ORCID Profile
0000-0001-7508-8033
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In Research Link Australia (RLA), "Research Topics" refer to ANZSRC FOR and SEO codes. These topics are either sourced from ANZSRC FOR and SEO codes listed in researchers' related grants or generated by a large language model (LLM) based on their publications.
Biochemistry and Cell Biology | Structural Biology (incl. Macromolecular Modelling) | Genetics | Analytical Biochemistry | Biophysics | Gene Expression (incl. Microarray and other genome-wide approaches) | Characterisation of Biological Macromolecules | Medicinal and Biomolecular Chemistry | Biologically Active Molecules | Structural Chemistry and Spectroscopy | Enzymes | Gene Expression | Characterisation Of Macromolecules | Pharmaceutical Sciences | Biomolecular Modelling and Design | Physical Chemistry (Incl. Structural) | Proteins and Peptides | Structural Chemistry | Condensed Matter Physics | Polymerisation Mechanisms | Chemical Characterisation of Materials | Receptors and Membrane Biology | Biochemistry and Cell Biology not elsewhere classified | Instruments And Techniques | Epigenetics (incl. Genome Methylation and Epigenomics) | Genome Structure and Regulation | Medical Biochemistry: Proteins And Peptides | Cellular Interactions (Incl. Adhesion, Matrix, Cell Wall) | Organic Semiconductors | Cell Neurochemistry | Medical Molecular Engineering of Nucleic Acids and Proteins | Pharmacology and Pharmaceutical Sciences | Solid State Chemistry | Macromolecular and Materials Chemistry | Other Physical Sciences | Medical Biochemistry and Metabolomics | Medical Biochemistry: Proteins and Peptides (incl. Medical Proteomics) | Agricultural Molecular Engineering of Nucleic Acids and Proteins | Electronic and Magnetic Properties of Condensed Matter; Superconductivity | Cell Development, Proliferation and Death
Expanding Knowledge in the Biological Sciences | Biological sciences | Expanding Knowledge in the Medical and Health Sciences | Expanding Knowledge in the Chemical Sciences | Human Pharmaceutical Treatments (e.g. Antibiotics) | Cardiovascular system and diseases | Cancer and related disorders | Scientific instrumentation | Expanding Knowledge in the Agricultural and Veterinary Sciences | Control of Animal Pests, Diseases and Exotic Species in Farmland, Arable Cropland and Permanent Cropland Environments | Clinical Health (Organs, Diseases and Abnormal Conditions) not elsewhere classified | Neurodegenerative Disorders Related to Ageing | Chemical sciences | Clinical health not specific to particular organs, diseases and conditions | Cancer and Related Disorders | Diagnostics | Treatments (e.g. chemicals, antibiotics) | Infectious Diseases | Other | Blood disorders | Crop Protection Chemicals | Diagnostic methods | Biofuel (Biomass) Energy | Solar-Photovoltaic Energy | Polymeric materials (e.g. paints) | Expanding Knowledge in the Physical Sciences |
Publisher: Maad Rayan Publishing Company
Date: 28-01-2018
DOI: 10.15171/BI.2018.19
Publisher: American Chemical Society (ACS)
Date: 28-02-2008
DOI: 10.1021/BI702390K
Abstract: Orotidine 5'-monophosphate (OMP) decarboxylase from Plasmodium falciparum (PfODCase, EC 4.1.1.23) has been overexpressed, purified, subjected to kinetic and biochemical analysis, and crystallized. The native enzyme is a homodimer with a subunit molecular mass of 38 kDa. The saturation curve for OMP as a substrate conformed to Michaelis-Menten kinetics with K m = 350 +/- 60 nM and V max = 2.70 +/- 0.10 micromol/min/mg protein. Inhibition patterns for nucleoside 5'-monophosphate analogues were linear competitive with respect to OMP with a decreasing potency of inhibition of PfODCase in the order: pyrazofurin 5'-monophosphate ( K i = 3.6 +/- 0.7 nM) > xanthosine 5'-monophosphate (XMP, K i = 4.4 +/- 0.7 nM) > 6-azauridine 5'-monophosphate (AzaUMP, K i = 12 +/- 3 nM) > allopurinol-3-riboside 5'-monophosphate ( K i = 240 +/- 20 nM). XMP is an approximately 150-fold more potent inhibitor of PfODCase compared with the human enzyme. The structure of PfODCase was solved in the absence of ligand and displays a classic TIM-barrel fold characteristic of the enzyme. Both the phosphate-binding loop and the betaalpha5-loop have conformational flexibility, which may be associated with substrate capture and product release along the reaction pathway.
Publisher: Elsevier BV
Date: 05-2012
DOI: 10.1016/J.TIBS.2012.02.001
Abstract: Classical zinc fingers (ZFs) are one of the most common protein domains in higher eukaryotes and have been known for almost 30 years to act as sequence-specific DNA-binding domains. This knowledge has come, however, from the study of a small number of archetypal proteins, and a larger picture is beginning to emerge that ZF functions are far more erse than originally suspected. Here, we review the evidence that a subset of ZF proteins live double lives, binding to both DNA and RNA targets and frequenting both the cytoplasm and the nucleus. This duality can create an important additional level of gene regulation that serves to connect transcriptional and post-transcriptional control.
Publisher: Bentham Science Publishers Ltd.
Date: 11-2009
DOI: 10.2174/138161209789271861
Abstract: LIM (Lin-11, Isl-1, Mec-3), RING (Really interesting new gene), PHD (Plant homology domain) and MYND (myeloid, Nervy, DEAF-1) domains are all zinc-binding domains that ligate two zinc ions. Unlike the better known classical zinc fingers, these domains do not bind DNA, but instead mediate interactions with other proteins. LIM-domain containing proteins have erse functions as regulators of gene expression, cell adhesion and motility and signal transduction. RING finger proteins are generally associated with ubiquitination the presence of such a domain is the defining feature of a class of E3 ubiquitin protein ligases. PHD proteins have been associated with SUMOylation but most recently have emerged as a chromatin recognition motif that reads the methylation state of histones. The function of the MYND domain is less clear, but MYND domains are also found in proteins that have ubiquitin ligase and/or histone methyltransferase activity. Here we review the structure-function relationships for these domains and discuss strategies to modulate their activity.
Publisher: Springer Science and Business Media LLC
Date: 04-1997
DOI: 10.1007/BF02443517
Publisher: Elsevier BV
Date: 04-2005
Publisher: Wiley
Date: 18-11-2019
DOI: 10.1002/AJMG.C.31752
Abstract: The nucleosome remodeling and deacetylase (NuRD) complex is a major regulator of gene expression involved in pluripotency, lineage commitment, and corticogenesis. This important complex is composed of seven different proteins, with mutations in CHD3, CHD4, and GATAD2B being associated with neurodevelopmental disorders presenting with macrocephaly and intellectual disability similar to other overgrowth and intellectual disability (OGID) syndromes. Pathogenic variants in CHD3 and CHD4 primarily involve disruption of enzymatic function. GATAD2B variants include loss-of-function mutations that alter protein dosage and missense variants that involve either of two conserved domains (CR1 and CR2) known to interact with other NuRD proteins. In addition to macrocephaly and intellectual disability, CHD3 variants are associated with inguinal hernias and apraxia of speech whereas CHD4 variants are associated with skeletal anomalies, deafness, and cardiac defects. GATAD2B-associated neurodevelopmental disorder (GAND) has phenotypic overlap with both of these disorders. Of note, structural models of NuRD indicate that CHD3 and CHD4 require direct contact with the GATAD2B-CR2 domain to interact with the rest of the complex. Therefore, the phenotypic overlaps of CHD3- and CHD4-related disorders with GAND are consistent with a loss in the ability of GATAD2B to recruit CHD3 or CHD4 to the complex. The shared features of these neurodevelopmental disorders may represent a new class of OGID syndrome: the NuRDopathies.
Publisher: Elsevier BV
Date: 11-2004
DOI: 10.1016/J.CELL.2004.11.025
Abstract: Hemoglobin A (HbA), the oxygen delivery system in humans, comprises two alpha and two beta subunits. Free alpha-hemoglobin (alphaHb) is unstable, and its precipitation contributes to the pathophysiology of beta thalassemia. In erythrocytes, the alpha-hemoglobin stabilizing protein (AHSP) binds alphaHb and inhibits its precipitation. The crystal structure of AHSP bound to Fe(II)-alphaHb reveals that AHSP specifically recognizes the G and H helices of alphaHb through a hydrophobic interface that largely recapitulates the alpha1-beta1 interface of hemoglobin. The AHSP-alphaHb interactions are extensive but suboptimal, explaining why beta-hemoglobin can competitively displace AHSP to form HbA. Remarkably, the Fe(II)-heme group in AHSP bound alphaHb is coordinated by the distal but not the proximal histidine. Importantly, binding to AHSP facilitates the conversion of oxy-alphaHb to a deoxygenated, oxidized [Fe(III)], nonreactive form in which all six coordinate positions are occupied. These observations reveal the molecular mechanisms by which AHSP stabilizes free alphaHb.
Publisher: Elsevier BV
Date: 10-2016
DOI: 10.1016/J.JMB.2016.08.028
Abstract: The packaging of eukaryotic DNA into nucleosomes, and the organisation of these nucleosomes into chromatin, plays a critical role in regulating all DNA-associated processes. Chromodomain helicase DNA-binding protein 1 (CHD1) is an ATP-dependent chromatin remodelling protein that is conserved throughout eukaryotes and has an ability to assemble and organise nucleosomes both in vitro and in vivo. This activity is involved in the regulation of transcription and is implicated in mammalian development and stem cell biology. CHD1 is classically depicted as possessing a pair of tandem chromodomains that directly precede a core catalytic helicase-like domain that is then followed by a SANT-SLIDE DNA-binding domain. Here, we have identified an additional conserved domain C-terminal to the SANT-SLIDE domain and determined its structure by multidimensional heteronuclear NMR spectroscopy. We have termed this domain the CHD1 helical C-terminal (CHCT) domain as it is comprised of five α-helices arranged in a variant helical bundle topology. CHCT has a conserved, positively charged surface and is able to bind DNA and nucleosomes. In addition, we have identified another group of proteins, the as yet uncharacterised C17orf64 proteins, as also containing a conserved CHCT domain. Our data provide new structural insights into the CHD1 enzyme family.
Publisher: Wiley
Date: 15-02-2002
DOI: 10.1046/J.1432-1033.2002.02766.X
Abstract: The solution structure of a synthetic mutant type I antifreeze protein (AFP I) was determined in aqueous solution at pH 7.0 using nuclear magnetic resonance (NMR) spectroscopy. The mutations comprised the replacement of the four Thr residues by Val and the introduction of two additional Lys-Glu salt bridges. The antifreeze activity of this mutant peptide, VVVV2KE, has been previously shown to be similar to that of the wild type protein, HPLC6 (defined here as TTTT). The solution structure reveals an alphahelix bent in the same direction as the more bent conformer of the published crystal structure of TTTT, while the side chain chi1 rotamers of VVVV2KE are similar to those of the straighter conformer in the crystal of TTTT. The Val side chains of VVVV2KE assume the same orientations as the Thr side chains of TTTT, confirming the conservative nature of this mutation. The combined data suggest that AFP I undergoes an equilibrium between straight and bent helices in solution, combined with independent equilibria between different side chain rotamers for some of the amino acid residues. The present study presents the first complete sequence-specific resonance assignments and the first complete solution structure determination by NMR of any AFP I protein.
Publisher: Wiley
Date: 02-07-2010
DOI: 10.1002/PROT.22752
Abstract: Prostate apoptosis response factor-4 (Par-4) is a pro-apoptotic and tumor-suppressive protein. A highly conserved heptad repeat sequence at the Par-4 C-terminus suggests the presence of a leucine zipper (LZ). This C-terminal region is essential for Par-4 self-association and interaction with various effector proteins. We have used nuclear magnetic resonance (NMR) spectroscopy to fully assign the chemical shift resonances of a peptide comprising the LZ domain of Par-4 at neutral pH. Further, we have investigated the properties of the Par-4 LZ domain and two point mutants under a variety of conditions using NMR, circular dichroism (CD), light scattering, and bioinformatics. Results indicate an environment-dependent conformational equilibrium between a partially ordered monomer (POM) and a predominantly coiled coil dimer (CCD). The combination of techniques used allows the time scales of the equilibrium to be probed and also helps to identify features of the amino acid sequence that may influence the equilibrium.
Publisher: Wiley
Date: 09-07-2014
Abstract: The realization that gene transcription is much more pervasive than previously thought and that many erse RNA species exist in simple as well as complex organisms has triggered efforts to develop functionalized RNA-binding proteins (RBPs) that have the ability to probe and manipulate RNA function. Previously, we showed that the RanBP2-type zinc finger (ZF) domain is a good candidate for an addressable single-stranded-RNA (ssRNA) binding domain that can recognize ssRNA in a modular and specific manner. In the present study, we successfully engineered a sequence specificity change onto this ZF scaffold by using a combinatorial approach based on phage display. This work constitutes a foundation from which a set of RanBP2 ZFs might be developed that is able to recognize any given RNA sequence.
Publisher: Cold Spring Harbor Laboratory
Date: 25-11-2021
DOI: 10.1101/2021.11.25.469965
Abstract: The Nucleosome Remodeling and Deacetylase (NuRD) complex is a chromatin-modifying assembly that regulates gene expression and DNA damage repair. Despite its importance, limited structural information describing the complete NuRD complex is available and a detailed understanding of its mechanism is therefore lacking. Drawing on information from SEC-MALLS, DIA-MS, XLMS, negative-stain EM, X-ray crystallography, NMR spectroscopy, secondary structure predictions and homology models, we applied Bayesian integrative structure determination to investigate the molecular architecture of three NuRD sub-complexes: MTA1-HDAC1-RBBP4 (MHR), MTA1 N -HDAC1-MBD3 GATAD2CC (MHM), and MTA1-HDAC1-RBBP4-MBD3-GATAD2A (NuDe). The integrative structures were corroborated by examining independent crosslinks, cryo-EM maps, biochemical assays, known cancer-associated mutations, and structure predictions from AlphaFold. The robustness of the models was assessed by jack-knifing. Localization of the full-length MBD3, which connects the deacetylase and chromatin remodeling modules in NuRD, has not previously been possible our models indicate two different locations for MBD3, suggesting a mechanism by which MBD3 in the presence of GATAD2A asymmetrically bridges the two modules in NuRD. Further, our models uncovered three previously unrecognized subunit interfaces in NuDe: HDAC1 C -MTA1 BAH , MTA1 BAH -MBD3 MBD , and HDAC1 60-100 -MBD3 MBD . Our approach also allowed us to localize regions of unknown structure, such as HDAC1 C and MBD3 IDR , thereby resulting in the most complete and robustly cross-validated structural characterization of these NuRD sub-complexes so far.
Publisher: Royal Society of Chemistry (RSC)
Date: 2021
DOI: 10.1039/D1SC03127A
Abstract: New operationally simple platform for the chemoselective arylation of cysteine in peptides and proteins to access a variety of high value bioconjugates.
Publisher: Cold Spring Harbor Laboratory
Date: 10-11-2021
DOI: 10.1101/2021.11.09.467981
Abstract: Although ACE2 is the primary receptor for SARS-CoV-2 infection, a systematic assessment of host factors that regulate binding to SARS-CoV-2 spike protein has not been described. Here we use whole genome CRISPR activation to identify host factors controlling cellular interactions with SARS-CoV-2. Our top hit was a TLR -related cell surface receptor called leucine-rich repeat-containing protein 15 ( LRRC15 ). LRRC15 expression was sufficient to promote SARS-CoV-2 Spike binding where they form a cell surface complex. LRRC15 mRNA is expressed in human collagen-producing lung myofibroblasts and LRRC15 protein is induced in severe COVID-19 infection where it can be found lining the airways. Mechanistically, LRRC15 does not itself support SARS-CoV-2 infection, but fibroblasts expressing LRRC15 can suppress both pseudotyped and authentic SARS-CoV-2 infection in trans . Moreover, LRRC15 expression in fibroblasts suppresses collagen production and promotes expression of IFIT, OAS, and MX-family antiviral factors. Overall, LRRC15 is a novel SARS-CoV-2 spike-binding receptor that can help control viral load and regulate antiviral and antifibrotic transcriptional programs in the context of COVID-19 infection.
Publisher: Springer Science and Business Media LLC
Date: 28-01-2023
DOI: 10.1038/S41467-023-36114-X
Abstract: Specialized chromatin-binding proteins are required for DNA-based processes during development. We recently established PWWP2A as a direct histone variant H2A.Z interactor involved in mitosis and craniofacial development. Here, we identify the H2A.Z/PWWP2A-associated protein HMG20A as part of several chromatin-modifying complexes, including NuRD, and show that it localizes to distinct genomic regulatory regions. Hmg20a depletion causes severe head and heart developmental defects in Xenopus laevis . Our data indicate that craniofacial malformations are caused by defects in neural crest cell (NCC) migration and cartilage formation. These developmental failures are phenocopied in Hmg20a -depleted mESCs, which show inefficient differentiation into NCCs and cardiomyocytes (CM). Consequently, loss of HMG20A, which marks open promoters and enhancers, results in chromatin accessibility changes and a striking deregulation of transcription programs involved in epithelial-mesenchymal transition (EMT) and differentiation processes. Collectively, our findings implicate HMG20A as part of the H2A.Z/PWWP2A/NuRD-axis and reveal it as a key modulator of intricate developmental transcription programs that guide the differentiation of NCCs and CMs.
Publisher: Proceedings of the National Academy of Sciences
Date: 27-05-2020
Abstract: Blood-feeding arthropods produce antiinflammatory salivary proteins called evasins that function through inhibition of chemokine-receptor signaling in the host. Herein, we show that the evasin ACA-01 from the Amblyomma cajennense tick can be posttranslationally sulfated at two tyrosine residues, albeit as a mixture of sulfated variants. Homogenously sulfated variants of the proteins were efficiently assembled via a semisynthetic native chemical ligation strategy. Sulfation significantly improved the binding affinity of ACA-01 for a range of proinflammatory chemokines and enhanced the ability of ACA-01 to inhibit chemokine signaling through cognate receptors. Comparisons of evasin sequences and structural data suggest that tyrosine sulfation serves as a receptor mimetic strategy for recognizing and suppressing the proinflammatory activity of a wide variety of mammalian chemokines. As such, the incorporation of this posttranslational modification (PTM) or mimics thereof into evasins may provide a strategy to optimize tick salivary proteins for antiinflammatory applications.
Publisher: American Chemical Society (ACS)
Date: 1996
DOI: 10.1021/BI952817O
Publisher: Elsevier BV
Date: 09-2006
DOI: 10.1016/J.JMB.2006.07.010
Abstract: The 22 kDa haem-binding protein, p22HBP, is highly expressed in erythropoietic tissues and binds to a range of metallo- and non-metalloporphyrin molecules with similar affinities, suggesting a role in haem regulation or synthesis. We have determined the three-dimensional solution structure of p22HBP and mapped the porphyrin-binding site, which comprises a number of loops and a alpha-helix all located on a single face of the molecule. The structure of p22HBP is related to the bacterial multi-drug resistance protein BmrR, and is the first protein with this fold to be identified in eukaryotes. Strikingly, the porphyrin-binding site in p22HBP is located in a similar position to the drug-binding site of BmrR. These similarities suggest that the broad ligand specificity observed for both BmrR and p22HBP may result from a conserved ligand interaction mechanism. Taken together, these data suggest that the both the fold and its associated function, that of binding to a broad range of small hydrophobic molecules, are ancient, and have been adapted throughout evolution for a variety of purposes.
Publisher: Elsevier BV
Date: 10-2001
Publisher: Elsevier BV
Date: 12-2005
DOI: 10.1016/J.BIOCEL.2005.06.013
Abstract: CSL (CBF1, Suppressor of Hairless, Lag-1) is a transcription factor that is responsible for activating the genes downstream of the Notch signalling pathway, a pathway that is essential for the development of the nervous system and the differentiation of the haematopoietic system among others. In the absence of Notch signalling, CSL represses transcription of Notch target genes, and following activation by Notch, CSL is converted into a transcriptional activator and activates transcription of the same genes. These two opposing functions of CSL are mediated through interactions with distinct protein complexes. The Notch signalling pathway and its crucial cofactor CSL can maintain cells in an undifferentiated state, and have therefore been associated with a growing list of cancers. In addition, CSL has been co-opted by Epstein-Barr virus to mediate viral and host gene transcription following infection.
Publisher: Wiley
Date: 21-08-2012
DOI: 10.1002/PRO.2124
Publisher: Elsevier BV
Date: 2006
DOI: 10.1016/J.BIOCEL.2005.06.017
Abstract: GATA-1, the founding member of the GATA transcription factor family, is essential for cell maturation and differentiation within the erythroid and megakaryocytic lineages. GATA-1 regulates the expression of many genes within these lineages and its functionality depends upon its ability to bind both DNA and protein partners. Disruption of either of these functions causes severe hematopoietic dysfunction and results in blood disorders, such as thrombocytopenia and anemia. Within this review, we will focus on the structural aspects of GATA-1 with regard to interactions with its many partners and the identification of several mutations that disrupt these interactions.
Publisher: Oxford University Press (OUP)
Date: 03-04-2012
DOI: 10.1093/NAR/GKS285
Publisher: Elsevier BV
Date: 10-2002
DOI: 10.1016/S1357-2725(02)00070-5
Abstract: Ikaros is an essential transcription factor for normal lymphocyte development. Because of its interaction with a number of closely related factors, Ikaros is required for correct regulation of differentiation and cell proliferation in T- and B-cell lineages. Interestingly, Ikaros appears to function both as a transcriptional repressor and as an activator through its ability to bind a large number of nuclear factors, including components of both histone deacetylase and ATP-dependent chromatin remodelling complexes. In addition, nuclear localisation is important for Ikaros function--unlike most transcription factors, Ikaros is localised to discrete nuclear foci in lymphoid cells, suggesting it employs novel mechanisms to regulate transcription.
Publisher: Elsevier BV
Date: 2016
Publisher: Wiley
Date: 11-2005
Abstract: Hemoglobin (Hb) synthesis is coordinated by homeostatic mechanisms to limit the accumulation of free alpha or beta subunits, which are cytotoxic. Alpha hemoglobin-stabilizing protein (AHSP) is an abundant erythroid protein that specifically binds free alphaHb, stabilizes its structure, and limits its ability to participate in chemical reactions that generate reactive oxygen species. Gene ablation studies in mice demonstrate that AHSP is required for normal erythropoiesis. AHSP-null erythrocytes are short-lived, contain Hb precipitates, and exhibit signs of oxidative damage. Loss of AHSP exacerbates beta-thalassemia in mice, indicating that altered AHSP expression or function could modify thalassemia phenotypes in humans, a topic that is beginning to be explored in clinical studies. We used biochemical, spectroscopic, and crystallographic methods to examine how AHSP stabilizes alphaHb. AHSP binds the G and H helices of alphaHb on a surface that largely overlaps with the alpha1-beta1 interface of HbA. This result explains previous findings that betaHb can competitively displace AHSP from alphaHb to form HbA tetramer. Remarkably, binding of AHSP to oxygenated alphaHb induces dramatic conformational changes and converts the heme-bound iron to an oxidized hemichrome state in which all six coordinate positions are occupied. This structure limits the reactivity of heme iron, providing a mechanism by which AHSP stabilizes alphaHb. These findings suggest a biochemical pathway through which AHSP might participate in normal Hb synthesis and modulate the severity of thalassemias. Moreover, understanding how AHSP stabilizes alphaHb provides a theoretical basis for new strategies to inhibit the damaging effects of free alphaHb that accumulates in beta-thalassemia.
Publisher: Elsevier BV
Date: 03-2014
DOI: 10.1016/J.BMC.2015.01.023
Abstract: We have developed an approach for directly isolating an intact multi-protein chromatin remodeling complex from mammalian cell extracts using synthetic peptide affinity reagent 4. FOG1(1-15), a short peptide sequence known to target subunits of the nucleosome remodeling and deacetylase (NuRD) complex, was joined via a 35-atom hydrophilic linker to the StreptagII peptide. Loading this peptide onto Streptactin beads enabled capture of the intact NuRD complex from MEL cell nuclear extract. Gentle biotin elution yielded the desired intact complex free of significant contaminants and in a form that was catalytically competent in a nucleosome remodeling assay. The efficiency of 4 in isolating the NuRD complex was comparable to other reported methods utilising recombinantly produced GST-FOG1(1-45).
Publisher: Elsevier BV
Date: 2018
DOI: 10.1111/JTH.13900
Abstract: Essentials Three dominant variants for the autosomal recessive bleeding disorder type-8 have been described. To date, there has been no phenotype/genotype correlation explaining their dominant transmission. Proline plays an important role in P2Y12R ligand binding and signaling defects. P2Y12R homodimer formation is critical for the receptor function and signaling. Background Although inherited platelet disorders are still underdiagnosed worldwide, advances in molecular techniques are improving disease diagnosis and patient management. Objective To identify and characterize the mechanism underlying the bleeding phenotype in a Caucasian family with an autosomal dominant P2RY12 variant. Methods Full blood counts, platelet aggregometry, flow cytometry and western blotting were performed before next-generation sequencing (NGS). Detailed molecular analysis of the identified variant of the P2Y12 receptor (P2Y12R) was subsequently performed in mammalian cells overexpressing receptor constructs. Results All three referred in iduals had markedly impaired ADP-induced platelet aggregation with primary wave only, despite normal total and surface P2Y12R expression. By NGS, a single P2RY12:c.G794C substitution (p.R265P) was identified in all affected in iduals, and this was confirmed by Sanger sequencing. Mammalian cell experiments with the R265P-P2Y12R variant showed normal receptor surface expression versus wild-type (WT) P2Y12R. Agonist-stimulated R265P-P2Y12R function (both signaling and surface receptor loss) was reduced versus WT P2Y12R. Critically, R265P-P2Y12R acted in a dominant negative manner, with agonist-stimulated WT P2Y12R activity being reduced by variant coexpression, suggesting dramatic loss of WT homodimers. Importantly, platelet P2RY12 cDNA cloning and sequencing in two affected in iduals also revealed three-fold mutant mRNA overexpression, decreasing even further the likelihood of WT homodimer formation. R265 located within extracellular loop 3 (EL3) is one of four residues that are important for receptor functional integrity, maintaining the binding pocket conformation and allowing rotation following ligand binding. Conclusion This novel dominant negative variant confirms the important role of R265 in EL3 in the functional integrity of P2Y12R, and suggests that pathologic heterodimer formation may underlie this family bleeding phenotype.
Publisher: MDPI AG
Date: 18-11-2020
DOI: 10.3390/MOLECULES25225392
Abstract: Proteases catalyse irreversible posttranslational modifications that often alter a biological function of the substrate. The protease dipeptidyl peptidase 4 (DPP4) is a pharmacological target in type 2 diabetes therapy primarily because it inactivates glucagon-like protein-1. DPP4 also has roles in steatosis, insulin resistance, cancers and inflammatory and fibrotic diseases. In addition, DPP4 binds to the spike protein of the MERS virus, causing it to be the human cell surface receptor for that virus. DPP4 has been identified as a potential binding target of SARS-CoV-2 spike protein, so this question requires experimental investigation. Understanding protein structure and function requires reliable protocols for production and purification. We developed such strategies for baculovirus generated soluble recombinant human DPP4 (residues 29–766) produced in insect cells. Purification used differential ammonium sulphate precipitation, hydrophobic interaction chromatography, dye affinity chromatography in series with immobilised metal affinity chromatography, and ion-exchange chromatography. The binding affinities of DPP4 to the SARS-CoV-2 full-length spike protein and its receptor-binding domain (RBD) were measured using surface plasmon resonance and ELISA. This optimised DPP4 purification procedure yielded 1 to 1.8 mg of pure fully active soluble DPP4 protein per litre of insect cell culture with specific activity U/mg, indicative of high purity. No specific binding between DPP4 and CoV-2 spike protein was detected by surface plasmon resonance or ELISA. In summary, a procedure for high purity high yield soluble human DPP4 was achieved and used to show that, unlike MERS, SARS-CoV-2 does not bind human DPP4.
Publisher: Proceedings of the National Academy of Sciences
Date: 18-04-2023
Abstract: Significant recent advances in structural biology, particularly in the field of cryoelectron microscopy, have dramatically expanded our ability to create structural models of proteins and protein complexes. However, many proteins remain refractory to these approaches because of their low abundance, low stability, or—in the case of complexes—simply not having yet been analyzed. Here, we demonstrate the power of using cross-linking mass spectrometry (XL-MS) for the high-throughput experimental assessment of the structures of proteins and protein complexes. This included those produced by high-resolution but in vitro experimental data, as well as in silico predictions based on amino acid sequence alone. We present the largest XL-MS dataset to date, describing 28,910 unique residue pairs captured across 4,084 unique human proteins and 2,110 unique protein–protein interactions. We show that models of proteins and their complexes predicted by AlphaFold2, and inspired and corroborated by the XL-MS data, offer opportunities to deeply mine the structural proteome and interactome and reveal mechanisms underlying protein structure and function.
Publisher: American Chemical Society (ACS)
Date: 06-1994
DOI: 10.1021/JA00090A005
Publisher: American Chemical Society (ACS)
Date: 06-1994
DOI: 10.1021/JA00090A006
Publisher: Elsevier BV
Date: 09-2002
Publisher: Proceedings of the National Academy of Sciences
Date: 02-05-2011
Abstract: Acetylation of histones triggers association with bromodomain-containing proteins that regulate erse chromatin-related processes. Although acetylation of transcription factors has been appreciated for some time, the mechanistic consequences are less well understood. The hematopoietic transcription factor GATA1 is acetylated at conserved lysines that are required for its stable association with chromatin. We show that the BET family protein Brd3 binds via its first bromodomain (BD1) to GATA1 in an acetylation-dependent manner in vitro and in vivo. Mutation of a single residue in BD1 that is involved in acetyl-lysine binding abrogated recruitment of Brd3 by GATA1, demonstrating that acetylation of GATA1 is essential for Brd3 association with chromatin. Notably, Brd3 is recruited by GATA1 to both active and repressed target genes in a fashion seemingly independent of histone acetylation. Anti-Brd3 ChIP followed by massively parallel sequencing in GATA1-deficient erythroid precursor cells and those that are GATA1 replete revealed that GATA1 is a major determinant of Brd3 recruitment to genomic targets within chromatin. A pharmacologic compound that occupies the acetyl-lysine binding pockets of Brd3 bromodomains disrupts the Brd3-GATA1 interaction, diminishes the chromatin occupancy of both proteins, and inhibits erythroid maturation. Together these findings provide a mechanism for GATA1 acetylation and suggest that Brd3 “reads” acetyl marks on nuclear factors to promote their stable association with chromatin.
Publisher: American Chemical Society (ACS)
Date: 02-03-2009
DOI: 10.1021/BI900043P
Publisher: Wiley
Date: 05-10-1997
DOI: 10.1002/(SICI)1097-0282(19971005)42:4<387::AID-BIP2>3.0.CO;2-M
Publisher: Wiley
Date: 05-2002
DOI: 10.1046/J.1365-2958.2002.02920.X
Abstract: The bacterial septum appears to comprise a macromolecular assembly of essential cell ision proteins (the 'septasome') that are responsible for physically iding the cell during cytokinesis. FtsL and DivIC are essential components of this ision machinery in Bacillus subtilis. We used yeast two-hybrid analysis as well as a variety of biochemical and biophysical methods to examine the proposed interaction between Bacillus subtilis FtsL and DivIC. We show that FtsL and DivIC are thermodynamically unstable proteins that are likely to be unfolded and therefore targeted for degradation unless stabilized by interactions with other components of the septasome. However, we show that this stabilization does not result from a direct interaction between FtsL and DivIC. We propose that the observed interdependence of DivIC and FtsL stability is a result of indirect interactions that are mediated by other septasomal proteins.
Publisher: Portland Press Ltd.
Date: 15-01-0011
DOI: 10.1042/BJ20100642
Abstract: To produce functional Hb (haemoglobin), nascent α-globin (αo) and β-globin (βo) chains must each bind a single haem molecule (to form αh and βh) and interact together to form heterodimers. The precise sequence of binding events is unknown, and it has been suggested that additional factors might enhance the efficiency of Hb folding. AHSP (α-haemoglobin-stabilizing protein) has been shown previously to bind αh and regulate redox activity of the haem iron. In the present study, we used a combination of classical and dynamic light scattering and NMR spectroscopy to demonstrate that AHSP forms a heterodimeric complex with αo that inhibits αo aggregation and promotes αo folding in the absence of haem. These findings indicate that AHSP may function as an αo-specific chaperone, and suggest an important role for αo in guiding Hb assembly by stabilizing βo and inhibiting off-pathway self-association of βh.
Publisher: Proceedings of the National Academy of Sciences
Date: 07-04-2009
Abstract: The alternative splicing of mRNA is a critical process in higher eukaryotes that generates substantial proteomic ersity. Many of the proteins that are essential to this process contain arginine/serine-rich (RS) domains. ZRANB2 is a widely-expressed and highly-conserved RS-domain protein that can regulate alternative splicing but lacks canonical RNA-binding domains. Instead, it contains 2 RanBP2-type zinc finger (ZnF) domains. We demonstrate that these ZnFs recognize ssRNA with high affinity and specificity. Each ZnF binds to a single AGGUAA motif and the 2 domains combine to recognize AGGUAA(N x )AGGUAA double sites, suggesting that ZRANB2 regulates alternative splicing via a direct interaction with pre-mRNA at sites that resemble the consensus 5′ splice site. We show using X-ray crystallography that recognition of an AGGUAA motif by a single ZnF is dominated by side-chain hydrogen bonds to the bases and formation of a guanine-tryptophan-guanine “ladder.” A number of other human proteins that function in RNA processing also contain RanBP2 ZnFs in which the RNA-binding residues of ZRANB2 are conserved. The ZnFs of ZRANB2 therefore define another class of RNA-binding domain, advancing our understanding of RNA recognition and emphasizing the versatility of ZnF domains in molecular recognition.
Publisher: Springer Science and Business Media LLC
Date: 04-2011
Publisher: Springer Science and Business Media LLC
Date: 1999
Abstract: Zinc fingers (ZnFs) are generally regarded as DNA-binding motifs. However, a number of recent reports have implicated particular ZnFs in the mediation of protein-protein interactions. The N-terminal ZnF of GATA-1 (NF) is one such finger, having been shown to interact with a number of other proteins, including the recently discovered transcriptional co-factor FOG. Here we solve the three-dimensional structure of the NF in solution using multidimensional 1H/15N NMR spectroscopy, and we use 1H/15N spin relaxation measurements to investigate its backbone dynamics. The structure consists of two distorted beta-hairpins and a single alpha-helix, and is similar to that of the C-terminal ZnF of chicken GATA-1. Comparisons of the NF structure with those of other C4-type zinc binding motifs, including hormone receptor and LIM domains, also reveal substantial structural homology. Finally, we use the structure to map the spatial locations of NF residues shown by mutagenesis to be essential for FOG binding, and demonstrate that these residues all lie on a single face of the NF. Notably, this face is well removed from the putative DNA-binding face of the NF, an observation which is suggestive of simultaneous roles for the NF that is, stabilisation of GATA-1 DNA complexes and recruitment of FOG to GATA-1-controlled promoter regions.
Publisher: Wiley
Date: 02-06-2011
DOI: 10.1002/PROT.23068
Abstract: One of the most common ways to demonstrate a direct protein-protein interaction in vitro is the glutathione-S-transferse (GST)-pulldown. Here we report the detailed characterization of a putative interaction between two transcription factor proteins, GATA-1 and Krüppel-like factor 3 (KLF3/BKLF) that show robust interactions in GST-pulldown experiments. Attempts to map the interaction interface of GATA-1 on KLF3 using a mutagenic screening approach did not yield a contiguous binding face on KLF3, suggesting that the interaction might be non-specific. NMR experiments showed that the proteins do not interact at protein concentrations of 50-100 μM. Rather, the GST tag can cause part of KLF3 to misfold. In addition to misfolding, the fact that both proteins are DNA-binding domains appears to introduce binding artifacts (possibly nucleic acid bridging) that cannot be resolved by the addition of nucleases or ethidium bromide (EtBr). This study emphasizes the need for caution in relying on GST-pulldown results and related methods, without convincing confirmation from different approaches.
Publisher: Springer Science and Business Media LLC
Date: 19-08-2016
DOI: 10.1007/S12104-015-9631-1
Abstract: Chromatin remodelling proteins are an essential family of eukaryotic proteins. They harness the energy from ATP hydrolysis and apply it to alter chromatin structure in order to regulate all aspects of genome biology. Chromodomain helicase DNA-binding protein 1 (CHD1) is one such remodelling protein that has specialised nucleosome organising abilities and is conserved across eukaryotes. CHD1 possesses a pair of tandem chromodomains that directly precede the core catalytic Snf2 helicase-like domain, and a C-terminal SANT-SLIDE DNA-binding domain. We have identified an additional conserved domain in the C-terminal region of CHD1. Here, we report the backbone and side chain resonance assignments for this domain from human CHD1 at pH 6.5 and 25 °C (BMRB No. 25638).
Publisher: Elsevier BV
Date: 06-2008
Publisher: Springer Science and Business Media LLC
Date: 2001
Publisher: Elsevier BV
Date: 05-2005
DOI: 10.1016/J.JSB.2005.02.005
Abstract: Elastin is the protein responsible for the elastic properties of vertebrate tissue. Very little is currently known about the structure of elastin or of its soluble precursor tropoelastin. We have used high-resolution solution NMR methods to probe the conformational preferences of a conserved hydrophobic region in tropoelastin, domain 26 (D26). Using a combination of homonuclear, 15N-separated and triple resonance experiments, we have obtained essentially full chemical shift assignments for D26 at 278K. An analysis of secondary chemical shift changes, as well as NOE and 15N relaxation data, leads us to conclude that this domain is essentially unstructured in solution and does not interact with intact tropoelastin. D26 does not display exposed hydrophobic clusters, as expected for a fully unfolded protein and commensurate with an absence of flexible structural motifs, as identified by lack of binding of the fluorescent probe 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid. Sedimentation equilibrium data establish that this domain is strictly monomeric in solution. NMR spectra recorded at 278 and 308K indicate that no significant structural changes occur for this domain over the temperature range 278-308K, in contrast to the characteristic coacervation behavior that is observed for the full-length protein.
Publisher: Elsevier BV
Date: 12-2007
Publisher: Royal Society of Chemistry (RSC)
Date: 2021
DOI: 10.1039/D1CS00548K
Abstract: This tutorial review provides an introduction to a wide range of methods used to quantify protein interactions, outlining their strengths, weaknesses and requirements.
Publisher: Elsevier BV
Date: 05-2000
Publisher: Wiley
Date: 11-11-2019
DOI: 10.1002/PRO.3752
Abstract: A key learning outcome for undergraduate biochemistry classes is a thorough understanding of the principles of protein structure. Traditional approaches to teaching this material, which include two‐dimensional (2D) images on paper, physical molecular modeling kits, and projections of 3D structures into 2D, are unable to fully capture the dynamic 3D nature of proteins. We have built a virtual reality application, Peppy, aimed at facilitating teaching of the principles of protein secondary structure. Rather than attempt to model molecules with the same fidelity to the underlying physical chemistry as existing, research‐oriented molecular modelling approaches, we took the more straightforward approach of harnessing the Unity video game physics engine. Indeed, the simplicity and limitations of our model are strengths in a teaching context, provoking questions and thus deeper understanding. Peppy allows exploration of the relative effects of hydrogen bonding (and electrostatic interactions more generally), backbone φ/ψ angles, basic chemical structure, and steric effects on a polypeptide structure in an accessible format that is novel, dynamic, and fun to use. Apart from describing the implementation and use of Peppy, we discuss the outcomes of deploying Peppy in undergraduate biochemistry courses.
Publisher: Springer Science and Business Media LLC
Date: 11-1995
DOI: 10.1007/BF00197813
Abstract: Changes in dopaminergic neural function can be induced by an acute inflammatory state that, by altering the integrity of the neurovasculature, induces neuronal stress, cell death and causes functional deficits. Effectively blocking these effects of inflammation could, therefore, reduce both neuronal and functional decline. To test this hypothesis, we inhibited vascular adhesion protein 1 (VAP-1), a membrane-bound protein expressed on the endothelial cell surface, that mediates leukocyte extravasation and induces oxidative stress. We induced dopaminergic neuronal loss by infusing lipopolysaccharide (LPS) directly into the substantia nigra (SN) in rats and administered the VAP-1 inhibitor, PXS-4681A, daily. LPS produced: an acute inflammatory response, the loss of dopaminergic neurons in the SN, reduced the dopaminergic projection to SN target regions, particularly the dorsolateral striatum (DLS), and a deficit in habit learning, a key function of the DLS. In an attempt to protect SN neurons from this inflammatory response we found that VAP-1 inhibition not only reduced neutrophil infiltration in the SN and striatum, but also reduced the associated striatal microglia and astrocyte response. We found VAP-1 inhibition protected dopamine neurons in the SN, their projections to the striatum and promoted the functional recovery of habit learning. Thus, we reversed the loss of habitual actions, a function usually dependent on dopamine release in DLS and sensitive to striatal dysfunction. We establish, therefore, that VAP-1 inhibition has an anti-inflammatory profile that may be beneficial in the treatment of dopamine neuron dysfunction caused by an acute inflammatory state in the brain.
Publisher: American Chemical Society (ACS)
Date: 09-05-1995
DOI: 10.1021/BI00018A020
Abstract: Leucine zippers constitute a widely observed structural motif which serves to promote both homo- and heterodimerization in a number of DNA-binding proteins. As part of our ongoing efforts to characterize both the structure and the dynamical properties of this dimerization domain as they relate to biological function, we report here the secondary structure in solution of a recombinant dimeric peptide (rJunLZ) comprising residues Arg276-Asn314 of the leucine zipper domain of c-Jun. Two- and three-dimensional homo- and heteronuclear NMR experiments have allowed definition of the secondary structure of rJunLZ and have provided a total of approximately 1500 interproton distance and 62 phi dihedral angle constraints for tertiary structure calculations. Amide proton protection factors, calculated from hydrogen-deuterium exchange experiments, have identified 62 hydrogen bonds in the rJunLZ dimer. We have also examined the role of Asn22, the only polar residue situated at the hydrophobic dimer interface. Virtually all leucine zipper sequences contain such a polar residue (usually Asn) near the center of the motif. X-ray crystallographic studies showed that, in the case of the GCN4 homodimer, the polar residue (Asn) adopts an asymmetric conformation in an otherwise essentially symmetric structure. In contrast, all NMR studies of leucine zipper homodimers to date have suggested that the dimers are completely symmetric in solution. We present evidence that the side-chain amide protons of Asn22 are hydrogen-bonded in solution and that this side chain exchanges rapidly between two distinct conformations. On the basis of these observations, we propose a dynamic model which can explain the apparent differences in symmetry observed in NMR and X-ray crystallographic studies of leucine zipper homodimers. We show that mutation of Asn22 to a hydrophobic Leu residue markedly increases the thermal stability of the rJunLZ homodimer, consistent with a destabilizing role for this residue. However, at temperatures below 30 degrees C, the Asn22-->Leu mutant rearranges to form oligomers larger than the dimer, as was previously observed for the corresponding Asn-->Val mutation in the GCN4 leucine zipper. These results are consistent with the hypothesis that the polar Asn residue commonly observed at the interface of leucine zippers imposes specificity for the dimer structure at the expense of stability [Harbury, P.B., Zhang, T., Kim, P.S., & Alber, T. (1993) Science 262, 1401-1407].
Publisher: Elsevier BV
Date: 06-2004
Publisher: Wiley
Date: 09-2008
Publisher: Springer New York
Date: 2012
DOI: 10.1007/978-1-4614-3229-6_2
Abstract: There are many different techniques available to biologists and biochemists that can be used to detect and characterize the self-association of proteins. Each technique has strengths and weaknesses and it is often useful to combine several approaches to maximize the former and minimize the latter. Here we review a range of methodologies that identify protein self-association and/or allow the stoichiometry and affinity of the interaction to be determined, placing an emphasis on what type of information can be obtained and outlining the advantages and disadvantages involved. In general, in vitro biophysical techniques, such as size exclusion chromatography, analytical ultracentrifugation, scattering techniques, NMR spectroscopy, isothermal titration calorimetry, fluorescence anisotropy and mass spectrometry, provide information on stoichiometry and/or binding affinities. Other approaches such as cross-linking, fluorescence methods (e.g., fluorescence correlation spectroscopy, FCS Förster resonance energy transfer, FRET fluorescence recovery after photobleaching, FRAP and proximity imaging, PRIM) and complementation approaches (e.g., yeast two hybrid assays and bimolecular fluorescence complementation, BiFC) can be used to detect protein self-association in a cellular context.
Publisher: American Chemical Society (ACS)
Date: 25-03-2020
DOI: 10.26434/CHEMRXIV.12026952
Abstract: The development of low-affinity fragment hits into higher affinity leads is a major hurdle in fragment-based drug design. Here we demonstrate an approach for the Rapid Elaboration of Fragments into Leads (REFiL) applying an integrated workflow that provides a systematic approach to generate higher-affinity binders without the need for structural information. The workflow involves the selection of commercial analogues of fragment hits to generate preliminary structure-activity relationships. This is followed by parallel microscale chemistry using chemoinformatically designed reagent libraries to rapidly explore chemical ersity. Upon completion of a fragment screen against Bromodomain-3 extra terminal (BRD3-ET) domain we applied the REFiL workflow, which allowed us to develop a series of tetrahydrocarbazole ligands that bind to the peptide binding site of BRD3-ET. With REFiL we were able to rapidly improve binding affinity -fold. The REFiL workflow can be applied readily to a broad range of protein targets without the need of a structure, allowing the efficient evolution of low-affinity fragments into higher affinity leads and chemical probes. br
Publisher: American Chemical Society (ACS)
Date: 25-03-2020
DOI: 10.26434/CHEMRXIV.12026952.V1
Abstract: The development of low-affinity fragment hits into higher affinity leads is a major hurdle in fragment-based drug design. Here we demonstrate an approach for the Rapid Elaboration of Fragments into Leads (REFiL) applying an integrated workflow that provides a systematic approach to generate higher-affinity binders without the need for structural information. The workflow involves the selection of commercial analogues of fragment hits to generate preliminary structure-activity relationships. This is followed by parallel microscale chemistry using chemoinformatically designed reagent libraries to rapidly explore chemical ersity. Upon completion of a fragment screen against Bromodomain-3 extra terminal (BRD3-ET) domain we applied the REFiL workflow, which allowed us to develop a series of tetrahydrocarbazole ligands that bind to the peptide binding site of BRD3-ET. With REFiL we were able to rapidly improve binding affinity -fold. The REFiL workflow can be applied readily to a broad range of protein targets without the need of a structure, allowing the efficient evolution of low-affinity fragments into higher affinity leads and chemical probes.
Publisher: Wiley
Date: 16-11-2017
DOI: 10.1111/MPP.12597
Publisher: Elsevier BV
Date: 11-2017
DOI: 10.1016/J.CHEMBIOL.2017.08.014
Abstract: Small post-translationally modified peptide hormones mediate crucial developmental and regulatory processes in plants. CLAVATA/ENDOSPERM-SURROUNDING REGION (CLE) genes are found throughout the plant kingdom and encode for 12-13 amino acid peptides that must often undergo post-translational proline hydroxylation and glycosylation with O-β1,2-triarabinose moieties before they become functional. Apart from a few recent ex les, a detailed understanding of the structure and function of most CLE hormones is yet to be uncovered. This is mainly owing to difficulties in isolating mature homogeneously modified CLE peptides from natural plant sources. In this study, we describe the efficient synthesis of a synthetic Araf
Publisher: Informa UK Limited
Date: 07-2011
DOI: 10.1128/MCB.05413-11
Publisher: Wiley
Date: 26-06-2008
Publisher: IEEE
Date: 11-2016
Publisher: American Society for Clinical Investigation
Date: 03-04-2023
DOI: 10.1172/JCI162685
Publisher: Elsevier BV
Date: 2011
Publisher: Proceedings of the National Academy of Sciences
Date: 15-08-2011
Abstract: The control of red blood cell and megakaryocyte development by the regulatory protein GATA1 is a paradigm for transcriptional regulation of gene expression in cell lineage differentiation and maturation. Most GATA1-regulated events require GATA1 to bind FOG1, and essentially all GATA1-activated genes are cooccupied by a TAL1/E2A/LMO2/LDB1 complex however, it is not known whether FOG1 and TAL1/E2A/LMO2/LDB1 are simultaneously recruited by GATA1. Our structural data reveal that the FOG1-binding domain of GATA1, the N finger, can also directly contact LMO2 and show that, despite the small size ( 50 residues) of the GATA1 N finger, both FOG1 and LMO2 can simultaneously bind this domain. LMO2 in turn can simultaneously contact both GATA1 and the DNA-binding protein TAL1/E2A at bipartite E-box/WGATAR sites. Taken together, our data provide the first structural snapshot of multiprotein complex formation at GATA1-dependent genes and support a model in which FOG1 and TAL1/E2A/LMO2/LDB1 can cooccupy E-box/WGATAR sites to facilitate GATA1-mediated activation of gene activation.
Publisher: American Chemical Society (ACS)
Date: 06-03-2013
DOI: 10.1021/BI4000172
Abstract: Troponin C (TnC) is the calcium-binding subunit of the troponin complex responsible for initiating striated muscle contraction in response to calcium influx. In the skeletal TnC isoform, calcium binding induces a structural change in the regulatory N-domain of TnC that involves a transition from a closed to open structural state and accompanying exposure of a large hydrophobic patch for troponin I (TnI) to subsequently bind. However, little is understood about how calcium primes the N-domain of the cardiac isoform (cTnC) for interaction with the TnI subunit as the open conformation of the regulatory domain of cTnC has been observed only in the presence of bound TnI. Here we use paramagnetic relaxation enhancement (PRE) to characterize the closed to open transition of isolated cTnC in solution, a process that cannot be observed by traditional nuclear magnetic resonance methods. Our PRE data from four spin-labeled monocysteine constructs of isolated cTnC reveal that calcium binding triggers movement of the N-domain helices toward an open state. Fitting of the PRE data to a closed to open transition model reveals the presence of a small population of cTnC molecules in the absence of calcium that possess an open conformation, the level of which increases substantially upon Ca(2+) binding. These data support a model in which calcium binding creates a dynamic equilibrium between the closed and open structural states to prime cTnC for interaction with its target peptide. We also used PRE data to assess the structural effects of a familial hypertrophic cardiomyopathy point mutation located within the N-domain of cTnC (A8V). The PRE data show that the Ca(2+) switch mechanism is perturbed by the A8V mutation, resulting in a more open N-domain conformation in both the apo and holo states.
Publisher: American Chemical Society (ACS)
Date: 20-01-2005
DOI: 10.1021/BI047782J
Abstract: We have determined the solution structure of rSS3, a recombinant form of the type I shorthorn sculpin antifreeze protein (AFP), at 278 and 268 K. This AFP contains an unusual sequence of N-terminal residues, together with two of the 11-residue repeats that are characteristic of the type I winter flounder AFP. The solution conformation of the N-terminal region of the sculpin AFP has been assumed to be the critical factor that results in recognition of different ice planes by the sculpin and flounder AFPs. At 278 K, the two repeats units (residues 11-20 and 21-32) in rSS3 form a continuous alpha-helix, with the residues 30-33 in the second repeat somewhat less well defined. Within the N-terminal region, residues 2-6 are well defined and helical and linked to the main helix by a more flexible region comprising residues A7-T11. At 268 K the AFP is overall more helical but retains the apparent hinge region. The helical conformation of the two repeats units is almost identical to the corresponding repeats in the type I winter flounder AFP. We also show that while tetracetylated rSS3 has antifreeze activity comparable to the natural AFP, its overall structure is the same as that of the unacetylated peptide. These data provide some insight into the structural determinants of antifreeze activity and should assist in the development of models that explain the recognition of different ice interfaces by the sculpin and flounder type I AFPs.
Publisher: Springer Science and Business Media LLC
Date: 16-02-2014
DOI: 10.1007/S12104-013-9470-X
Abstract: The transcriptional regulator LMO4 and the transcription factor DEAF-1 are both essential for brain and skeletal development. They are also implicated in human breast cancers overexpression of LMO4 is an indicator of poor prognosis, and overexpression of DEAF-1 promotes epithelial breast cell proliferation. We have generated a stable LMO4-DEAF-1 complex comprising the C-terminal LIM domain of LMO4 and an intrinsically disordered LMO4-interaction domain from DEAF-1 tethered by a glycine/serine linker. Here we report the (1)H, (15)N and (13)C assignments of this construct. Analysis of the assignments indicates the presence of structure in the DEAF-1 part of the complex supporting the presence of a physical interaction between the two proteins.
Publisher: Elsevier BV
Date: 06-2003
Publisher: American Association for the Advancement of Science (AAAS)
Date: 31-07-2020
Abstract: A thermostable drug suppresses a pERK-FosB/ΔFosB-VCAM-1 axis, endothelial activation, angiogenesis, and retinal permeability.
Publisher: Elsevier BV
Date: 07-2016
Publisher: Springer Science and Business Media LLC
Date: 27-02-2011
DOI: 10.1038/NSMB.2005
Abstract: Spectacular progress has been made in the design of proteins that recognize double-stranded DNA with a chosen specificity, to the point that designer DNA-binding proteins can be ordered commercially. This success raises the question of whether it will be possible to engineer libraries of proteins that can recognize RNA with tailored specificity. Given the recent explosion in the number and ersity of RNA species demonstrated to play roles in biology, designer RNA-binding proteins are set to become valuable tools, both in the research laboratory and potentially in the clinic. Here we discuss the prospects for the realization of this idea.
Publisher: Proceedings of the National Academy of Sciences
Date: 03-02-2022
Abstract: Bloom syndrome complex (BS complex) is necessary for maintenance of genome stability and suppression of cancer-causing mutations. Composed of a helicase, a topoisomerase, and two scaffolds, the BS complex is implicated in several steps that ensure the high fidelity of DNA repair by recombination. One step, called “double Holliday junction dissolution,” ensures untangling of DNA at the conclusion of repair. Here, we used cross-link mass spectrometry to show how the BS complex assembles. Using biochemical reactions and cell complementation with mutant proteins, our results reveal an important role of helicase dimerization (and tight coupling of proteins within the complex) for double Holliday junction dissolution and genome stability.
Publisher: Wiley
Date: 13-11-2017
DOI: 10.1111/FEBS.14301
Publisher: American Society of Hematology
Date: 27-06-2013
DOI: 10.1182/BLOOD-2013-03-488080
Abstract: Disease-causing mutations in GATA1 impair binding to the cofactors FOG1 or TAL1 but not DNA. Different substitutions at the same residue selectively disrupt FOG1 or TAL1 binding leading to distinct disease phenotypes.
Publisher: Wiley
Date: 28-01-2011
DOI: 10.1111/J.1742-4658.2011.08005.X
Abstract: A strength of NMR spectroscopy is its ability to monitor, on an atomic level, molecular changes and interactions. In this review, which is intended for non-spectroscopist, we describe major uses of NMR in protein science beyond solution structure determination. After first touching on how NMR can be used to quickly determine whether a mutation induces structural perturbations in a protein, we describe the unparalleled ability of NMR to monitor binding interactions over a wide range of affinities, molecular masses and solution conditions. We discuss the use of NMR to measure the dynamics of proteins at the atomic level and over a wide range of timescales. Finally, we outline new and expanding areas such as macromolecular structure determination in multicomponent systems, as well as in the solid state and in vivo.
Publisher: Elsevier BV
Date: 09-2001
Publisher: Cold Spring Harbor Laboratory
Date: 16-11-2022
DOI: 10.1101/2022.11.16.516813
Abstract: Significant recent advances in structural biology, particularly in the field of cryo-electron microscopy, have dramatically expanded our ability to create structural models of proteins and protein complexes. However, many proteins remain refractory to these approaches because of their low abundance, low stability or – in the case of complexes – simply not having yet been analysed. Here, we demonstrate the power of combining cross-linking mass spectrometry (XL-MS) with artificial intelligence-based structure prediction to discover and experimentally substantiate models for protein and protein complex structures at proteome scale. We present the deepest XL-MS dataset to date, describing 28,910 unique residue pairs captured across 4,084 unique human proteins and 2,110 unique protein-protein interactions. We show that integrative models of complexes driven by AlphaFold Multimer and inspired and corroborated by the XL-MS data offer new opportunities to deeply mine the structural proteome and interactome and reveal new mechanisms underlying protein structure and function.
Publisher: Wiley
Date: 1999
DOI: 10.1110/PS.8.6.1320
Abstract: The N-terminal 17 residues of ubiquitin have been shown by 1H NMR to fold autonomously into a beta-hairpin structure in aqueous solution. This structure has a specific, native-like register, though side-chain contacts differ in detail from those observed in the intact protein. An autonomously folding hairpin has previously been identified in the case of streptococcal protein G, which is structurally homologous with ubiquitin, but remarkably, the two are not in topologically equivalent positions in the fold. This suggests that the organization of folding may be quite different for proteins sharing similar tertiary structures. Two smaller peptides have also been studied, corresponding to the isolated arms of the N-terminal hairpin of ubiquitin, and significant differences from simple random coil predictions observed in the spectra of these subfragments, suggestive of significant limitation of the backbone conformational space s led, presumably as a consequence of the strongly beta-structure favoring composition of the sequences. This illustrates the ability of local sequence elements to express a propensity for beta-structure even in the absence of actual sheet formation. Attempts were made to estimate the population of the folded state of the hairpin, in terms of a simple two-state folding model. Using published "random coil" values to model the unfolded state, and values derived from native ubiquitin for the putative unique, folded state, it was found that the apparent population varied widely for different residues and with different NMR parameters. Use of the spectra of the subfragment peptides to provide a more realistic model of the unfolded state led to better agreement in the estimates that could be obtained from chemical shift and coupling constant measurements, while making it clear that some other approaches to population estimation could not give meaningful results, because of the tendency to populate the beta-region of conformational space even in the absence of the hairpin structure.
Publisher: Elsevier BV
Date: 02-2006
DOI: 10.1016/J.JMB.2006.11.058
Abstract: The THAP (Thanatos-associated protein) domain is a recently discovered zinc-binding domain found in proteins involved in transcriptional regulation, cell-cycle control, apoptosis and chromatin modification. It contains a single zinc atom ligated by cysteine and histidine residues within a Cys-X(2-4)-Cys-X(35-53)-Cys-X(2)-His consensus. We have determined the NMR solution structure of the THAP domain from Caenorhabditis elegans C-terminal binding protein (CtBP) and show that it adopts a fold containing a treble clef motif, bearing similarity to the zinc finger-associated domain (ZAD) from Drosophila Grauzone. The CtBP THAP domain contains a large, positively charged surface patch and we demonstrate that this domain can bind to double-stranded DNA in an electrophoretic mobility-shift assay. These data, together with existing reports, indicate that THAP domains might exhibit a functional ersity similar to that observed for classical and GATA-type zinc fingers.
Publisher: American Society for Clinical Investigation
Date: 15-09-2003
DOI: 10.1172/JCI19137
Publisher: American Chemical Society (ACS)
Date: 18-03-2008
DOI: 10.1021/OL8002767
Abstract: The glycosynthase derived from E. coli beta-glucuronidase catalyzes the glucuronylation of a range of primary, secondary, and aryl alcohols with moderate to excellent yields. The procedure provides an efficient, stereoselective, and scalable single-step synthesis of beta-glucuronides under mild conditions.
Publisher: Springer Science and Business Media LLC
Date: 25-09-2007
Abstract: Human g rowth factor r eceptor b ound protein 7 (Grb7) is an adapter protein that mediates the coupling of tyrosine kinases with their downstream signaling pathways. Grb7 is frequently overexpressed in invasive and metastatic human cancers and is implicated in cancer progression via its interaction with the ErbB2 receptor and focal adhesion kinase (FAK) that play critical roles in cell proliferation and migration. It is thus a prime target for the development of novel anti-cancer therapies. Recently, an inhibitory peptide (G7-18NATE) has been developed which binds specifically to the Grb7 SH2 domain and is able to attenuate cancer cell proliferation and migration in various cancer cell lines. As a first step towards understanding how Grb7 may be inhibited by G7-18NATE, we solved the crystal structure of the Grb7 SH2 domain to 2.1 Å resolution. We describe the details of the peptide binding site underlying target specificity, as well as the dimer interface of Grb 7 SH2. Dimer formation of Grb7 was determined to be in the μM range using analytical ultracentrifugation for both full-length Grb7 and the SH2 domain alone, suggesting the SH2 domain forms the basis of a physiological dimer. ITC measurements of the interaction of the G7-18NATE peptide with the Grb7 SH2 domain revealed that it binds with a binding affinity of K d = ~35.7 μM and NMR spectroscopy titration experiments revealed that peptide binding causes perturbations to both the ligand binding surface of the Grb7 SH2 domain as well as to the dimer interface, suggesting that dimerisation of Grb7 is impacted on by peptide binding. Together the data allow us to propose a model of the Grb7 SH2 domain/G7-18NATE interaction and to rationalize the basis for the observed binding specificity and affinity. We propose that the current study will assist with the development of second generation Grb7 SH2 domain inhibitors, potentially leading to novel inhibitors of cancer cell migration and invasion.
Publisher: Elsevier BV
Date: 06-2016
DOI: 10.1016/J.SBI.2016.05.005
Abstract: Emerging data suggest that the mechanisms by which RNA-binding proteins (RBPs) interact with RNA and the rules governing specificity might be substantially more complex than those underlying their DNA-binding counterparts. Even our knowledge of what constitutes the RNA-bound proteome is contentious recent studies suggest that 10-30% of RBPs contain no known RNA-binding domain. Adding to this situation is a growing disconnect between the avalanche of identified interactions between proteins and long noncoding RNAs and the absence of biophysical data on these interactions. RNA-protein interactions are also at the centre of what might emerge as one of the biggest shifts in thinking about cell and molecular biology this century, following from recent reports of ribonucleoprotein complexes that drive reversible membrane-free phase separation events within the cell. Unexpectedly, low-complexity motifs are important in the formation of these structures. Here we briefly survey recent advances in our understanding of the specificity of RBPs.
Publisher: Elsevier BV
Date: 08-2014
DOI: 10.1016/J.PEP.2014.05.005
Abstract: Spt4/5 is a hetero-dimeric transcription elongation factor that can both inhibit and promote transcription elongation by RNA polymerase II (RNAPII). However, Spt4/5's mechanism of action remains elusive. Spt5 is an essential protein and the only universally-conserved RNAP-associated transcription elongation factor. The protein contains multiple Kyrpides, Ouzounis and Woese (KOW) domains. These domains, in other proteins, are thought to bind RNA although there is little direct evidence in the literature to support such a function in Spt5. This could be due, at least in part, to difficulties in expressing and purifying recombinant Spt5. When expressed in Escherichia coli (E. coli), Spt5 is innately insoluble. Here we report a new approach for the successful expression and purification of milligram quantities of three different multi-KOW domain complexes of Saccharomyces cerevisiae Spt4/5 for use in future functional studies. Using the E. coli strain Rosetta2 (DE3) we have developed strategies for co-expression of Spt4 and multi-KOW domain Spt5 complexes from the bi-cistronic pET-Duet vector. In a second strategy, Spt4/5 was expressed via co-transformation of Spt4 in the vector pET-M11 with Spt5 ubiquitin fusion constructs in the vector pHUE. We characterized the multi-KOW domain Spt4/5 complexes by Western blot, limited proteolysis, circular dichroism, SDS-PAGE and size exclusion chromatography-multiangle light scattering and found that the proteins are folded with a Spt4:Spt5 hetero-dimeric stoichiometry of 1:1. These expression constructs encompass a larger region of Spt5 than has previously been reported, and will provide the opportunity to elucidate the biological function of the multi-KOW containing Spt5.
Publisher: Wiley
Date: 12-2009
Abstract: A comprehensive understanding of protein-protein interactions is an important next step in our quest to understand how the information contained in a genome is put into action. Although a number of experimental techniques can report on the existence of a protein- protein interaction, very few can provide detailed structural information. NMR spectroscopy is one of these, and in recent years several complementary NMR approaches, including residual dipolar couplings and the use of paramagnetic effects, have been developed that can provide insight into the structure of protein-protein complexes. In this article, we review these approaches and comment on their strengths and weaknesses.
Publisher: Elsevier BV
Date: 04-2014
Publisher: Elsevier BV
Date: 11-2000
DOI: 10.1016/S0969-2126(00)00527-X
Abstract: Zinc finger domains have traditionally been regarded as sequence-specific DNA binding motifs. However, recent evidence indicates that many zinc fingers mediate specific protein-protein interactions. For instance, several zinc fingers from FOG family proteins have been shown to interact with the N-terminal zinc finger of GATA-1. We have used NMR spectroscopy to determine the first structures of two FOG family zinc fingers that are involved in protein-protein interactions: fingers 1 and 9 from U-shaped. These fingers resemble classical TFIIIA-like zinc fingers, with the exception of an unusual extended portion of the polypeptide backbone prior to the fourth zinc ligand. [15N,(1)H]-HSQC titrations have been used to define the GATA binding surface of USH-F1, and comparison with other FOG family proteins indicates that the recognition mechanism is conserved across species. The surface of FOG-type fingers that interacts with GATA-1 overlaps substantially with the surface through which classical fingers typically recognize DNA. This suggests that these fingers could not contact both GATA and DNA simultaneously. In addition, results from NMR, gel filtration, and sedimentation equilibrium experiments suggest that the interactions are of moderate affinity. Our results demonstrate unequivocally that zinc fingers comprising the classical betabetaalpha fold are capable of mediating specific contacts between proteins. The existence of this alternative function has implications for the prediction of protein function from sequence data and for the evolution of protein function.
Publisher: Oxford University Press (OUP)
Date: 24-11-2017
DOI: 10.1093/NAR/GKW1014
Publisher: Elsevier BV
Date: 09-2006
Publisher: Elsevier BV
Date: 02-2007
DOI: 10.1016/J.TIBS.2006.12.007
Abstract: Zinc-fingers (ZnFs) are extremely abundant in higher eukaryotes. Once considered to function exclusively as sequence-specific DNA-binding motifs, ZnFs are now known to have additional activities such as the recognition of RNA and other proteins. Here we discuss recent advances in our understanding of ZnFs as specific modules for protein recognition. Structural studies of ZnF complexes reveal considerable ersity in terms of protein partners, binding modes and affinities, and highlight the often underestimated versatility of ZnF structure and function. An appreciation of the structural features of ZnF-protein interactions will contribute to our ability to engineer and to use ZnFs with tailored protein-binding properties.
Publisher: Elsevier BV
Date: 09-2002
DOI: 10.1016/S0092-8674(02)00932-7
Abstract: Hop is a small, ergent homeodomain protein that lacks certain conserved residues required for DNA binding. Hop gene expression initiates early in cardiogenesis and continues in cardiomyocytes throughout embryonic and postnatal development. Genetic and biochemical data indicate that Hop functions directly downstream of Nkx2-5. Inactivation of Hop in mice by homologous recombination results in a partially penetrant embryonic lethal phenotype with severe developmental cardiac defects involving the myocardium. Inhibition of Hop activity in zebrafish embryos likewise disrupts cardiac development and results in severely impaired cardiac function. Hop physically interacts with serum response factor (SRF) and inhibits activation of SRF-dependent transcription by inhibiting SRF binding to DNA. Hop encodes an unusual homeodomain protein that modulates SRF-dependent cardiac-specific gene expression and cardiac development.
Publisher: Public Library of Science (PLoS)
Date: 13-10-2014
Publisher: Springer Science and Business Media LLC
Date: 04-06-2012
DOI: 10.1007/S12104-011-9319-0
Abstract: LMO4 is a broadly expressed LIM-only protein that is involved in neural tube development and implicated in breast cancer. Here we report backbone and side chain NMR assignments for an engineered intramolecular complex of the N-terminal LIM domain from LMO4 tethered to residues 641-685 of C-terminal binding protein interacting protein (CtIP/RBBP8).
Publisher: Elsevier BV
Date: 03-2011
DOI: 10.1016/J.JMB.2010.12.041
Abstract: The recognition of single-stranded RNA (ssRNA) is an important aspect of gene regulation, and a number of different classes of protein domains that recognize ssRNA in a sequence-specific manner have been identified. Recently, we demonstrated that the RanBP2-type zinc finger (ZnF) domains from the human splicing factor ZnF Ran binding domain-containing protein 2 (ZRANB2) can bind to a sequence containing the consensus AGGUAA. Six other human proteins, namely, Ewing's sarcoma (EWS), translocated in liposarcoma (TLS)/FUS, RNA-binding protein 56 (RBP56), RNA-binding motif 5 (RBM5), RNA-binding motif 10 (RBM10) and testis-expressed sequence 13A (TEX13A), each contains a single ZnF with homology to the ZRANB2 ZnFs, and several of these proteins have been implicated in the regulation of mRNA processing. Here, we show that all of these ZnFs are able to bind with micromolar affinities to ssRNA containing a GGU motif. NMR titration data reveal that binding is mediated by the corresponding surfaces on each ZnF, and we also show that sequence selectivity is largely limited to the GGU core motif and that substitution of the three flanking adenines that were selected in our original selection experiment has a minimal effect on binding affinity. These data establish a subset of RanBP2-type ZnFs as a new family of ssRNA-binding motifs.
Publisher: American Association for Cancer Research (AACR)
Date: 15-04-2004
DOI: 10.1158/0008-5472.CAN-03-2093
Abstract: A novel gene, thyroid cancer 1 (TC-1), was found recently to be overexpressed in thyroid cancer. TC-1 shows no homology to any of the known thyroid cancer-associated genes. We have produced stable transformants of normal thyroid cells that express the TC-1 gene, and these cells show increased proliferation rates and anchorage-independent growth in soft agar. Apoptosis rates also are decreased in the transformed cells. We also have expressed recombinant TC-1 protein and have undertaken a structural and functional characterization of the protein. The protein is monomeric and predominantly unstructured under conditions of physiologic salt and pH. This places it in the category of natively disordered proteins, a rapidly expanding group of proteins, many members of which play critical roles in cell regulation processes. We show that the protein can be phosphorylated by cyclic AMP-dependent protein kinase and protein kinase C, and the activity of both of these kinases is up-regulated when cells are stably transfected with TC-1. These results suggest that overexpression of TC-1 may be important in thyroid carcinogenesis.
Publisher: Elsevier BV
Date: 02-2020
Publisher: Elsevier BV
Date: 03-2004
DOI: 10.1016/J.STR.2003.12.002
Abstract: Superantigens are a class of microbial proteins with the ability to excessively activate T cells by binding to the T cell receptor. The staphylococcal and streptococcal superantigens are closely related in structure and possess an N-terminal domain that resembles an OB fold and a C-terminal domain similar to a beta-grasp fold. Yersinia pseudotuberculosis produces superantigens, YPMa, YPMb, and YPMc, which have no significant amino acid similarity to other proteins. We have determined the crystal and solution structures of YPMa, which show that the protein has a jelly-roll fold. The closest structural neighbors to YPMa are viral capsid proteins and members of the tumor necrosis factor superfamily. In the crystal structure, YPMa packs as a trimer, another feature shared with viral capsid proteins and TNF superfamily proteins. However, in solution YPMa behaves as a monomer, and any functional relevance of the trimer observed in the crystals is yet to be established.
Publisher: American Chemical Society (ACS)
Date: 11-08-2006
DOI: 10.1021/BI060641S
Abstract: Homeodomain-only protein (HOP) is an 8-kDa transcriptional corepressor that is essential for the normal development of the mammalian heart. Previous studies have shown that HOP, which consists entirely of a putative homeodomain, acts downstream of Nkx2.5 and associates with the serum response factor (SRF), repressing transcription from SRF-responsive genes. HOP is also able to recruit histone deacetylase (HDAC) activity, consistent with its ability to repress transcription. Unlike other classic homeodomain proteins, HOP does not appear to interact with DNA, although it has been unclear if this is because of an overall ergent structure or because of specific amino acid differences between HOP and other homeodomains. To work toward an understanding of HOP function, we have determined the 3D structure of full-length HOP and used a range of biochemical assays to define the parts of the protein that are functionally important for its repression activity. We show that HOP forms a classical homeodomain fold but that it cannot recognize double stranded DNA, a result that emphasizes the importance of caution in predicting protein function from sequence homology alone. We also demonstrate that two distinct regions on the surface of HOP are required for its ability to repress an SRF-driven reporter gene, and it is likely that these motifs direct interactions between HOP and partner proteins such as SRF- and HDAC-containing complexes. Our results demonstrate that the homeodomain fold has been co-opted during evolution for functions other than sequence-specific DNA binding and suggest that HOP functions as an adaptor protein to mediate transcriptional repression.
Publisher: Elsevier BV
Date: 04-2013
DOI: 10.1016/J.JMB.2013.01.017
Abstract: LIM-only protein 4 (LMO4) is strongly linked to the progression of breast cancer. Although the mechanisms underlying this phenomenon are not well understood, a role is emerging for LMO4 in regulation of the cell cycle. We determined the solution structure of LMO4 in complex with CtIP (C-terminal binding protein interacting protein)/RBBP8, a tumour suppressor protein that is involved in cell cycle progression, DNA repair and transcriptional regulation. Our data reveal that CtIP and the essential LMO cofactor LDB1 (LIM-domain binding protein 1) bind to the same face on LMO4 and cannot simultaneously bind to LMO4. We hypothesise that overexpression of LMO4 may disrupt some of the normal tumour suppressor activities of CtIP, thereby contributing to breast cancer progression.
Publisher: Elsevier BV
Date: 09-2013
Abstract: One method commonly used to characterize protein-DNA interactions is surface plasmon resonance (SPR). In a typical SPR experiment, chip-bound DNA is exposed to increasing concentrations of protein the resulting binding data are used to calculate a dissociation constant for the interaction. However, in cases in which knowledge of the specificity of the interaction is required, a large set of DNA variants has to be tested this is time consuming and costly, in part because of the requirement for multiple SPR chips. We have developed a new protocol that uses steady-state binding levels in SPR competition experiments to determine protein-binding dissociation constants for a set of DNA variants. This approach is rapid and straightforward and requires the use of only a single SPR chip. Additionally, in contrast to other methods, our approach does not require prior knowledge of parameters such as on or off rates, using an estimate of the wild-type interaction as the sole input. Utilizing relative steady-state responses, our protocol also allows for the rapid, reliable, and simultaneous determination of protein-binding dissociation constants of a large series of DNA mutants in a single experiment in a semiquantitative fashion. We compare our approach to existing methods, highlighting specific advantages as well as limitations.
Publisher: Wiley
Date: 28-01-2011
DOI: 10.1111/J.1742-4658.2011.08004.X
Abstract: NMR spectroscopy is a powerful tool for studying the structure, function and dynamics of biological macromolecules. However, non-spectroscopists often find NMR theory daunting and data interpretation nontrivial. As the first of two back-to-back reviews on NMR spectroscopy aimed at non-spectroscopists, the present review first provides an introduction to the basics of macromolecular NMR spectroscopy, including a discussion of typical s le requirements and what information can be obtained from simple NMR experiments. We then review the use of NMR spectroscopy for determining the 3D structures of macromolecules and examine how to judge the quality of NMR-derived structures.
Publisher: Informa UK Limited
Date: 17-11-2017
Publisher: Wiley
Date: 11-02-1970
Publisher: Elsevier BV
Date: 10-2008
DOI: 10.1016/J.JMB.2008.07.034
Abstract: Class I hydrophobins are fungal proteins that self-assemble into robust hipathic rodlet monolayers on the surface of aerial structures such as spores and fruiting bodies. These layers share many structural characteristics with amyloid fibrils and belong to the growing family of functional amyloid-like materials produced by microorganisms. Although the three-dimensional structure of the soluble monomeric form of a class I hydrophobin has been determined, little is known about the molecular structure of the rodlets or their assembly mechanism. Several models have been proposed, some of which suggest that the Cys3-Cys4 loop has a critical role in the initiation of assembly or in the polymeric structure. In order to provide insight into the relationship between hydrophobin sequence and rodlet assembly, we investigated the role of the Cys3-Cys4 loop in EAS, a class I hydrophobin from Neurospora crassa. Remarkably, deletion of up to 15 residues from this 25-residue loop does not impair rodlet formation or reduce the surface activity of the protein, and the physicochemical properties of rodlets formed by this mutant are indistinguishable from those of its full-length counterpart. In addition, the core structure of the truncation mutant is essentially unchanged. Molecular dynamics simulations carried out on the full-length protein and this truncation mutant binding to an air-water interface show that, although it is hydrophobic, the loop does not play a role in positioning the protein at the surface. These results demonstrate that the Cys3-Cys4 loop does not have an integral role in the formation or structure of the rodlets and that the major determinant of the unique properties of these proteins is the hipathic core structure, which is likely to be preserved in all hydrophobins despite the high degree of sequence variation across the family.
Publisher: Public Library of Science (PLoS)
Date: 13-11-2014
Publisher: Wiley
Date: 08-12-2020
DOI: 10.1002/PRO.4006
Publisher: Elsevier BV
Date: 12-2021
Publisher: Springer Science and Business Media LLC
Date: 2002
Publisher: Wiley
Date: 05-2003
DOI: 10.1093/EMBOJ/CDG196
Publisher: Royal Society of Chemistry (RSC)
Date: 1997
DOI: 10.1039/A704001F
Publisher: Springer Science and Business Media LLC
Date: 23-03-2020
DOI: 10.1038/S41467-020-15183-2
Abstract: Chromatin remodellers hydrolyse ATP to move nucleosomal DNA against histone octamers. The mechanism, however, is only partially resolved, and it is unclear if it is conserved among the four remodeller families. Here we use single-molecule assays to examine the mechanism of action of CHD4, which is part of the least well understood family. We demonstrate that the binding energy for CHD4-nucleosome complex formation—even in the absence of nucleotide—triggers significant conformational changes in DNA at the entry side, effectively priming the system for remodelling. During remodelling, flanking DNA enters the nucleosome in a continuous, gradual manner but exits in concerted 4–6 base-pair steps. This decoupling of entry- and exit-side translocation suggests that ATP-driven movement of entry-side DNA builds up strain inside the nucleosome that is subsequently released at the exit side by DNA expulsion. Based on our work and previous studies, we propose a mechanism for nucleosome sliding.
Publisher: Springer Science and Business Media LLC
Date: 12-11-2022
DOI: 10.1038/S41467-022-34530-Z
Abstract: The importance of modified peptides and proteins for applications in drug discovery, and for illuminating biological processes at the molecular level, is fueling a demand for efficient methods that facilitate the precise modification of these biomolecules. Herein, we describe the development of a photocatalytic method for the rapid and efficient dimerization and site-specific functionalization of peptide and protein diselenides. This methodology, dubbed the photocatalytic diselenide contraction, involves irradiation at 450 nm in the presence of an iridium photocatalyst and a phosphine and results in rapid and clean conversion of diselenides to reductively stable selenoethers. A mechanism for this photocatalytic transformation is proposed, which is supported by photoluminescence spectroscopy and density functional theory calculations. The utility of the photocatalytic diselenide contraction transformation is highlighted through the dimerization of selenopeptides, and by the generation of two families of protein conjugates via the site-selective modification of calmodulin containing the 21 st amino acid selenocysteine, and the C-terminal modification of a ubiquitin diselenide.
Publisher: American Chemical Society (ACS)
Date: 11-1998
DOI: 10.1021/BI981163R
Publisher: Elsevier BV
Date: 12-1998
Publisher: Elsevier BV
Date: 02-2008
Publisher: Springer Science and Business Media LLC
Date: 06-12-2022
DOI: 10.1038/S41467-022-35002-0
Abstract: CHD4 is an essential, widely conserved ATP-dependent translocase that is also a broad tumour dependency. In common with other SF2-family chromatin remodelling enzymes, it alters chromatin accessibility by repositioning histone octamers. Besides the helicase and adjacent tandem chromodomains and PHD domains, CHD4 features 1000 residues of N- and C-terminal sequence with unknown structure and function. We demonstrate that these regions regulate CHD4 activity through different mechanisms. An N-terminal intrinsically disordered region (IDR) promotes remodelling integrity in a manner that depends on the composition but not sequence of the IDR. The C-terminal region harbours an auto-inhibitory region that contacts the helicase domain. Auto-inhibition is relieved by a previously unrecognized C-terminal SANT-SLIDE domain split by ~150 residues of disordered sequence, most likely by binding of this domain to substrate DNA. Our data shed light on CHD4 regulation and reveal strong mechanistic commonality between CHD family members, as well as with ISWI-family remodellers.
Publisher: Elsevier BV
Date: 12-2020
Publisher: Elsevier BV
Date: 12-2013
Publisher: Wiley
Date: 15-07-2016
DOI: 10.1002/PRO.2976
Publisher: Elsevier BV
Date: 02-2005
DOI: 10.1016/J.STR.2004.12.007
Abstract: Zinc binding motifs have received much attention in the area of protein design. Here, we have tested the suitability of a recently discovered nonnative zinc binding structure as a protein design scaffold. A series of multiple alanine mutants was created to investigate the minimal requirements for folding, and solution structures of these mutants showed that the original fold was maintained, despite changes in approximately 50% of the sequence. We next attempted to transplant binding faces from chosen bimolecular interactions onto one of these mutants, and many of the resulting "chimeras" were shown to adopt a native-like fold. These results both highlight the robust nature of small zinc binding domains and underscore the complexity of designing functional proteins, even using such small, highly ordered scaffolds as templates.
Publisher: Elsevier BV
Date: 02-2009
Publisher: Elsevier BV
Date: 09-2014
Publisher: Research Square Platform LLC
Date: 05-01-2022
DOI: 10.21203/RS.3.RS-1179181/V1
Abstract: Current vaccines against SARS-CoV-2 substantially reduce mortality, but protection against infection is less effective. Enhancing immunity in the respiratory tract, via mucosal vaccination, may provide protection against infection and minimise viral spread. We tested a novel subunit vaccine in mice, consisting of SARS-CoV-2 Spike protein with a TLR2-stimulating adjuvant, delivered to mice parenterally or mucosally. Both routes of vaccination induced substantial neutralising antibody (nAb) titres, however, mucosal vaccination uniquely generated anti-Spike IgA, increased nAb in the serum and airways, and increased lung CD4 + T-cell responses. TLR2 is expressed by respiratory epithelia and immune cells. Using TLR2 deficient chimeric mice, we determined that TLR2 expression in either compartment facilitated early innate responses to mucosal vaccination. By contrast, TLR2 on hematopoietic cells was essential for optimal lung-localised, antigen-specific responses. In a K18-hACE2 mice, vaccination provided complete protection against disease and sterilising lung immunity against SARS-CoV-2. These data support mucosal vaccination as a strategy to improve protection in the respiratory tract against SARS-CoV-2 and other respiratory viruses.
Publisher: American Chemical Society (ACS)
Date: 28-03-2012
DOI: 10.1021/OL300557G
Abstract: The synthesis of sublancin 168, a unique S-glucosylated bacteriocin antibiotic, is described. The natural product and two S-glycosylated variants were successfully prepared via native chemical ligation followed by folding. The synthetic glycopeptides were shown to possess primarily an α-helical secondary structure by CD and NMR studies.
Publisher: American Chemical Society (ACS)
Date: 16-03-2001
DOI: 10.1021/BI001655F
Abstract: The molecular recognition of oligonucleotides by chiral ruthenium complexes has been probed by NMR spectroscopy using the template Delta-cis-alpha- and Delta-cis-beta-[Ru(RR-picchxnMe(2)) (bidentate)](2+), where the bidentate ligand is one of phen (1,10-phenanthroline), dpq (dipyrido[3,2-f:2',3'-h]quinoxaline), or phi (9,10-phenanthrenequinone diimine) and picchxnMe(2)() is N,N'-dimethyl-N,N'-di(2-picolyl)-1,2-diaminocyclohexane. By varying only the bidentate ligand in a series of complexes, it was shown that the bidentate alone can alter binding modes. DNA binding studies of the Delta-cis-alpha-[Ru(RR-picchxnMe(2))(phen)](2+) complex indicate fast exchange kinetics on the chemical shift time scale and a "partial intercalation" mode of binding. This complex binds to [d(CGCGATCGCG)](2) and [d(ATATCGATAT)](2) at AT, TA, and GA sites from the minor groove, as well as to the ends of the oligonucleotide at low temperature. Studies of the Delta-cis-beta-[Ru(RR-picchxnMe(2))(phen)](2+) complex with [d(CGCGATCGCG)](2) showed that the complex binds only weakly to the ends of the oligonucleotide. The interaction of Delta-cis-alpha-[Ru(RR-picchxnMe(2))(dpq)](2+) with [d(CGCGATCGCG)](2) showed intermediate exchange kinetics and evidence of minor groove intercalation at the GA base step. In contrast to the phen and dpq complexes, Delta-cis-alpha- and Delta-cis-beta-[Ru(RR-picchxnMe(2))(phi)](2+) showed evidence of major groove binding independent of the metal ion configuration. DNA stabilization induced by complex binding to [d(CGCGATCGCG)](2) (measured as DeltaT(m)) increases in the order phen < dpq and DNA affinity in the order phen < dpq < phi. The groove binding preferences exhibited by the different bidentate ligands is explained with the aid of molecular modeling experiments.
Publisher: Elsevier BV
Date: 04-2013
Publisher: Oxford University Press (OUP)
Date: 04-07-2017
DOI: 10.1093/NAR/GKX578
Publisher: Springer Science and Business Media LLC
Date: 11-2005
Publisher: American Chemical Society (ACS)
Date: 1993
DOI: 10.1021/JA00054A033
Publisher: Elsevier BV
Date: 07-2013
Publisher: Proceedings of the National Academy of Sciences
Date: 23-01-2012
Abstract: The hydrophobin EAS from the fungus Neurospora crassa forms functional amyloid fibrils called rodlets that facilitate spore formation and dispersal. Self-assembly of EAS into fibrillar rodlets occurs spontaneously at hydrophobic:hydrophilic interfaces and the rodlets further associate laterally to form hipathic monolayers. We have used site-directed mutagenesis and peptide experiments to identify the region of EAS that drives intermolecular association and formation of the cross-β rodlet structure. Transplanting this region into a nonamyloidogenic hydrophobin enables it to form rodlets. We have also determined the structure and dynamics of an EAS variant with reduced rodlet-forming ability. Taken together, these data allow us to pinpoint the conformational changes that take place when hydrophobins self-assemble at an interface and to propose a model for the hipathic EAS rodlet structure.
Publisher: Elsevier BV
Date: 10-2003
Publisher: Elsevier BV
Date: 05-2020
Publisher: Elsevier BV
Date: 04-2011
DOI: 10.1016/J.JMB.2011.02.048
Abstract: The (βα)(8) barrel is one of the most common protein folds, and enzymes with this architecture display a remarkable range of catalytic activities. Many of these functions are associated with ancient metabolic pathways, and phylogenetic reconstructions suggest that the (βα)(8) barrel was one of the very first protein folds to emerge. Consequently, there is considerable interest in understanding the evolutionary processes that gave rise to this fold. In particular, much attention has been focused on the plausibility of (βα)(8) barrel evolution from homodimers of half barrels. However, we previously isolated a three-quarter-barrel-sized fragment of a (βα)(8) barrel, termed truncated phosphoribosylanthranilate isomerase (trPRAI), that is soluble and almost as thermostable as full-length N-(5'-phosphoribosyl)anthranilate isomerase (PRAI). Here, we report the NMR-derived structure of trPRAI. The subdomain is monomeric, is well ordered and adopts a native-like structure in solution. Side chains from strands β(1) (Glu3 and Lys5), β(2) (Tyr25) and β(6) (Lys122) of trPRAI repack to shield the hydrophobic core from the solvent. This result demonstrates that three-quarter barrels were viable intermediates in the evolution of the (βα)(8) barrel fold. We propose a unified model for (βα)(8) barrel evolution that combines our data, previously published work and plausible scenarios for the emergence of (initially error-prone) genetic systems. In this model, the earliest proto-cells contained erse pools of part-barrel subdomains. Combinatorial assembly of these subdomains gave rise to many distinct lineages of (βα)(8) barrel proteins, that is, our model excludes the possibility that there was a single (βα)(8) barrel from which all present ex les are descended.
Publisher: Royal Society of Chemistry (RSC)
Date: 17-12-2003
DOI: 10.1039/B208846K
Publisher: Springer Science and Business Media LLC
Date: 20-04-2005
DOI: 10.1007/S00249-005-0480-1
Abstract: Growth factor receptor bound protein 7 (Grb7) is an adaptor protein that is co-overexpressed and forms a tight complex with the ErbB2 receptor in a number of breast tumours and breast cancer cell lines. The interaction of Grb7 with the ErbB2 receptor is mediated via its Src homology 2 (SH2) domain. Whilst most SH2 domains exist as monomers, recently reported studies have suggested that the Grb7-SH2 domain exists as a homodimer. The self-association properties of the Grb7-SH2 domain were therefore studied using sedimentation equilibrium ultracentrifugation. Analysis of the data demonstrated that the Grb7-SH2 domain is dimeric with a dissociation constant of approximately 11 muM. We also demonstrate, using size-exclusion chromatography, that mutation of phenylalanine 511 to an arginine produces a monomeric form of the Grb7-SH2 domain. This mutation represents the first step in the engineering of a Grb7-SH2 domain with good solution properties for further biophysical and structural investigation.
Publisher: MDPI AG
Date: 20-10-2022
DOI: 10.3390/BIOM12101526
Abstract: New antifungals with unique modes of action are urgently needed to treat the increasing global burden of invasive fungal infections. The fungal inositol polyphosphate kinase (IPK) pathway, comprised of IPKs that convert IP3 to IP8, provides a promising new target due to its impact on multiple, critical cellular functions and, unlike in mammalian cells, its lack of redundancy. Nearly all IPKs in the fungal pathway are essential for virulence, with IP3-4 kinase (IP3-4K) the most critical. The dibenzylaminopurine compound, N2-(m-trifluorobenzylamino)-N6-(p-nitrobenzylamino)purine (TNP), is a commercially available inhibitor of mammalian IPKs. The ability of TNP to be adapted as an inhibitor of fungal IP3-4K has not been investigated. We purified IP3-4K from the human pathogens, Cryptococcus neoformans and Candida albicans, and optimised enzyme and surface plasmon resonance (SPR) assays to determine the half inhibitory concentration (IC50) and binding affinity (KD), respectively, of TNP and 38 analogues. A novel chemical route was developed to efficiently prepare TNP analogues. TNP and its analogues demonstrated inhibition of recombinant IP3-4K from C. neoformans (CnArg1) at low µM IC50s, but not IP3-4K from C. albicans (CaIpk2) and many analogues exhibited selectivity for CnArg1 over the human equivalent, HsIPMK. Our results provide a foundation for improving potency and selectivity of the TNP series for fungal IP3-4K.
Publisher: Oxford University Press (OUP)
Date: 07-2016
DOI: 10.1093/NAR/GKW590
Publisher: Elsevier BV
Date: 09-2004
Publisher: American Association for the Advancement of Science (AAAS)
Date: 18-11-2016
Abstract: Alzheimer's disease presents with amyloid-β (Aβ) plaques and tau tangles. The prevailing idea in the field is that Aβ induces phosphorylation of tau, which in turn mediates neuronal dysfunction. Working in Alzheimer's disease mouse models, Ittner et al. found evidence for a protective role of tau in early Alzheimer's disease. This protection involves specific tau phosphorylation at threonine 205 at the postsynapse. A protective role of phosphorylated tau in disease challenges the dogma that tau phosphorylation only mediates toxic processes. Science , this issue p. 904
Publisher: Proceedings of the National Academy of Sciences
Date: 03-01-2012
Abstract: CHD4 is a catalytic subunit of the NuRD (nucleosome remodeling and deacetylase) complex essential in transcriptional regulation, chromatin assembly and DNA damage repair. CHD4 contains tandem plant homeodomain (PHD) fingers connected by a short linker, the biological function of which remains unclear. Here we explore the combinatorial action of the CHD4 PHD1/2 fingers and detail the molecular basis for their association with chromatin. We found that PHD1/2 targets nucleosomes in a multivalent manner, concomitantly engaging two histone H3 tails. This robust synergistic interaction displaces HP1 γ from pericentric sites, inducing changes in chromatin structure and leading to the dispersion of the heterochromatic mark H3K9me3. We demonstrate that recognition of the histone H3 tails by the PHD fingers is required for repressive activity of the CHD4/NuRD complex. Together, our data elucidate the molecular mechanism of multivalent association of the PHD fingers with chromatin and reveal their critical role in the regulation of CHD4 functions.
Publisher: Springer Science and Business Media LLC
Date: 16-10-2018
DOI: 10.1038/S41467-018-06665-5
Abstract: Chromatin structure and function is regulated by reader proteins recognizing histone modifications and/or histone variants. We recently identified that PWWP2A tightly binds to H2A.Z-containing nucleosomes and is involved in mitotic progression and cranial–facial development. Here, using in vitro assays, we show that distinct domains of PWWP2A mediate binding to free linker DNA as well as H3K36me3 nucleosomes. In vivo, PWWP2A strongly recognizes H2A.Z-containing regulatory regions and weakly binds H3K36me3-containing gene bodies. Further, PWWP2A binds to an MTA1-specific subcomplex of the NuRD complex (M1HR), which consists solely of MTA1, HDAC1, and RBBP4/7, and excludes CHD, GATAD2 and MBD proteins. Depletion of PWWP2A leads to an increase of acetylation levels on H3K27 as well as H2A.Z, presumably by impaired chromatin recruitment of M1HR. Thus, this study identifies PWWP2A as a complex chromatin-binding protein that serves to direct the deacetylase complex M1HR to H2A.Z-containing chromatin, thereby promoting changes in histone acetylation levels.
Publisher: Elsevier BV
Date: 1998
DOI: 10.1016/S0968-0004(97)01168-7
Abstract: Tree lycopsids prospered in the Late Devonian and constituted a major part of the Late Paleozoic forest ecosystem that deeply impacted the Earth's climate. However, the fertile organs of these early tree lycopsids display low morphological disparity, which has h ered further knowledge about their ecological habit. Here, we report Omprelostrobus gigas gen. et sp. nov. from the Upper Devonian (Famennian) Wutong Formation at Changxing, Zhejiang, China. The collection includes aerial axes, strobili and associated roots. The strobili are the largest among coeval lycopsids to our knowledge, and are ided into proximal and distal portions by dimorphic sporophylls with differentiated laminae and probable strong photosynthetic capacity. The associated but not attached roots displaying multiple isotomous branches lack rootlets and typical rootlet scars. The varied strobili sizes of early tree lycopsids were relatively independent of their body plan, but the large strobili could suggest increased reproductive investment to overcome the disadvantages of the disturbed flooded habitat.
Publisher: Springer Science and Business Media LLC
Date: 15-11-2022
DOI: 10.1038/S41467-022-34297-3
Abstract: Current vaccines against SARS-CoV-2 substantially reduce mortality, but protection against infection is less effective. Enhancing immunity in the respiratory tract, via mucosal vaccination, may provide protection against infection and minimise viral spread. Here, we report testing of a subunit vaccine in mice, consisting of SARS-CoV-2 Spike protein with a TLR2-stimulating adjuvant (Pam 2 Cys), delivered to mice parenterally or mucosally. Both routes of vaccination induce substantial neutralising antibody (nAb) titres, however, mucosal vaccination uniquely generates anti-Spike IgA, increases nAb in the serum and airways, and increases lung CD4 + T-cell responses. TLR2 is expressed by respiratory epithelia and immune cells. Using TLR2 deficient chimeric mice, we determine that TLR2 expression in either compartment facilitates early innate responses to mucosal vaccination. By contrast, TLR2 on hematopoietic cells is essential for optimal lung-localised, antigen-specific responses. In K18-hACE2 mice, vaccination provides complete protection against disease and sterilising lung immunity against SARS-CoV-2, with a short-term non-specific protective effect from mucosal Pam 2 Cys alone. These data support mucosal vaccination as a strategy to improve protection in the respiratory tract against SARS-CoV-2 and other respiratory viruses.
Publisher: Springer Science and Business Media LLC
Date: 06-2005
DOI: 10.1038/NATURE03609
Abstract: The synthesis of haemoglobin A (HbA) is exquisitely coordinated during erythrocyte development to prevent damaging effects from in idual alpha- and beta-subunits. The alpha-haemoglobin-stabilizing protein (AHSP) binds alpha-haemoglobin (alphaHb), inhibits the ability of alphaHb to generate reactive oxygen species and prevents its precipitation on exposure to oxidant stress. The structure of AHSP bound to ferrous alphaHb is thought to represent a transitional complex through which alphaHb is converted to a non-reactive, hexacoordinate ferric form. Here we report the crystal structure of this ferric alphaHb-AHSP complex at 2.4 A resolution. Our findings reveal a striking bis-histidyl configuration in which both the proximal and the distal histidines coordinate the haem iron atom. To attain this unusual conformation, segments of alphaHb undergo drastic structural rearrangements, including the repositioning of several alpha-helices. Moreover, conversion to the ferric bis-histidine configuration strongly and specifically inhibits redox chemistry catalysis and haem loss from alphaHb. The observed structural changes, which impair the chemical reactivity of haem iron, explain how AHSP stabilizes alphaHb and prevents its damaging effects in cells.
Publisher: Wiley
Date: 19-03-2019
DOI: 10.1111/FEBS.14800
Abstract: The nucleosome remodelling and deacetylase complex (NuRD) is a widely conserved regulator of gene expression. The determination of the subunit composition of the complex and identification of its binding partners are important steps towards understanding its architecture and function. The question of how these properties of the complex vary across different cell types has not been addressed in detail to date. Here, we set up a two-step purification protocol coupled to liquid chromatography-tandem mass spectrometry to assess NuRD composition and interaction partners in three different cancer cell lines, using label-free intensity-based absolute quantification (iBAQ). Our data indicate that the stoichiometry of the NuRD complex is preserved across our three different cancer cell lines. In addition, our interactome data suggest ZNF219 and SLC25A5 as possible interaction partners of the complex. To corroborate this latter finding, in vitro and cell-based pull-down experiments were carried out. These experiments indicated that ZNF219 can interact with RBBP4, GATAD2A/B and chromodomain helicase DNA binding 4, whereas SLC25A5 might interact with MTA2 and GATAD2A.
Publisher: Proceedings of the National Academy of Sciences
Date: 15-02-1993
Abstract: An approach toward the estimation of binding constants for organic molecules in aqueous solution is presented, based upon a partitioning of the free energy of binding. Consideration is given to polar and hydrophobic contributions and to the entropic cost of rotor restrictions and bimolecular associations. Several parameters (derived from an analysis of entropy changes upon the melting of crystals and from the binding of cell wall peptide analogues to the antibiotic ristocetin A) which may be useful guides to a crude understanding of binding phenomena are presented: (i) amide-amide hydrogen bond strengths of -(1 to 7) +/- 2 kJ.mol-1, (ii) a hydrophobic effect of -0.2 +/- 0.05 kJ.mol-1.A-2 of hydrocarbon removed from exposure to water in the binding process, and (iii) free energy costs for rotor restrictions of 3.5-5.0 kJ.mol-1. The validity of the parameters for hydrogen bond strengths is dependent on the validity of the other two parameters. The phenomenon of entropy/enthalpy compensation is considered, with the conclusion that enthalpic barriers to dissociations will result in larger losses in translational and rotational entropy in the association step. The dimerization of some vancomycin group antibiotics is strongly exothermic (-36 to -51 kJ.mol-1) and is promoted by a factor of 50-100 by a disaccharide attached to ring 4 (in vancomycin and eremomycin) and by a factor of ca. 1000 by an amino-sugar attached to the benzylic position of ring 6 in eremomycin. The dimerization process (which, as required for an exothermic association, appears to be costly in entropy) may be relevant to the mode of action of the antibiotics.
Publisher: Cold Spring Harbor Laboratory
Date: 26-04-2012
Abstract: The VapBC toxin-antitoxin (TA) family is the largest of nine identified TA families. The toxin, VapC, is a metal-dependent ribonuclease that is inhibited by its cognate antitoxin, VapB. Although the VapBCs are the largest TA family, little is known about their biological roles. Here we describe a new general method for the overexpression and purification of toxic VapC proteins and subsequent determination of their RNase sequence-specificity. Functional VapC was isolated by expression of the nontoxic VapBC complex, followed by removal of the labile antitoxin (VapB) using limited trypsin digestion. We have then developed a sensitive and robust method for determining VapC ribonuclease sequence-specificity. This technique employs the use of Pentaprobes as substrates for VapC. These are RNA sequences encoding every combination of five bases. We combine the RNase reaction with MALDI-TOF MS to detect and analyze the cleavage products and thus determine the RNA cut sites. Successful MALDI-TOF MS analysis of RNA fragments is acutely dependent on s le preparation methods. The sequence-specificity of four VapC proteins from two different organisms (VapC PAE0151 and VapC PAE2754 from Pyrobaculum aerophilum , and VapC Rv0065 and VapC Rv0617 from Mycobacterium tuberculosis ) was successfully determined using the described strategy. This rapid and sensitive method can be applied to determine the sequence-specificity of VapC ribonucleases along with other RNA interferases (such as MazF) from a range of organisms.
Publisher: Springer Science and Business Media LLC
Date: 17-12-2001
DOI: 10.1038/NSB743
Publisher: Wiley
Date: 26-08-2022
DOI: 10.1002/PRO.4387
Abstract: The nucleosome remodeling and deacetylase (NuRD) complex is a chromatin‐modifying assembly that regulates gene expression and DNA damage repair. Despite its importance, limited structural information describing the complete NuRD complex is available and a detailed understanding of its mechanism is therefore lacking. Drawing on information from SEC‐MALLS, DIA‐MS, XLMS, negative‐stain EM, X‐ray crystallography, NMR spectroscopy, secondary structure predictions, and homology models, we applied Bayesian integrative structure determination to investigate the molecular architecture of three NuRD sub‐complexes: MTA1‐HDAC1‐RBBP4, MTA1 N ‐HDAC1‐MBD3 GATAD2CC , and MTA1‐HDAC1‐RBBP4‐MBD3‐GATAD2A [nucleosome deacetylase (NuDe)]. The integrative structures were corroborated by examining independent crosslinks, cryo‐EM maps, biochemical assays, known cancer‐associated mutations, and structure predictions from AlphaFold. The robustness of the models was assessed by jack‐knifing. Localization of the full‐length MBD3, which connects the deacetylase and chromatin remodeling modules in NuRD, has not previously been possible our models indicate two different locations for MBD3, suggesting a mechanism by which MBD3 in the presence of GATAD2A asymmetrically bridges the two modules in NuRD. Further, our models uncovered three previously unrecognized subunit interfaces in NuDe: HDAC1 C ‐MTA1 BAH , MTA1 BAH ‐MBD3 MBD , and HDAC1 60–100 ‐MBD3 MBD . Our approach also allowed us to localize regions of unknown structure, such as HDAC1 C and MBD3 IDR , thereby resulting in the most complete and robustly cross‐validated structural characterization of these NuRD sub‐complexes so far.
Publisher: Elsevier BV
Date: 11-1998
Publisher: Humana Press
Date: 2010
DOI: 10.1007/978-1-60761-753-2_29
Abstract: Over the last 25 years, we have learned that many structural classes of zinc-binding domains (zinc fingers, ZFs) exist and it has become clear that the molecular functions of these domains are by no means limited to the sequence-specific recognition of double-stranded DNA. For ex le, ZFs can act as protein recognition or RNA-binding modules, and some domains can exhibit more than one function. In this chapter we describe the progress that has been made in understanding the role of ZF domains as RNA-recognition modules, and we speculate about both the prevalence of such functions and the prospects for creating designer ZFs that target RNA.
Publisher: Elsevier BV
Date: 02-2015
Publisher: Research Square Platform LLC
Date: 06-01-2022
DOI: 10.21203/RS.3.RS-1172665/V1
Abstract: Although ACE2 is the primary receptor for SARS-CoV-2 infection, a systematic assessment of factors controlling SARS-CoV-2 host interactions has not been described. Here we used whole genome CRISPR activation to identify host factors controlling SARS-CoV-2 Spike binding. The top hit was a Toll-like receptor-related cell surface receptor called leucine-rich repeat-containing protein 15 (LRRC15). LRRC15 expression was sufficient to promote SARS-CoV-2 Spike binding where it forms a cell surface complex with LRRC15 but does not support infection. Instead, LRRC15 functioned as a negative receptor suppressing both pseudotyped and live SARS-CoV-2 infection. LRRC15 is expressed in collagen-producing lung myofibroblasts where it can sequester virus and reduce infection in trans. Mechanistically LRRC15 is regulated by TGF-β, where moderate LRRC15 expression drives collagen production but high levels suppress it, revealing a novel lung fibrosis feedback circuit. Overall, LRRC15 is a master regulator of SARS-CoV-2, suppressing infection and controlling collagen production associated with “long-haul” COVID-19.
Publisher: Elsevier BV
Date: 04-1996
Publisher: Public Library of Science (PLoS)
Date: 27-08-2014
Publisher: Wiley
Date: 02-2000
DOI: 10.1046/J.1432-1327.2000.01095.X
Abstract: Zinc fingers (ZnFs) are extremely common protein domains. Several classes of ZnFs are distinguished by the nature and spacing of their zinc-coordinating residues. While the structure and function of some ZnFs are well characterized, many others have been identified only through their amino acid sequence. A number of proteins contain a conserved C-X2-C-X12-H-X1-5-C sequence, which is similar to the spacing observed for the 'classic' CCHH ZnFs. Although these domains have been implicated in protein-protein (and not protein-nucleic acid) interactions, nothing is known about their structure or function at a molecular level. Here, we address this problem through the expression and biophysical characterization of several CCHC-type zinc fingers from the erythroid transcription factor FOG and the related Drosophila protein U-shaped. Each of these domains does indeed fold in a zinc-dependent fashion, coordinating the metal in a tetrahedral manner through the sidechains of one histidine and three cysteine residues, and forming extremely thermostable structures. Analysis of CD spectra suggests an overall fold similar to that of the CCHH fingers, and indeed a point mutant of FOG-F1 in which the final cysteine residue is replaced by histidine remains capable of folding. However, the CCHC (as opposed to CCHH) motif is a prerequisite for GATA-1 binding activity, demonstrating that CCHC and CCHH topologies are not interchangeable. This demonstration that members of a structurally distinct subclass of genuine zinc finger domains are involved in the mediation of protein-protein interactions has implications for the prediction of protein function from nucleotide sequences.
Publisher: Public Library of Science (PLoS)
Date: 25-07-2012
Publisher: Elsevier BV
Date: 10-2009
Publisher: Wiley
Date: 19-04-2012
Publisher: Elsevier BV
Date: 05-2002
DOI: 10.1016/S0969-2126(02)00757-8
Abstract: Many different zinc binding modules have been identified. Their abundance and variety suggests that the formation of zinc binding folds might be relatively common. We have determined the structure of CH1(1), a 27-residue peptide derived from the first cysteine/histidine-rich region (CH1) of CREB binding protein (CBP). This peptide forms a highly ordered zinc-dependent fold that is distinct from known folds. The structure differs from a subsequently determined structure of a larger region from the CH3 region of CBP, and the CH1(1) fold probably represents a nonphysiologically active form. Despite this, the fold is thermostable and tolerant to both multiple alanine mutations and changes in the zinc-ligand spacing. Our data support the idea that zinc binding domains may arise frequently. Additionally, such structures may prove useful as scaffolds for protein design, given their stability and robustness.
Publisher: Wiley
Date: 02-1999
DOI: 10.1046/J.1365-2958.1999.01256.X
Abstract: Correct placement of the ision septum in Escherichia coli requires the co-ordinated action of three proteins, MinC, MinD and MinE. MinC and MinD interact to form a non-specific ision inhibitor that blocks septation at all potential ision sites. MinE is able to antagonize MinCD in a topologically sensitive manner, as it restricts MinCD activity to the unwanted ision sites at the cell poles. Here, we show that the topological specificity function of MinE residues in a structurally autonomous, trypsin-resistant domain comprising residues 31-88. Nuclear magnetic resonance (NMR) and circular dichroic spectroscopy indicate that this domain includes both alpha and beta secondary structure, while analytical ultracentrifugation reveals that it also contains a region responsible for MinE homodimerization. While trypsin digestion indicates that the anti-MinCD domain of MinE (residues 1-22) does not form a tightly folded structural domain, NMR analysis of a peptide corresponding to MinE1-22 indicates that this region forms a nascent helix in which the peptide rapidly interconverts between disordered (random coil) and alpha-helical conformations. This suggests that the N-terminal region of MinE may be poised to adopt an alpha-helical conformation when it interacts with the target of its anti-MinCD activity, presumably MinD.
Publisher: American Society for Microbiology
Date: 07-2000
DOI: 10.1128/JVI.74.13.5911-5920.2000
Abstract: Entry into the host cell by enveloped viruses is mediated by fusion (F) or transmembrane glycoproteins. Many of these proteins share a fold comprising a trimer of antiparallel coiled-coil heterodimers, where the heterodimers are formed by two discontinuous heptad repeat motifs within the proteolytically processed chain. The F protein of human respiratory syncytial virus (RSV the major cause of lower respiratory tract infections in infants) contains two corresponding regions that are predicted to form coiled coils (HR1 and HR2), together with a third predicted heptad repeat (HR3) located in a nonhomologous position. In order to probe the structures of these three domains and ascertain the nature of the interactions between them, we have studied the isolated HR1, HR2, and HR3 domains of RSV F by using a range of biophysical techniques, including circular dichroism, nuclear magnetic resonance spectroscopy, and sedimentation equilibrium. HR1 forms a symmetrical, trimeric coiled coil in solution ( K 3 ≈ 2.2 × 10 11 M −2 ) which interacts with HR2 to form a 3:3 hexamer. The HR1-HR2 interaction domains have been mapped using limited proteolysis, reversed-phase high-performance liquid chromatography, and electrospray-mass spectrometry. HR2 in isolation exists as a largely unstructured monomer, although it exhibits a tendency to form aggregates with β-sheet-like characteristics. Only a small increase in α-helical content was observed upon the formation of the hexamer. This suggests that the RSV F glycoprotein contains a domain that closely resembles the core structure of the simian parainfluenza virus 5 fusion protein (K. A. Baker, R. E. Dutch, R. A. Lamb, and T. S. Jardetzky, Mol. Cell 3:309–319, 1999). Finally, HR3 forms weak α-helical homodimers that do not appear to interact with HR1, HR2, or the HR1-HR2 complex. The results of these studies support the idea that viral fusion proteins have a common core architecture.
Publisher: Wiley
Date: 25-09-2012
DOI: 10.1002/PRO.2153
Publisher: Elsevier BV
Date: 04-2011
Publisher: Proceedings of the National Academy of Sciences
Date: 12-10-2020
Abstract: Large DNA-encoded libraries of cyclic peptides are emerging as powerful sources of molecules to tackle challenging drug targets. The structural and functional ersity contained within these libraries is, however, little explored. Here we demonstrate that one such library contains members that use unexpectedly erse mechanisms to recognize the same surface on the same target proteins with high affinity and specificity. This range of binding modes is much larger than observed in natural ligands of the same proteins, demonstrating the power and versatility of the technology. Our data also reveal opportunities for the development of more sophisticated approaches to achieving specificity when trying to selectively target one member of a family of closely related proteins.
Publisher: Springer Science and Business Media LLC
Date: 1997
Publisher: Springer Science and Business Media LLC
Date: 21-02-2019
DOI: 10.1038/S41598-019-38655-Y
Abstract: Transcriptomes consist of several classes of RNA that have wide-ranging but often poorly described functions and the deregulation of which leads to numerous diseases. Engineering of functionalized RNA-binding proteins (RBPs) could therefore have many applications. Our previous studies suggested that the RanBP2-type Zinc Finger (ZF) domain is a suitable scaffold to investigate the design of single-stranded RBPs. In the present work, we have analyzed the natural sequence specificity of various members of the RanBP2-type ZF family and characterized the interaction with their target RNA. Surprisingly, our data showed that natural RanBP2-type ZFs with different RNA-binding residues exhibit a similar sequence specificity and therefore no simple recognition code can be established. Despite this finding, different discriminative abilities were observed within the family. In addition, in order to target a long RNA sequence and therefore gain in specificity, we generated a 6-ZF array by combining ZFs from the RanBP2-type family but also from different families, in an effort to achieve a wider target sequence repertoire. We showed that this chimeric protein recognizes its target sequence (20 nucleotides), both in vitro and in living cells. Altogether, our results indicate that the use of ZFs in RBP design remains attractive even though engineering of specificity changes is challenging.
Publisher: Elsevier BV
Date: 10-2008
DOI: 10.1016/J.MICRON.2007.08.003
Abstract: Hydrophobins are a remarkable class of small cysteine-rich proteins found exclusively in fungi. They self-assemble to form robust polymeric monolayers that are highly hipathic and play numerous roles in fungal biology, such as in the formation and dispersal of aerial spores and in pathogenic and mutualistic interactions. The polymeric form can be reversibly disassembled and is able to reverse the wettability of a surface, leading to many proposals for nanotechnological applications over recent years. The surprising properties of hydrophobins and their potential for commercialization have led to substantial efforts to delineate their morphology and molecular structure. In this review, we summarize the progress that has been made using a variety of spectroscopic and microscopic approaches towards understanding the molecular mechanisms underlying hydrophobin structure.
Publisher: Elsevier BV
Date: 11-2011
Publisher: Springer International Publishing
Date: 2018
Publisher: Elsevier BV
Date: 08-2008
DOI: 10.1016/J.SBI.2008.04.009
Abstract: The design of metal-binding functionality in proteins is expanding into many different areas with a wide range of practical and research applications. Here we review several developing areas of metal-related protein design, including the use of metals to induce protein-protein interactions or facilitate the assembly of extended nanostructures the design of metallopeptides that bind metal and other inorganic surfaces, an area with potential in erse applications ranging from nanoelectronics and photonics to biotechnology and biomedicine and, the creation of sensitive and selective metal sensors for use both in vivo and in vitro.
Publisher: Springer Science and Business Media LLC
Date: 19-06-2010
DOI: 10.1007/S12104-010-9240-Y
Abstract: Lmo2 is a LIM-only protein involved in hematopoiesis and the development of T-cell acute lymphoblastic leukaemia. Here we report backbone and side chain NMR assignments for an engineered intramolecular complex of the C-terminal LIM domain from Lmo2 tethered to the LIM interaction domain (LID) from LIM domain binding protein 1 (Ldb1).
Publisher: Proceedings of the National Academy of Sciences
Date: 11-01-2005
Abstract: GATA-1 and friend of GATA (FOG) are zinc-finger transcription factors that physically interact to play essential roles in erythroid and megakaryocytic development. Several naturally occurring mutations in the GATA-1 gene that alter the FOG-binding domain have been reported. The mutations are associated with familial anemias and thrombocytopenias of differing severity. To elucidate the molecular basis for the GATA-1/FOG interaction, we have determined the three-dimensional structure of a complex comprising the interaction domains of these proteins. The structure reveals how zinc fingers can act as protein recognition motifs. Details of the architecture of the contact domains and their physical properties provide a molecular explanation for how the GATA-1 mutations contribute to distinct but related genetic diseases.
Publisher: Elsevier BV
Date: 11-1997
DOI: 10.1016/S0969-2126(97)00301-8
Abstract: Versutoxin (delta-ACTX-Hv1) is the major component of the venom of the Australian Blue Mountains funnel web spider, Hadronyche versuta. delta-ACTX-Hv1 produces potentially fatal neurotoxic symptoms in primates by slowing the inactivation of voltage-gated sodium channels delta-ACTX-Hv1 is therefore a useful tool for studying sodium channel function. We have determined the three-dimensional structure of delta-ACTX-Hv1 as the first step towards understanding the molecular basis of its interaction with these channels. The solution structure of delta-ACTX-Hv1, determined using NMR spectroscopy, comprises a core beta region containing a triple-stranded antiparallel beta sheet, a thumb-like extension protruding from the beta region and a C-terminal 310 helix that is appended to the beta domain by virtue of a disulphide bond. The beta region contains a cystine knot motif similar to that seen in other neurotoxic polypeptides. The structure shows homology with mu-agatoxin-I, a spider toxin that also modifies the inactivation kinetics of vertebrate voltage-gated sodium channels. More surprisingly, delta-ACTX-Hv1 shows both sequence and structural homology with gurmarin, a plant polypeptide. This similarity leads us to suggest that the sweet-taste suppression elicited by gurmarin may result from an interaction with one of the downstream ion channels involved in sweet-taste transduction. delta-ACTX-Hv1 shows no structural homology with either sea anemone or alpha-scorpion toxins, both of which also modify the inactivation kinetics of voltage-gated sodium channels by interacting with channel recognition site 3. However, we have shown that delta-ACTX-Hv1 contains charged residues that are topologically related to those implicated in the binding of sea anemone and alpha-scorpion toxins to mammalian voltage-gated sodium channels, suggesting similarities in their mode of interaction with these channels.
Publisher: Elsevier BV
Date: 07-2003
Publisher: Elsevier BV
Date: 2002
Publisher: Wiley
Date: 02-12-2019
DOI: 10.1111/MMI.14426
Abstract: Commensal bacteria serve as an important line of defense against colonisation by opportunisitic pathogens, but the underlying molecular mechanisms remain poorly explored. Here, we show that strains of a commensal bacterium, Haemophilus haemolyticus, make hemophilin, a heme-binding protein that inhibits growth of the opportunistic pathogen, non-typeable Haemophilus influenzae (NTHi) in culture. We purified the NTHi-inhibitory protein from H. haemolyticus and identified the hemophilin gene using proteomics and a gene knockout. An x-ray crystal structure of recombinant hemophilin shows that the protein does not belong to any of the known heme-binding protein folds, suggesting that it evolved independently. Biochemical characterisation shows that heme can be captured in the ferrous or ferric state, and with a variety of small heme-ligands bound, suggesting that hemophilin could function under a range of physiological conditions. Hemophilin knockout bacteria show a limited capacity to utilise free heme for growth. Our data suggest that hemophilin is a hemophore and that inhibition of NTHi occurs by heme starvation, raising the possibility that competition from hemophilin-producing H. haemolyticus could antagonise NTHi colonisation in the respiratory tract.
Publisher: Elsevier BV
Date: 05-2006
DOI: 10.1016/J.JMB.2006.02.074
Abstract: The overexpression of LIM-only protein 2 (LMO2) in T-cells, as a result of chromosomal translocations, retroviral insertion during gene therapy, or in transgenic mice models, leads to the onset of T-cell leukemias. LMO2 comprises two protein-binding LIM domains that allow LMO2 to interact with multiple protein partners, including LIM domain-binding protein 1 (Ldb1, also known as CLIM2 and NLI), an essential cofactor for LMO proteins. Sequestration of Ldb1 by LMO2 in T-cells may prevent it binding other key partners, such as LMO4. Here, we show using protein engineering and enzyme-linked immunosorbent assay (ELISA) methodologies that LMO2 binds Ldb1 with a twofold lower affinity than does LMO4. Thus, excess LMO2 rather than an intrinsically higher binding affinity would lead to sequestration of Ldb1. Both LIM domains of LMO2 are required for high-affinity binding to Ldb1 (K(D) = 2.0 x 10(-8) M). However, the first LIM domain of LMO2 is primarily responsible for binding to Ldb1 (K(D) = 2.3 x 10(-7) M), whereas the second LIM domain increases binding by an order of magnitude. We used mutagenesis in combination with yeast two-hybrid analysis, and phage display selection to identify LMO2-binding "hot spots" within Ldb1 that locate to the LIM1-binding region. The delineation of this region reveals some specific differences when compared to the equivalent LMO4:Ldb1 interaction that hold promise for the development of reagents to specifically bind LMO2 in the treatment of leukemia.
Publisher: Elsevier BV
Date: 08-2007
DOI: 10.1086/519530
Publisher: Portland Press Ltd.
Date: 25-09-2009
DOI: 10.1042/BJ20090870
Abstract: CHD4 (chromodomain helicase DNA-binding protein 4) ATPase is a major subunit of the repressive NuRD (nucleosome remodelling and deacetylase) complex, which is involved in transcriptional regulation and development. CHD4 contains two PHD (plant homeodomain) fingers of unknown function. Here we show that the second PHD finger (PHD2) of CHD4 recognizes the N-terminus of histone H3 and that this interaction is facilitated by acetylation or methylation of Lys9 (H3K9ac and H3K9me respectively) but is inhibited by methylation of Lys4 (H3K4me) or acetylation of Ala1 (H3A1ac). An 18 μM binding affinity toward unmodified H3 rises to 0.6 μM for H3K9ac and to 0.9 μM for H3K9me3, whereas it drops to 2.0 mM for H3K4me3, as measured by tryptophan fluorescence and NMR. A peptide library screen further shows that phosphorylation of Thr3, Thr6 or Ser10 abolishes this interaction. A model of the PHD2–H3 complex, generated using a combination of NMR, data-driven docking and mutagenesis data, reveals an elongated site on the PHD2 surface where the H3 peptide is bound. Together our findings suggest that the PHD2 finger plays a role in targeting of the CHD4/NuRD complex to chromatin.
Publisher: Elsevier BV
Date: 09-2013
Publisher: Elsevier BV
Date: 08-2014
Publisher: American Society of Hematology
Date: 22-10-2015
DOI: 10.1182/BLOOD-2015-06-650887
Abstract: The platelet defect associated with Paris-Trousseau thrombocytopenia and Jacobsen syndrome is caused by an abnormal transcription factor FLI1. FLI1 DNA–binding ETS domain mutations cause bleeding disorders with both autosomal dominant and recessive patterns of inheritance.
Publisher: Elsevier BV
Date: 07-2003
DOI: 10.1016/S0969-2126(03)00122-9
Abstract: The design of proteins with tailored functions remains a relatively elusive goal. Small size, a well-defined structure, and the ability to maintain structural integrity despite multiple mutations are all desirable properties for such designer proteins. Many zinc binding domains fit this description. We determined the structure of a PHD finger from the transcriptional cofactor Mi2beta and investigated the suitability of this domain as a scaffold for presenting selected binding functions. The two flexible loops in the structure were mutated extensively by either substitution or expansion, without affecting the overall fold of the domain. A binding site for the corepressor CtBP2 was also grafted onto the domain, creating a new PHD domain that can specifically bind CtBP2 both in vitro and in the context of a eukaryotic cell nucleus. These results represent a step toward designing new regulatory proteins for modulating aberrant gene expression in vivo.
Publisher: American Society for Clinical Investigation
Date: 15-11-2004
DOI: 10.1172/JCI21982
Publisher: Elsevier BV
Date: 07-2008
DOI: 10.1016/J.JMB.2008.05.038
Abstract: Dihydrodipicolinate synthase (DHDPS) is an essential enzyme in (S)-lysine biosynthesis and an important antibiotic target. All X-ray crystal structures solved to date reveal a homotetrameric enzyme. In order to explore the role of this quaternary structure, dimeric variants of Escherichia coli DHDPS were engineered and their properties were compared to those of the wild-type tetrameric form. X-ray crystallography reveals that the active site is not disturbed when the quaternary structure is disrupted. However, the activity of the dimeric enzymes in solution is substantially reduced, and a tetrahedral adduct of a substrate analogue is observed to be trapped at the active site in the crystal form. Remarkably, heating the dimeric enzymes increases activity. We propose that the homotetrameric structure of DHDPS reduces dynamic fluctuations present in the dimeric forms and increases specificity for the first substrate, pyruvate. By restricting motion in a key catalytic motif, a competing, non-productive reaction with a substrate analogue is avoided. Small-angle X-ray scattering and mutagenesis data, together with a B-factor analysis of the crystal structures, support this hypothesis and lead to the suggestion that in at least some cases, the evolution of quaternary enzyme structures might serve to optimise the dynamic properties of the protein subunits.
Publisher: MDPI AG
Date: 17-11-2020
DOI: 10.20944/PREPRINTS202009.0390.V2
Abstract: Proteases catalyse irreversible posttranslational modifications that often alter a biological function of the substrate. The protease dipeptidyl peptidase 4 (DPP4) is a pharmacological target in type 2 diabetes therapy primarily because it inactivates glucagon-like protein-1. DPP4 also has roles in steatosis, insulin resistance, cancers and inflammatory and fibrotic diseases. In addition, DPP4 binds to the spike protein of MERS virus, causing it to be the human cell surface receptor for that virus. DPP4 has been identified as a potential binding target of SARS-CoV-2 spike protein, so this question requires experimental investigation. Understanding protein structure and function requires reliable protocols for production and purification. We developed such strategies for baculovirus generated soluble recombinant human DPP4 (residues 29-766) produced in insect cells. Purification used differential ammonium sulphate precipitation, hydrophobic interaction chromatography, dye affinity chromatography in series with immobilised metal affinity chromatography, and ion exchange chromatography. The binding affinities of DPP4 to the SARS-CoV-2 full-length spike protein and its receptor binding domain (RBD) were measured using surface plasmon resonance and ELISA. This optimised DPP4 purification procedure yielded 1 to 1.8 mg of pure fully active soluble DPP4 protein per litre of insect cell culture with specific activity & U/mg, indicative of high purity. No specific binding between DPP4 and CoV-2 spike protein was detected by surface plasmon resonance or ELISA. In summary, a procedure for high purity high yield soluble human DPP4 was achieved and used to show that, unlike MERS-CoV, SARS-CoV-2 does not bind human DPP4.
Publisher: International Union of Crystallography (IUCr)
Date: 28-11-2008
Publisher: Elsevier BV
Date: 02-1999
DOI: 10.1016/S1097-2765(00)80312-3
Abstract: GATA-1 and FOG (Friend of GATA-1) are each essential for erythroid and megakaryocyte development. FOG, a zinc finger protein, interacts with the amino (N) finger of GATA-1 and cooperates with GATA-1 to promote differentiation. To determine whether this interaction is critical for GATA-1 action, we selected GATA-1 mutants in yeast that fail to interact with FOG but retain normal DNA binding, as well a compensatory FOG mutant that restores interaction. These novel GATA-1 mutants do not promote erythroid differentiation of GATA-1- erythroid cells. Differentiation is rescued by the second-site FOG mutant. Thus, interaction of FOG with GATA-1 is essential for the function of GATA-1 in erythroid differentiation. These findings provide a paradigm for dissecting protein-protein associations involved in mammalian development.
Publisher: MDPI AG
Date: 17-09-2020
DOI: 10.20944/PREPRINTS202009.0390.V1
Abstract: Proteases catalyse irreversible posttranslational modifications that often alter a biological function of the substrate. The protease dipeptidyl peptidase 4 (DPP4) is a pharmacological target in type 2 diabetes therapy primarily because it inactivates glucagon-like protein-1. DPP4 also has roles in steatosis, insulin resistance, cancers and inflammatory and fibrotic diseases. In addition, DPP4 binds to the spike protein of MERS virus, causing it to be the human cell surface receptor for that virus. DPP4 has been identified as a potential binding target of SARS-CoV-2 spike protein, so this question requires experimental investigation. Understanding protein structure and function requires reliable protocols for production and purification. We developed such strategies for baculovirus generated soluble recombinant human DPP4 (residues 29-766) produced in insect cells. Purification used differential ammonium sulfate precipitation, hydrophobic interaction chromatography, dye affinity chromatography in series with immobilised metal affinity chromatography, and ion exchange chromatography. The binding affinities of DPP4 to the SARS-CoV-2 full-length spike protein and its receptor binding domain (RBD) were measured using surface plasmon resonance. This optimised DPP4 purification procedure yielded 1 to 1.8 mg of pure fully active soluble DPP4 protein per litre of insect cell culture with specific activity & U/mg, indicative of high purity. No specific binding between DPP4 and CoV-2 spike protein was detected. In summary, a procedure for high purity high yield soluble human DPP4 was achieved and used to show that, unlike MERS, SARS-CoV-2 does not bind human DPP4.
Publisher: Elsevier BV
Date: 12-2000
Publisher: Royal Society of Chemistry (RSC)
Date: 2009
DOI: 10.1039/B904751D
Abstract: ESI mass spectrometry was used to assess the ability of metal complexes to inhibit binding of a transcription factor to a DNA molecule containing its recognition sequence.
Publisher: Wiley
Date: 12-2006
Publisher: Oxford University Press (OUP)
Date: 09-2007
Abstract: The gene-for-gene mechanism of plant disease resistance involves direct or indirect recognition of pathogen avirulence (Avr) proteins by plant resistance (R) proteins. Flax rust (Mel sora lini) AvrL567 avirulence proteins and the corresponding flax (Linum usitatissimum) L5, L6, and L7 resistance proteins interact directly. We determined the three-dimensional structures of two members of the AvrL567 family, AvrL567-A and AvrL567-D, at 1.4- and 2.3-Å resolution, respectively. The structures of both proteins are very similar and reveal a β-sandwich fold with no close known structural homologs. The polymorphic residues in the AvrL567 family map to the surface of the protein, and polymorphisms in residues associated with recognition differences for the R proteins lead to significant changes in surface chemical properties. Analysis of single amino acid substitutions in AvrL567 proteins confirm the role of in idual residues in conferring differences in recognition and suggest that the specificity results from the cumulative effects of multiple amino acid contacts. The structures also provide insights into possible pathogen-associated functions of AvrL567 proteins, with nucleic acid binding activity demonstrated in vitro. Our studies provide some of the first structural information on avirulence proteins that bind directly to the corresponding resistance proteins, allowing an examination of the molecular basis of the interaction with the resistance proteins as a step toward designing new resistance specificities.
Publisher: Elsevier BV
Date: 07-2002
Publisher: Elsevier BV
Date: 05-2018
Publisher: Springer Science and Business Media LLC
Date: 17-02-2013
DOI: 10.1007/S12104-013-9469-3
Abstract: Chromatin-remodeling proteins have a pivotal role in normal cell function and development, catalyzing conformational changes in DNA that ultimately result in changes in gene expression patterns. Chromodomain helicase DNA-binding protein 4 (CHD4), the defining subunit of the nucleosome remodeling and deacetylase (NuRD) complex, is a nucleosome-remodeling protein of the SNF2/ISWI2 family, members of which contain two chromo domains and an ATP-dependent helicase module. CHD3, CHD4 and CHD5 also contain two contiguous PHD domains and have an extended N-terminal region that has not previously been characterized. We have identified a stable domain in the N-terminal region of CHD4 and report here the backbone and side chain resonance assignments for this domain at pH 7.5 and 25 °C (BMRB No. 18906).
Publisher: Springer Science and Business Media LLC
Date: 04-08-2012
DOI: 10.1007/S12104-011-9330-5
Abstract: Hydrophobins are proteins secreted by filamentous fungi that are able to self-assemble into monolayers at hydrophobic:hydrophilic interfaces. The layers are hipathic and can reverse the wettability of surfaces. Hydrophobins have several roles in fungal development, including the formation of coatings on fungal structures to render them hydrophobic. Here we report the backbone and sidechain assignments for the class I hydrophobin DewA from the fungus Aspergillus nidulans.
Publisher: Wiley
Date: 22-04-2019
DOI: 10.1002/PROT.25687
Abstract: InterPro family IPR020489 comprises ~1000 uncharacterized bacterial proteins. Previously we showed that overexpressing the Escherichia coli representative of this family, EcYejG, conferred low-level resistance to aminoglycoside antibiotics. In an attempt to shed light on the biochemical function of EcYejG, we have solved its structure using multinuclear solution NMR spectroscopy. The structure most closely resembles that of domain III from elongation factor G (EF-G). EF-G catalyzes ribosomal translocation and mutations in EF-G have also been associated with aminoglycoside resistance. While we were unable to demonstrate a direct interaction between EcYejG and the ribosome, the protein might play a role in translation.
Publisher: Springer Science and Business Media LLC
Date: 04-06-2010
DOI: 10.1007/S12104-010-9237-6
Abstract: KipI is a sporulation inhibitor in Bacillus subtilis which acts by binding to the dimerisation and histidine phosphotransfer (DHp) domain of KinA, the principle input kinase in the phosphorelay responsible for sporulation. The (15)N, (13)C and (1)H chemical shift assignments of the N-terminal domain of KipI were determined using multidimensional, multinuclear NMR experiments. The N-terminal domain has two conformers and resonance assignments have been made for both conformers.
Publisher: Elsevier BV
Date: 03-2014
Location: United Kingdom of Great Britain and Northern Ireland
Start Date: 05-2009
End Date: 09-2012
Amount: $420,000.00
Funder: Australian Research Council
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End Date: 02-2012
Amount: $670,000.00
Funder: Australian Research Council
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End Date: 12-2019
Amount: $444,800.00
Funder: Australian Research Council
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End Date: 08-2025
Amount: $4,163,359.00
Funder: Australian Research Council
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End Date: 12-2017
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Funder: Australian Research Council
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Amount: $530,000.00
Funder: Australian Research Council
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Amount: $365,000.00
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Funder: Australian Research Council
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End Date: 12-2005
Amount: $579,000.00
Funder: Australian Research Council
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End Date: 12-2022
Amount: $1,000,000.00
Funder: Australian Research Council
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End Date: 03-2015
Amount: $515,000.00
Funder: Australian Research Council
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End Date: 09-2016
Amount: $380,000.00
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End Date: 12-2010
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Funder: Australian Research Council
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End Date: 02-2025
Amount: $435,000.00
Funder: Australian Research Council
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End Date: 10-2011
Amount: $110,000.00
Funder: Australian Research Council
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Amount: $860,000.00
Funder: Australian Research Council
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Amount: $600,000.00
Funder: Australian Research Council
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End Date: 12-2019
Amount: $407,609.00
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End Date: 12-2010
Amount: $1,300,000.00
Funder: Australian Research Council
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End Date: 12-2020
Amount: $178,839.00
Funder: Australian Research Council
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End Date: 06-2023
Amount: $772,676.00
Funder: Australian Research Council
View Funded ActivityStart Date: 01-2004
End Date: 12-2005
Amount: $369,697.00
Funder: Australian Research Council
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End Date: 06-2018
Amount: $650,000.00
Funder: Australian Research Council
View Funded ActivityStart Date: 2018
End Date: 12-2020
Amount: $598,198.00
Funder: Australian Research Council
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