could lead to the validation of new drug targets for chronic obstructive pulmonary disease and arthritis.

A Glutathione-Dependent Detoxification System Is Required for Formaldehyde Resistance and Optimal Survival ofNeisseria meningitidisin Biofilms

Publisher: Mary Ann Liebert Inc

Date: 03-2013

DOI: 10.1089/ARS.2012.4749

Evolutionary model of protein secondary structure capable of revealing new biological relationships

Publisher: Wiley

Date: 23-06-2020

DOI: 10.1002/PROT.25898

Probing the Specificity of Binding to the Major Nuclear Localization Sequence-binding Site of Importin-α Using Oriented Peptide Library Screening

Publisher: Elsevier BV

Date: 06-2010

DOI: 10.1074/JBC.M109.079574

Crystallization of importin α, the nuclear-import receptor

Publisher: International Union of Crystallography (IUCr)

Date: 02-1999

DOI: 10.1107/S0907444998012943

Abstract: Crystals of recombinant importin α, the nuclear-import receptor, have been obtained at two different pH conditions by vapour diffusion using sodium citrate as precipitant and dithiothreitol as an additive. At pH 4–5, the crystals have the symmetry of the trigonal space group P 3 1 21 or P 3 2 21 ( a = b = 78.0, c = 255.8 Å, γ = 120°) at pH 6–7, the crystals have the symmetry of the orthorhombic space group P 2 1 2 1 2 1 ( a = 78.5, b = 89.7, c = 100.5 Å). In both cases, there is probably one molecule of importin α in the asymmetric unit. At least one of the crystal forms diffracts to a resolution higher than 3 Å using the laboratory X-ray source the crystals are suitable for crystal structure determination.

Conformations of wild-type and mutant OmpA signal sequences in membrane mimetic environments

Publisher: Springer Netherlands

Date: 1992

DOI: 10.1007/978-94-011-2264-1_94

Complex between Bovine Ribonuclease A and Porcine Ribonuclease Inhibitor Crystallizes in a Similar Unit Cell as Free Ribonuclease Inhibitor

Publisher: Elsevier BV

Date: 08-1994

DOI: 10.1006/JMBI.1994.1502

Abstract: We obtained three different morphologies of co-crystals of bovine ribonuclease A and porcine ribonuclease inhibitor. X-ray quality crystals were grown in 1.3M ammonium sulfate, 100 mM sodium acetate (pH 5.0) and 20 mM dithiothreitol at 21 degrees C. These crystals have the symmetry of the tetragonal space group I4 with a = 133.3 A and c = 86.7 A and diffract to 2.5 A resolution they have the same symmetry and only slightly different cell parameters than the crystals of free ribonuclease inhibitor. Polyacrylamide gel electrophoresis and the crystal density indicate that both ribonuclease inhibitor and ribonuclease A are present in the crystals. Although small, crystals are suitable for three-dimensional structural analysis.

A Proteome-Wide Immunoinformatics Tool to Accelerate T-Cell Epitope Discovery and Vaccine Design in the Context of Emerging Infectious Diseases: An Ethnicity-Oriented Approach

Publisher: Frontiers Media SA

Date: 26-02-2021

DOI: 10.3389/FIMMU.2021.598778

Abstract: Emerging infectious diseases (EIDs) caused by viruses are increasing in frequency, causing a high disease burden and mortality world-wide. The COVID-19 pandemic caused by the novel SARS-like coronavirus (SARS-CoV-2) underscores the need to innovate and accelerate the development of effective vaccination strategies against EIDs. Human leukocyte antigen (HLA) molecules play a central role in the immune system by determining the peptide repertoire displayed to the T-cell compartment. Genetic polymorphisms of the HLA system thus confer a strong variability in vaccine-induced immune responses and may complicate the selection of vaccine candidates, because the distribution and frequencies of HLA alleles are highly variable among different ethnic groups. Herein, we build on the emerging paradigm of rational epitope-based vaccine design, by describing an immunoinformatics tool (Pre ac-3.0) for proteome-wide T-cell epitope discovery that accounts for ethnic-level variations in immune responsiveness. Pre ac-3.0 implements both CD8+ and CD4+ T-cell epitope predictions based on HLA allele frequencies retrieved from the Allele Frequency Net Database. The tool was thoroughly assessed, proving comparable performances (AUC ~0.9) against four state-of-the-art pan-specific immunoinformatics methods capable of population-level analysis (NetMHCPan-4.0, Pickpocket, PSSMHCPan and SMM), as well as a strong accuracy on proteome-wide T-cell epitope predictions for HIV-specific immune responses in the Japanese population. The utility of the method was investigated for the COVID-19 pandemic, by performing in silico T-cell epitope mapping of the SARS-CoV-2 spike glycoprotein according to the ethnic context of the countries where the ChAdOx1 vaccine is currently initiating phase III clinical trials. Potentially immunodominant CD8+ and CD4+ T-cell epitopes and population coverages were predicted for each population (the Epitope Discovery mode), along with optimized sets of broadly recognized (promiscuous) T-cell epitopes maximizing coverage in the target populations (the Epitope Optimization mode). Population-specific epitope-rich regions (T-cell epitope clusters) were further predicted in protein antigens based on combined criteria of epitope density and population coverage. Overall, we conclude that Pre ac-3.0 holds potential to contribute in the understanding of ethnic-level variations of vaccine-induced immune responsiveness and to guide the development of epitope-based next-generation vaccines against emerging pathogens, whose geographic distributions and populations in need of vaccinations are often well-defined for regional epidemics.

Animal NLRs continue to inform plant NLR structure and function

Publisher: Elsevier BV

Date: 07-2019

DOI: 10.1016/J.ABB.2019.05.001

Abstract: Plant NLRs share many of the structural hallmarks of their animal counterparts. At a functional level, the central nucleotide-binding pocket appears to have binding and hydrolysis activities, similar to that of animal NLRs. The TIR domains of plant NLRs have been shown to self-associate, and there is emerging evidence that full-length plant NLRs may do so as well. It is therefore tempting to speculate that plant NLRs may form higher-order complexes similar to those of the mammalian inflammasome. Here we review the available knowledge on structure-function relationships in plant NLRs, focusing on how the information available on animal NLRs informs the mechanism of plant NLR function, and highlight the evidence that innate immunity signalling pathways in multicellular organisms often require the formation of higher-order protein complexes.

Self-association configures the NAD⁺-binding site of plant NLR TIR domains

Publisher: Cold Spring Harbor Laboratory

Date: 03-10-2021

DOI: 10.1101/2021.10.02.462850

Abstract: TIR domains are signalling domains present in plant nucleotide-binding leucine-rich repeat receptors (NLRs), with key roles in plant innate immunity. They are required for the induction of a hypersensitive response (HR) in effector-triggered immunity, but the mechanism by which this occurs is not yet fully understood. It has been recently shown that the TIR domains from several plant NLRs possess NADase activity. The oligomeric structure of TIR-containing NLRs ROQ1 and RPP1 reveals how the TIR domains arrange into an active conformation, but low resolution around the NAD + binding sites leaves questions unanswered about the molecular mechanisms linking self-association and NADase activity. In this study, a number of crystal structures of the TIR domain from the grapevine NLR RUN1 reveal how self-association and enzymatic activity may be linked. Structural features previously proposed to play roles involve the “AE interface” (mediated by helices A and E), the “BB-loop” (connecting β-strand B and helix B in the structure), and the “BE interface” (mediated by the BB-loop from one TIR and the “DE surface” of another). We demonstrate that self-association through the AE interface induces conformational changes in the NAD + -binding site, shifting the BB-loop away from the catalytic site and allowing NAD + to access the active site. We propose that an intact “DE surface” is necessary for production of the signalling product (variant cyclic ADPR), as it constitutes part of the active site. Addition of NAD + or NADP + is not sufficient to induce self-association, suggesting that NAD + binding occurs after TIR self-association. Our study identifies a mechanistic link between TIR self-association and NADase activity.

Hybrid Methods for Protein Structure Prediction

Publisher: Wiley

Date: 07-09-2010

DOI: 10.1002/9780470882207.CH12

TIR domain-associated nucleotides with functions in plant immunity and beyond

Publisher: Elsevier BV

Date: 06-2023

DOI: 10.1016/J.PBI.2023.102364

The Predikin webserver: improved prediction of protein kinase peptide specificity using structural information

Publisher: Oxford University Press (OUP)

Date: 19-05-2008

DOI: 10.1093/NAR/GKN279

Molecular basis for specificity of nuclear import and prediction of nuclear localization

Publisher: Elsevier BV

Date: 09-2011

DOI: 10.1016/J.BBAMCR.2010.10.013

Abstract: Although proteins are translated on cytoplasmic ribosomes, many of these proteins play essential roles in the nucleus, mediating key cellular processes including but not limited to DNA replication and repair as well as transcription and RNA processing. Thus, understanding how these critical nuclear proteins are accurately targeted to the nucleus is of paramount importance in biology. Interaction and structural studies in the recent years have jointly revealed some general rules on the specificity determinants of the recognition of nuclear targeting signals by their specific receptors, at least for two nuclear import pathways: (i) the classical pathway, which involves the classical nuclear localization sequences (cNLSs) and the receptors importin-α/karyopherin-α and importin-β/karyopherin-β1 and (ii) the karyopherin-β2 pathway, which employs the proline-tyrosine (PY)-NLSs and the receptor transportin-1/karyopherin-β2. The understanding of specificity rules allows the prediction of protein nuclear localization. We review the current understanding of the molecular determinants of the specificity of nuclear import, focusing on the importin-α•cargo recognition, as well as the currently available databases and predictive tools relevant to nuclear localization. This article is part of a Special Issue entitled: Regulation of Signaling and Cellular Fate through Modulation of Nuclear Protein Import.

Crystallization and preliminary X-ray diffraction analyses of the TIR domains of three TIR-NB-LRR proteins that are involved in disease resistance in Arabidopsis thaliana.

Publisher: International Union of Crystallography (IUCr)

Date: 30-10-2013

DOI: 10.1107/S1744309113026614

TRAF2 recruitment via T61 in CD30 drives NFκB activation and enhances hESC survival and proliferation

Publisher: American Society for Cell Biology (ASCB)

Date: 03-2015

DOI: 10.1091/MBC.E14-08-1290

Abstract: CD30 activates NFκB signaling in human embryonic stem cells. A single threonine residue in the CD30v protein is critical for this and recruitment of TRAF2. The data reveal the importance of this interaction for hESC survival and proliferation.

FHA domain boundaries of the Dun1p and Rad53p cell cycle checkpoint kinases

Publisher: Wiley

Date: 10-04-2000

DOI: 10.1016/S0014-5793(00)01392-2

Abstract: Dun1p and Rad53p of the budding yeast Saccharomyces cerevisiae are members of a conserved family of cell cycle checkpoint protein kinases that contain forkhead-associated (FHA) domains. Here, we demonstrate that these FHA domains contain 130-140 residues, and are thus considerably larger than previously predicted by sequence comparisons (55-75 residues). In vivo, expression of the proteolytically defined Dun1p FHA domain, but not a fragment containing only the predicted domain boundaries, inhibited the transcriptional induction of repair genes following replication blocks. This indicates that the non-catalytic FHA domain plays an important role in the transcriptional function of the Dun1p protein kinase.

Factors influencing nucleo-cytoplasmic trafficking: which matter? Response to Alvisi & Jans' comment onPhosphorylation adjacent to the nuclear localization signal of human dUTPase abolishes nuc