ORCID Profile
0000-0002-6919-1824
Current Organisation
University of Adelaide
Does something not look right? The information on this page has been harvested from data sources that may not be up to date. We continue to work with information providers to improve coverage and quality. To report an issue, use the Feedback Form.
Publisher: Elsevier BV
Date: 12-2023
Publisher: Portland Press Ltd.
Date: 13-08-2021
DOI: 10.1042/BCJ20210455
Abstract: Activation of enzymes by monovalent cations (M+) is a widespread phenomenon in biology. Despite this, there are few structure-based studies describing the underlying molecular details. Thiolases are a ubiquitous and highly conserved family of enzymes containing both K+-activated and K+-independent members. Guided by structures of naturally occurring K+-activated thiolases, we have used a structure-based approach to engineer K+-activation into a K+-independent thiolase. To our knowledge, this is the first demonstration of engineering K+-activation into an enzyme, showing the malleability of proteins to accommodate M+ ions as allosteric regulators. We show that a few protein structural features encode K+-activation in this class of enzyme. Specifically, two residues near the substrate-binding site are sufficient for K+-activation: A tyrosine residue is required to complete the K+ coordination sphere, and a glutamate residue provides a compensating charge for the bound K+ ion. Further to these, a distal residue is important for positioning a K+-coordinating water molecule that forms a direct hydrogen bond to the substrate. The stability of a cation–π interaction between a positively charged residue and the substrate is determined by the conformation of the loop surrounding the substrate-binding site. Our results suggest that this cation–π interaction effectively overrides K+-activation, and is, therefore, destabilised in K+-activated thiolases. Evolutionary conservation of these amino acids provides a promising signature sequence for predicting K+-activation in thiolases. Together, our structural, biochemical and bioinformatic work provide important mechanistic insights into how enzymes can be allosterically activated by M+ ions.
Publisher: Elsevier BV
Date: 08-2016
Publisher: Elsevier BV
Date: 05-2017
DOI: 10.1016/J.TIBS.2017.02.001
Abstract: Protein biotinylation is a key post-translational modification found throughout the living world. The covalent attachment of a biotin cofactor onto specific metabolic enzymes is essential for their activity. This modification is distinctive, in that it is carried out by a single enzyme: biotin protein ligase (BPL), an enzyme that is able to biotinylate multiple target substrates without aberrant-off target biotinylation. BPL achieves this target selectivity by recognizing a sequence motif in the context of a highly conserved tertiary structure. One structural class of BPLs has developed an additional 'substrate verification' mechanism to further enable appropriate protein selection. This is crucial for the precise and selective biotinylation required for efficient biotin management, especially in organisms that are auxotrophic for biotin.
Publisher: Springer Science and Business Media LLC
Date: 04-04-2020
Publisher: American Chemical Society (ACS)
Date: 17-05-2018
Publisher: Cold Spring Harbor Laboratory
Date: 06-09-2020
DOI: 10.1101/2020.09.05.284661
Abstract: The mechanistic target of rapamycin complex 1 (mTORC1) is an important regulator of cellular metabolism that is commonly hyperactivated in cancer. Recent cancer genome screens have identified multiple mutations in Ras-homolog enriched in brain (Rheb), the primary activator of mTORC1, that might act as driver oncogenes by causing hyperactivation of mTORC1. Here, we show that a number of recurrently occurring Rheb mutants drive hyperactive mTORC1 signalling through differing levels of insensitivity to the primary inactivator of Rheb, Tuberous Sclerosis Complex. We show that two activated mutants, Rheb-T23M and E40K, strongly drive increased cell growth, proliferation and anchorage-independent growth resulting in enhanced tumour growth in vivo . Proteomic analysis of cells expressing the mutations revealed, surprisingly, that these two mutants promote distinct oncogenic pathways with Rheb-T23M driving metabolic reprogramming and an increased rate of glycolysis, while Rheb-E40K regulates the translation factor eEF2 and autophagy, likely through a differential interaction with AMPK. Our findings suggest that unique ‘bespoke’ combination therapies may be utilised to treat cancers according to which Rheb mutant they harbour.
Publisher: American Chemical Society (ACS)
Date: 14-02-2020
Publisher: Springer Science and Business Media LLC
Date: 12-06-2015
DOI: 10.1038/NCOMMS8443
Publisher: Springer International Publishing
Date: 2021
Publisher: International Union of Crystallography (IUCr)
Date: 26-01-2022
DOI: 10.1107/S2059798321012031
Abstract: Purine biosynthesis is a fundamental cellular process that sustains life by maintaining the intracellular pool of purines for DNA/RNA synthesis and signal transduction. As an integral determinant of fungal survival and virulence, the enzymes in this metabolic pathway have been pursued as potential antifungal targets. Guanosine monophosphate (GMP) synthase has been identified as an attractive target as it is essential for virulence in the clinically prominent fungal pathogens Aspergillus fumigatus , Candida albicans and Cryptococcus neoformans . However, a lack of structural information on GMP synthase has hindered drug-design efforts. Here, the first structure of a GMP synthase of fungal origin, that from A. fumigatus (at 2.3 Å resolution), is presented. Structural analysis of GMP synthase shows a distinct absence of the D1 dimerization domain that is present in the human homologue. Interestingly, A. fumigatus GMP synthase adopts a dimeric state, as determined by native mass spectrometry and gel-filtration chromatography, in contrast to the monomeric human homologue. Analysis of the substrate-binding pockets of A. fumigatus GMP synthase reveals key differences in the ATP- and XMP-binding sites that can be exploited for species-specific inhibitor drug design. Furthermore, the inhibitory activities of the glutamine analogues acivicin (IC 50 = 16.6 ± 2.4 µ M ) and 6-diazo-5-oxo-L-norleucine (IC 50 = 29.6 ± 5.6 µ M ) against A. fumigatus GMP synthase are demonstrated. Together, these data provide crucial structural information required for specifically targeting A. fumigatus GMP synthase for future antifungal drug-discovery endeavours.
Publisher: Springer Science and Business Media LLC
Date: 20-08-2015
DOI: 10.1038/NCOMMS9013
Abstract: A subset of nuclear receptors (NRs) function as obligate heterodimers with retinoid X receptor (RXR), allowing integration of ligand-dependent signals across the dimer interface via an unknown structural mechanism. Using nuclear magnetic resonance (NMR) spectroscopy, x-ray crystallography and hydrogen/deuterium exchange (HDX) mass spectrometry, here we show an allosteric mechanism through which RXR co-operates with a permissive dimer partner, peroxisome proliferator-activated receptor (PPAR)-γ, while rendered generally unresponsive by a non-permissive dimer partner, thyroid hormone (TR) receptor. Amino acid residues that mediate this allosteric mechanism comprise an evolutionarily conserved network discovered by statistical coupling analysis (SCA). This SCA network acts as a signalling rheostat to integrate signals between dimer partners, ligands and coregulator-binding sites, thereby affecting signal transmission in RXR heterodimers. These findings define rules guiding how NRs integrate two ligand-dependent signalling pathways into RXR heterodimer-specific responses.
Publisher: American Chemical Society (ACS)
Date: 15-12-2021
DOI: 10.1021/ACS.INORGCHEM.1C02786
Abstract: The cytochrome P450 (CYP) superfamily of heme monooxygenases is involved in a range of important chemical biotransformations across nature. Azole-containing molecules have been developed as drugs that bind to the heme center of these enzymes, inhibiting their function. The optical spectrum of CYP enzymes after the addition of these inhibitors is used to assess how the molecules bind. Here we use the bacterial CYP199A4 enzyme, from
Publisher: Wiley
Date: 19-07-2018
Abstract: The human sliding cl (PCNA) controls access to DNA for many proteins involved in DNA replication and repair. Proteins are recruited to the PCNA surface by means of a short, conserved peptide motif known as the PCNA-interacting protein box (PIP-box). Inhibitors of these essential protein-protein interactions may be useful as cancer therapeutics by disrupting DNA replication and repair in these highly proliferative cells. PIP-box peptide mimetics have been identified as a potentially rapid route to potent PCNA inhibitors. Here we describe the rational design and synthesis of the first PCNA peptidomimetic ligands, based on the high affinity PIP-box sequence from the natural PCNA inhibitor p21. These mimetics incorporate covalent i,i+4 side-chain/side-chain lactam linkages of different lengths, designed to constrain the peptides into the 3
Publisher: Royal Society of Chemistry (RSC)
Date: 2016
DOI: 10.1039/C6OB01159D
Abstract: NMR and X-ray crystallography reveals covalent attachment of the macrocyclic aldehyde to serine195 of α-chymotrypsin and that its backbone binds as a β-strand.
Publisher: Springer Science and Business Media LLC
Date: 09-2011
DOI: 10.1038/NATURE10383
Publisher: Wiley
Date: 23-06-2021
Abstract: An i−i +4 or i−i +3 bimane‐containing linker was introduced into a peptide known to target Estrogen Receptor alpha (ERα), in order to stabilise an α‐helical geometry. These macrocycles were studied by CD and NMR to reveal the i−i +4 constrained peptide adopts a 3 10 ‐helical structure in solution, and an α‐helical conformation on interaction with the ERα coactivator recruitment surface in silico . An acyclic bimane‐modified peptide is also helical, when it includes a tryptophan or tyrosine residue but is significantly less helical with a phenylalanine or alanine residue, which indicates such a bimane modification influences peptide structure in a sequence dependent manner. The fluorescence intensity of the bimane appears influenced by peptide conformation, where helical peptides displayed a fluorescence increase when TFE was added to phosphate buffer, compared to a decrease for less helical peptides. This study presents the bimane as a useful modification to influence peptide structure as an acyclic peptide modification, or as a side‐chain constraint to give a macrocycle.
Publisher: MDPI AG
Date: 28-01-2023
DOI: 10.3390/IJMS24032519
Abstract: Platelets play a vital role in regulating hemostasis and thrombosis. Rho GTPases are well known as molecular switches that control various cellular functions via a balanced GTP-binding/GTP-hydrolysis cycle and signaling cascade through downstream effectors. In platelets, Rho GTPases function as critical regulators by mediating signal transduction that drives platelet activation and aggregation. Mostly by gene targeting and pharmacological inhibition approaches, Rho GTPase family members RhoA, Rac1, and Cdc42 have been shown to be indispensable in regulating the actin cytoskeleton dynamics in platelets, affecting platelet shape change, spreading, secretion, and aggregation, leading to thrombus formation. Additionally, studies of Rho GTPase function using platelets as a non-transformed model due to their anucleated nature have revealed valuable information on cell signaling principles. This review provides an updated summary of recent advances in Rho GTPase signaling in platelet regulation. We also highlight pharmacological approaches that effectively inhibited platelet activation to explore their possible development into future antiplatelet therapies.
Publisher: Springer Science and Business Media LLC
Date: 10-04-2011
DOI: 10.1038/NSMB.2046
Publisher: American Society for Microbiology
Date: 03-2019
DOI: 10.1128/AAC.02281-18
Abstract: Aspergillus fumigatus infections are associated with high mortality rates and high treatment costs. Limited available antifungals and increasing antifungal resistance highlight an urgent need for new antifungals.
Publisher: American Society for Clinical Investigation
Date: 17-06-2013
DOI: 10.1172/JCI68016
Publisher: American Chemical Society (ACS)
Date: 13-08-2019
DOI: 10.1021/ACSCHEMBIO.9B00463
Abstract: Here, we report the design, synthesis, and evaluation of a series of inhibitors of
Publisher: Cold Spring Harbor Laboratory
Date: 27-03-2023
DOI: 10.1101/2023.03.26.531482
Abstract: We previously reported potent ligands and inhibitors of Mycobacterium tuberculosis dethiobiotin synthetase ( Mt DTBS), a promising target for antituberculosis drug development (Schumann et al., ACS Chem Biol. 2021, 16, 2339-2347) here the unconventional origin of the fragment compound they were derived from is described for the first time. Compound 1 (9b-hydroxy-6b,7,8,9,9a,9b-hexahydrocyclopenta[3,4]cyclobuta[1,2-c]chromen-6(6a H )-one), identified by in silico fragment screen, was subsequently shown by surface plasmon resonance to have dose-responsive binding ( K D 0.6 mM). Clear electron density was revealed in the DAPA substrate binding pocket, when 1 was soaked into Mt DTBS crystals, but the density was inconsistent with the structure of 1 . Here we show the lactone of 1 hydrolyses to carboxylic acid 2 under basic conditions, including those of the crystallography soak, with subsequent ring-opening of the component cyclobutane ring to form cyclopentylacetic acid 3 . Crystals soaked directly with authentic 3 produced electron density that matched that of crystals soaked with presumed 1 , confirming the identity of the bound ligand. The synthetic utility of fortuitously formed 3 enabled subsequent compound development into nanomolar inhibitors. Our findings represent an ex le of chemical modification within drug discovery assays and demonstrate the value of high-resolution structural data in the fragment hit validation process. A molecule flagged in an in silico docking screen against Mt DTBS, was inadvertently hydrolysed in the crystal conditions used for hit validation. The resulting fragment-sized molecule bound to the DAPA substrate binding pocket of the target enzyme ( Mt DTBS) with millimolar affinity, as measured by surface plasmon resonance, but was later modified to a highly potent (nanomolar) ligand and promising lead for the development of novel tuberculosis treatments.
Publisher: American Chemical Society (ACS)
Date: 31-08-2023
Publisher: American Chemical Society (ACS)
Date: 11-01-2012
DOI: 10.1021/BI201548P
Publisher: Wiley
Date: 02-08-2023
Abstract: The cytochrome P450 (CYP) superfamily of monooxygenase enzymes play important roles in the metabolism of molecules which contain heterocyclic, aromatic functional groups. Here we study how oxygen‐ and sulfur‐containing heterocyclic groups interact with and are oxidized using the bacterial enzyme CYP199A4. This enzyme oxidized both 4‐(thiophen‐2‐yl)benzoic acid and 4‐(thiophen‐3‐yl)benzoic acid almost exclusively via sulfoxidation. The thiophene oxides produced were activated towards Diels‐Alder dimerization after sulfoxidation, forming dimeric metabolites. Despite X‐ray crystal structures demonstrating that the aromatic carbon atoms of the thiophene ring were located closer to the heme than the sulfur, sulfoxidation was still favoured with 4‐(thiophen‐3‐yl)benzoic acid. These results highlight a preference of this cytochrome P450 enzyme for sulfoxidation over aromatic hydroxylation. Calculations predict a strong preference for homodimerization of the enantiomers of the thiophene oxides and the formation of a single major product, in broad agreement with the experimental data. 4‐(Furan‐2‐yl)benzoic acid was oxidized to 4‐(4′‐hydroxybutanoyl)benzoic acid using a whole‐cell system. This reaction proceeded via a γ‐keto‐α,β‐unsaturated aldehyde species which could be trapped in vitro using semicarbazide to generate a pyridazine species. The combination of the enzyme structures, the biochemical data and theoretical calculations provides detailed insight into the formation of the metabolites formed from these heterocyclic compounds.
Publisher: American Society for Microbiology
Date: 2011
DOI: 10.1128/AAC.00558-10
Abstract: d -Alanine: d -alanine ligase (EC 6.3.2.4 Ddl) catalyzes the ATP-driven ligation of two d -alanine ( d -Ala) molecules to form the d -alanyl: d -alanine dipeptide. This molecule is a key building block in peptidoglycan biosynthesis, making Ddl an attractive target for drug development. d -Cycloserine (DCS), an analog of d -Ala and a prototype Ddl inhibitor, has shown promise for the treatment of tuberculosis. Here, we report the crystal structure of Mycobacterium tuberculosis Ddl at a resolution of 2.1 Å. This structure indicates that Ddl is a dimer and consists of three discrete domains the ligand binding cavity is at the intersection of all three domains and conjoined by several loop regions. The M. tuberculosis apo Ddl structure shows a novel conformation that has not yet been observed in Ddl enzymes from other species. The nucleotide and d -alanine binding pockets are flexible, requiring significant structural rearrangement of the bordering regions for entry and binding of both ATP and d -Ala molecules. Solution affinity and kinetic studies showed that DCS interacts with Ddl in a manner similar to that observed for d -Ala. Each ligand binds to two binding sites that have significant differences in affinity, with the first binding site exhibiting high affinity. DCS inhibits the enzyme, with a 50% inhibitory concentration (IC 50 ) of 0.37 mM under standard assay conditions, implicating a preferential and weak inhibition at the second, lower-affinity binding site. Moreover, DCS binding is tighter at higher ATP concentrations. The crystal structure illustrates potential drugable sites that may result in the development of more-effective Ddl inhibitors.
Publisher: American Chemical Society (ACS)
Date: 05-05-2023
Publisher: Wiley
Date: 28-08-2023
DOI: 10.1111/FEBS.16932
Abstract: Pseudomonas aeruginosa is a major human pathogen in the healthcare setting. The emergence of multi‐drug‐resistant and extensive drug‐resistant P. aeruginosa is of great concern, and clearly indicates that new alternatives to current first‐line antibiotics are required in the future. Inhibition of d ‐alanine– d ‐alanine production presents as a promising avenue as it is a key component in the essential process of cell wall biosynthesis. In P. aeruginosa , d ‐alanine– d ‐alanine production is facilitated by two isoforms, d ‐alanine– d ‐alanine ligase A ( Pa DdlA) and d ‐alanine– d ‐alanine ligase B ( Pa DdlA), but neither enzyme has been in idually characterised to date. Here, we present the functional and structural characterisation of Pa DdlA and Pa DdlB, and assess their potential as antibiotic targets. This was achieved using a combination of in vitro enzyme‐activity assays and X‐ray crystallography. The former revealed that both isoforms effectively catalyse d ‐alanine– d ‐alanine production with near identical efficiency, and that this is effectively disrupted by the model d ‐alanine– d ‐alanine ligase inhibitor, d ‐cycloserine. Next, each isoform was co‐crystallised with ATP and either d ‐alanine– d ‐alanine or d ‐cycloserine, allowing direct comparison of the key structural features. Both isoforms possess the same structural architecture and share a high level of conservation within the active site. Although residues forming the d ‐alanine pocket are completely conserved, the ATP‐binding pocket possesses several amino acid substitutions resulting in a differing chemical environment around the ATP adenine base. Together, these findings support that the discovery of dual Pa DdlA/ Pa DdlB competitive inhibitors is a viable approach for developing new antibiotics against P. aeruginosa .
Publisher: Elsevier BV
Date: 2017
DOI: 10.1016/J.IBMB.2016.11.008
Abstract: Bt toxins ingested by insect pests can bind to midgut receptors and cause death, although several steps in this process remain unclear. Multiple Bt toxin receptors have been identified in Lepidoptera, including a cadherin-like protein (CaLP), which is central to several models explaining Bt toxins' mode of action. Mutations in the Plutella xylostella ATP-dependent binding cassette transporter C2 (Px-abcc2), rather than CaLP, are genetically linked with Bt Cry1Ac resistance. Here we expressed Px-abcc2 in Drosophila and performed larval bioassays to determine whether this protein acts as an effective Bt receptor. Cry1Ac had no effect on larvae expressing Px-abcc2 in salivary glands, yet larvae expressing Px-abcc2 in the midgut were highly susceptible to both Cry1Ac protoxin and trypsin activated toxin. Furthermore, the CaLP orthologue has been lost from the Drosophila genome, making this a useful system for investigating the role of CaLP peptides from Manduca sexta (CR12-MPED), which are known to act as Bt synergists in larval feeding assays. Drosophila larvae expressing Px-ABCC2 in the midgut were fed LD
Publisher: American Chemical Society (ACS)
Date: 05-04-2019
DOI: 10.1021/ACSCHEMBIO.9B00236
Abstract: Obesity and rheumatic disease are mechanistically linked via chronic inflammation. The orphan receptor TREM-1 (triggering receptor expressed on myeloid cells-1) is a potent lifier of proinflammatory and noninfectious immune responses. Here, we show that the pan modulator SR1903 effectively blocks TREM-1 activation. SR1903 emerged from a chemical series of potent RORγ inverse agonists, although unlike close structural analogues, it has modest agonist activity on LXR and weak repressive activity (inverse agonism) of PPARγ, three receptors that play essential roles in inflammation and metabolism. The anti-inflammatory and antidiabetic efficacy of this unique modulator in collagen-induced arthritis and diet-induced obesity mouse models is demonstrated. Interestingly, in the context of obesity, SR1903 aided in the maintenance of the thymic homeostasis unlike selective RORγ inverse agonists. SR1903 was well-tolerated following chronic administration, and combined, these data suggest that it may represent a viable strategy for treatment of both metabolic and inflammatory disease. More importantly, the ability of SR1903 to block LPS signaling suggests the potential utility of this unique polypharmacological modulator for treatment of innate immune response disorders.
Publisher: American Chemical Society (ACS)
Date: 09-10-2018
Publisher: Wiley
Date: 06-11-2020
DOI: 10.1111/FEBS.15607
Publisher: Elsevier BV
Date: 07-2018
Publisher: American Society for Clinical Investigation
Date: 17-06-2013
DOI: 10.1172/JCI68035
Publisher: Elsevier BV
Date: 09-2021
Publisher: American Chemical Society (ACS)
Date: 12-01-2022
Publisher: American Chemical Society (ACS)
Date: 03-12-2016
Publisher: Elsevier BV
Date: 08-2021
Publisher: Frontiers Media SA
Date: 12-01-2021
DOI: 10.3389/FMOLB.2020.621366
Abstract: The impact of invasive fungal infections on human health is a serious, but largely overlooked, public health issue. Commonly affecting the immunocompromised community, fungal infections are predominantly caused by species of Candida, Cryptococcus , and Aspergillus . Treatments are reliant on the aggressive use of pre-existing antifungal drug classes that target the fungal cell wall and membrane. Despite their frequent use, these drugs are subject to unfavorable drug-drug interactions, can cause undesirable side-effects and have compromised efficacy due to the emergence of antifungal resistance. Hence, there is a clear need to develop novel classes of antifungal drugs. A promising approach involves exploiting the metabolic needs of fungi by targeted interruption of essential metabolic pathways. This review highlights potential antifungal targets including enolase, a component of the enolase-plasminogen complex, and enzymes from the mannitol biosynthesis and purine nucleotide biosynthesis pathways. There has been increased interest in the enzymes that comprise these particular pathways and further investigation into their merits as antifungal targets and roles in fungal survival and virulence are warranted. Disruption of these vital processes by targeting unconventional pathways with small molecules or antibodies may serve as a promising approach to discovering novel classes of antifungals.
Publisher: Elsevier BV
Date: 11-2018
Publisher: American Chemical Society (ACS)
Date: 21-02-2023
Publisher: Royal Society of Chemistry (RSC)
Date: 2021
DOI: 10.1039/D1CB00113B
Abstract: A small, inherently fluorescent macrocyclic peptide constrained with a bimane-linker is cell permeable, and binds the human sliding cl protein, PCNA, in a 3 10 -helical conformation with nanomolar affinity.
Publisher: Wiley
Date: 09-11-2022
Abstract: Cytochrome P450 (CYP) heme‐thiolate monooxygenases catalyze the hydroxylation of the C−H bonds of organic molecules. This reaction is initiated by a ferryl‐oxo heme radical cation (Cpd I). These enzymes can also catalyze sulfoxidation reactions and the ferric‐hydroperoxy complex (Cpd 0) and the Fe(III)‐H 2 O 2 complex have been proposed as alternative oxidants for this transformation. To investigate this, the oxidation of 4‐alkylthiobenzoic acids and 4‐methoxybenzoic acid by the CYP199A4 enzyme from Rhodopseudomonas palustris HaA2 was compared using both monooxygenase and peroxygenase pathways. By examining mutants at the mechanistically important, conserved acid alcohol‐pair (D251N, T252A and T252E) the relative amounts of the reactive intermediates that would form in these reactions were disturbed. Substrate binding and X‐ray crystal structures helped to understand changes in the activity and enabled an attempt to evaluate whether multiple oxidants can participate in these reactions. In peroxygenase reactions the T252E mutant had higher activity towards sulfoxidation than O ‐demethylation but in the monooxygenase reactions with the WT enzyme the activity of both reactions was similar. The peroxygenase activity of the T252A mutant was greater for sulfoxidation reactions than the WT enzyme, which is the reverse of the activity changes observed for O ‐demethylation. The monooxygenase activity and coupling efficiency of sulfoxidation and oxidative demethylation were reduced by similar degrees with the T252A mutant. These observations infer that while Cpd I is required for O ‐dealkylation, another oxidant may contribute to sulfoxidation. Based on the activity of the CYP199A4 mutants it is proposed that this is the Fe(III)‐H 2 O 2 complex which would be more abundant in the peroxide‐driven reactions.
Publisher: American Chemical Society (ACS)
Date: 31-07-2023
Publisher: MIT Press - Journals
Date: 2020
DOI: 10.1162/JOCN_A_01471
Abstract: Throughout the brain, information from in idual sources converges onto higher order neurons. For ex le, information from the two eyes first converges in binocular neurons in area V1. Some neurons are tuned to similarities between sources of information, which makes intuitive sense in a system striving to match multiple sensory signals to a single external cause—that is, establish causal inference. However, there are also neurons that are tuned to dissimilar information. In particular, some binocular neurons respond maximally to a dark feature in one eye and a light feature in the other. Despite compelling neurophysiological and behavioral evidence supporting the existence of these neurons [Katyal, S., Vergeer, M., He, S., He, B., & Engel, S. A. Conflict-sensitive neurons gate interocular suppression in human visual cortex. Scientific Reports, 8, 1239, 2018 Kingdom, F. A. A., Jennings, B. J., & Georgeson, M. A. Adaptation to interocular difference. Journal of Vision, 18, 9, 2018 Janssen, P., Vogels, R., Liu, Y., & Orban, G. A. At least at the level of inferior temporal cortex, the stereo correspondence problem is solved. Neuron, 37, 693–701, 2003 Tsao, D. Y., Conway, B. R., & Livingstone, M. S. Receptive fields of disparity-tuned simple cells in macaque V1. Neuron, 38, 103–114, 2003 Cumming, B. G., & Parker, A. J. Responses of primary visual cortical neurons to binocular disparity without depth perception. Nature, 389, 280–283, 1997], their function has remained opaque. To determine how neural mechanisms tuned to dissimilarities support perception, here we use electroencephalography to measure human observers' steady-state visually evoked potentials in response to change in depth after prolonged viewing of anticorrelated and correlated random-dot stereograms (RDS). We find that adaptation to anticorrelated RDS results in larger steady-state visually evoked potentials, whereas adaptation to correlated RDS has no effect. These results are consistent with recent theoretical work suggesting “what not” neurons play a suppressive role in supporting stereopsis [Goncalves, N. R., & Welchman, A. E. “What not” detectors help the brain see in depth. Current Biology, 27, 1403–1412, 2017] that is, selective adaptation of neurons tuned to binocular mismatches reduces suppression resulting in increased neural excitability.
Publisher: Wiley
Date: 22-03-2022
DOI: 10.1002/PROT.26331
Abstract: The metabolic enzyme, enolase, plays a crucial role in the cytoplasm where it maintains cellular energy production within the process of glycolysis. The main role of enolase in glycolysis is to convert 2‐phosphoglycerate to phosphoenolpyruvate however, enolase can fulfill roles that deviate from this function. In pathogenic bacteria and fungi, enolase is also located on the cell surface where it functions as a virulence factor. Surface‐expressed enolase is a receptor for human plasma proteins, including plasminogen, and this interaction facilitates nutrient acquisition and tissue invasion. A novel approach to developing antifungal drugs is to inhibit the formation of this complex. To better understand the structure of enolase and the interactions that may govern complex formation, we have solved the first X‐ray crystal structure of enolase from Aspergillus fumigatus (2.0 Å) and have shown that it preferentially adopts a dimeric quaternary structure using native mass spectrometry. Two additional X‐ray crystal structures of A. fumigatus enolase bound to the endogenous substrate 2‐phosphoglycerate and product phosphoenolpyruvate were determined and kinetic characterization was carried out to better understand the details of its canonical function. From these data, we have produced a model of the A. fumigatus enolase and human plasminogen complex to provide structural insights into the mechanisms of virulence and aid future development of small molecules or peptidomimetics for antifungal drug design.
Publisher: Elsevier BV
Date: 11-2019
Publisher: Elsevier BV
Date: 03-2011
Publisher: Portland Press Ltd.
Date: 13-07-2022
DOI: 10.1042/BCJ20220209
Abstract: Single-minded 2 (SIM2) is a neuron-enriched basic Helix–Loop–Helix/PER–ARNT–SIM (bHLH/PAS) transcription factor essential for mammalian survival. SIM2 is located within the Down syndrome critical region (DSCR) of chromosome 21, and manipulation in mouse models suggests Sim2 may play a role in brain development and function. During the screening of a clinical exome sequencing database, nine SIM2 non-synonymous mutations were found which were subsequently investigated for impaired function using cell-based reporter gene assays. Many of these human variants attenuated abilities to activate transcription and were further characterized to determine the mechanisms underpinning their deficiencies. These included impaired partner protein dimerization, reduced DNA binding, and reduced expression and nuclear localization. This study highlighted several SIM2 variants found in patients with disabilities and validated a candidate set as potentially contributing to pathology.
Publisher: American Chemical Society (ACS)
Date: 06-01-2022
Publisher: Wiley
Date: 19-07-2018
Publisher: EMBO
Date: 27-04-2007
Publisher: Elsevier BV
Date: 08-2021
Publisher: Wiley
Date: 12-01-2021
DOI: 10.1002/MGG3.1593
Abstract: Vanishing white matter (VWM) is a leukodystrophy, caused by recessive mutations in eukaryotic initiation factor 2B (eIF2B)‐subunit genes ( EIF2B1–EIF2B5 ) 80% are missense mutations. Clinical severity is highly variable, with a strong, unexplained genotype–phenotype correlation. With information from a recent natural history study, we severity‐graded 97 missense mutations. Using in silico modeling, we created a new human eIF2B model structure, onto which we mapped the missense mutations. Mutated residues were assessed for location in subunits, eIF2B complex, and functional domains, and for information on biochemical activity. Over 50% of mutations have (ultra‐)severe phenotypic effects. About 60% affect the ε‐subunit, containing the catalytic domain, mostly with (ultra‐)severe effects. About 55% affect subunit cores, with variable clinical severity. About 36% affect subunit interfaces, mostly with severe effects. Very few mutations occur on the external eIf2B surface, perhaps because they have minor functional effects and are tolerated. One external surface mutation affects eIF2B‐substrate interaction and is associated with ultra‐severe phenotype. Mutations that lead to (ultra‐)severe disease mostly affect amino acids with pivotal roles in complex formation and function of eIF2B. Therapies for VWM are emerging and reliable mutation‐based phenotype prediction is required for propensity score matching for trials and in the future for in idualized therapy decisions.
Publisher: Elsevier BV
Date: 10-2007
DOI: 10.1016/J.STR.2007.07.014
Abstract: Binding to helix 12 of the ligand-binding domain of PPARgamma is required for full agonist activity. Previously, the degree of stabilization of the activation function 2 (AF-2) surface was thought to correlate with the degree of agonism and transactivation. To examine this mechanism, we probed structural dynamics of PPARgamma with agonists that induced graded transcriptional responses. Here we present crystal structures and amide H/D exchange (HDX) kinetics for six of these complexes. Amide HDX revealed each ligand induced unique changes to the dynamics of the ligand-binding domain (LBD). Full agonists stabilized helix 12, whereas intermediate and partial agonists did not at all, and rather differentially stabilized other regions of the binding pocket. The gradient of PPARgamma transactivation cannot be accounted for solely through changes to the dynamics of AF-2. Thus, our understanding of allosteric signaling must be extended beyond the idea of a dynamic helix 12 acting as a molecular switch.
Publisher: KenzPub
Date: 2018
DOI: 10.11131/2018/101350
Publisher: Elsevier BV
Date: 02-2020
DOI: 10.1016/J.JINORGBIO.2019.110913
Abstract: The cytochrome P450 metalloenzyme (CYP) CYP199A4 from Rhodopseudomonas palustris HaA2 catalyzes the highly efficient oxidation of para-substituted benzoic acids. Here we determined crystal structures of CYP199A4, and the binding and turnover parameters, with different meta-substituted benzoic acids in order to establish which criteria are important for efficient catalysis. When compared to the para isomers, the meta-substituted benzoic acids were less efficiently oxidized. For ex le, 3-formylbenzoic acid was oxidized with lower activity than the equivalent para isomer and 3-methoxybenzoic acid did not undergo O-demethylation by CYP199A4. The structural data highlighted that the meta-substituted benzoic acids bound in the enzyme active site in a modified position with incomplete loss of the distal water ligand of the heme moiety. However, for both sets of isomers the meta- or para-substituent pointed towards, and was in close proximity, to the heme iron. The absence of oxidation activity with 3-methoxybenzoic acid was assigned to the observation that the CH bonds of this molecule point away from the heme iron. In contrast, in the para isomer they are in an ideal location for abstraction. These findings were confirmed by using the bulkier 3-ethoxybenzoic acid as a substrate which removed the water ligand and reoriented the meta-substituent so that the methylene hydrogens pointed towards the heme, enabling more efficient oxidation. Overall we show relatively small changes in substrate structure and position in the active site can have a dramatic effect on the activity.
Publisher: Elsevier BV
Date: 12-2004
DOI: 10.1016/J.STR.2004.09.018
Abstract: Human Proliferating Cellular Nuclear Antigen (hPCNA), a member of the sliding cl family of proteins, makes specific protein-protein interactions with DNA replication and repair proteins through a small peptide motif termed the PCNA-interacting protein, or PIP-box. We solved the structure of hPCNA bound to PIP-box-containing peptides from the p66 subunit of the human replicative DNA polymerase-delta (452-466) at 2.6 A and of the flap endonuclease (FEN1) (331-350) at 1.85 A resolution. Both structures demonstrate that the pol-delta p66 and FEN1 peptides interact with hPCNA at the same site shown to bind the cdk-inhibitor p21(CIP1). Binding studies indicate that peptides from the p66 subunit of the pol-delta holoenzyme and FEN1 bind hPCNA from 189- to 725-fold less tightly than those of p21. Thus, the PIP-box and flanking regions provide a small docking peptide whose affinities can be readily adjusted in accord with biological necessity to mediate the binding of DNA replication and repair proteins to hPCNA.
Publisher: American Chemical Society (ACS)
Date: 17-09-2021
Publisher: Hindawi Limited
Date: 2015
DOI: 10.1155/2015/816856
Abstract: PPAR γ (peroxisome proliferator activated receptor γ ) is a ligand activated transcription factor of the nuclear receptor superfamily that controls the expression of a variety of genes involved in fatty acid metabolism, adipogenesis, and insulin sensitivity. While endogenous ligands of PPAR γ include fatty acids and eicosanoids, synthetic full agonists of the receptor, including members of the thiazolidinedione (TZD) class, have been widely prescribed for the treatment of type II diabetes mellitus (T2DM). Unfortunately, the use of full agonists has been h ered by harsh side effects with some removed from the market in many countries. In contrast, partial agonists of PPAR γ have been shown to retain favourable insulin sensitizing effects while exhibiting little to no side effects and thus represent a new potential class of therapeutics for the treatment of T2DM. Partial agonists have been found to not only display differences in transcriptional and cellular outcomes, but also act through distinct structural and dynamic mechanisms within the ligand binding cavity compared to full agonists.
Publisher: American Chemical Society (ACS)
Date: 22-05-2017
Publisher: American Society for Microbiology
Date: 10-2014
DOI: 10.1128/AAC.02574-13
Abstract: In Mycobacterium tuberculosis , the carboxylation of acetyl coenzyme A (acetyl-CoA) to produce malonyl-CoA, a building block in long-chain fatty acid biosynthesis, is catalyzed by two enzymes working sequentially: a biotin carboxylase (AccA) and a carboxyltransferase (AccD). While the exact roles of the three different biotin carboxylases (AccA1 to -3) and the six carboxyltransferases (AccD1 to -6) in M. tuberculosis are still not clear, AccD6 in complex with AccA3 can synthesize malonyl-CoA from acetyl-CoA. A series of 10 herbicides that target plant acetyl-CoA carboxylases (ACC) were tested for inhibition of AccD6 and for whole-cell activity against M. tuberculosis . From the tested herbicides, haloxyfop, an arylophenoxypropionate, showed in vitro inhibition of M. tuberculosis AccD6, with a 50% inhibitory concentration (IC 50 ) of 21.4 ± 1 μM. Here, we report the crystal structures of M. tuberculosis AccD6 in the apo form (3.0 Å) and in complex with haloxyfop- R (2.3 Å). The structure of M. tuberculosis AccD6 in complex with haloxyfop- R shows two molecules of the inhibitor bound on each AccD6 subunit. These results indicate the potential for developing novel therapeutics for tuberculosis based on herbicides with low human toxicity.
Publisher: American Chemical Society (ACS)
Date: 31-07-2013
DOI: 10.1021/BI4002503
Publisher: Springer Science and Business Media LLC
Date: 12-04-2015
Publisher: American Chemical Society (ACS)
Date: 03-02-2018
Publisher: Elsevier BV
Date: 2021
Publisher: MDPI AG
Date: 10-12-2018
DOI: 10.3390/CRYST8120460
Abstract: Chymotrypsin is a protease that is commonly used as a standard for protein crystallization and as a model system for studying serine proteases. Unliganded bovine α-chymotrypsin was crystallized at neutral pH using ammonium sulphate as the precipitant, resulting in crystals that conform to P65 symmetry with unit cell parameters that have not been reported previously. Inspection of crystallographic interfaces revealed that the major interface between any two molecules in the crystal lattice represents the interface of the biological dimer, as previously observed for crystals of unliganded α-chymotrypsin grown at low pH in space group P21.
Publisher: Elsevier BV
Date: 06-2021
Publisher: American Chemical Society (ACS)
Date: 27-05-2015
DOI: 10.1021/ACS.BIOCHEM.5B00241
Abstract: Proliferating cell nuclear antigen (PCNA, processivity factor, sliding cl ) is a ring-shaped protein that tethers proteins to DNA in processes, including DNA replication, DNA repair, and cell-cycle control. Often used as a marker for cell proliferation, PCNA is overexpressed in cancer cells, making it an appealing pharmaceutical target. PCNA interacts with proteins through a PCNA interacting protein (PIP)-box, an eight-amino acid consensus sequence different binding partners display a wide range of affinities based on function. Of all biological PIP-boxes, p21 has the highest known affinity for PCNA, allowing for inhibition of DNA replication and cell growth under cellular stress. As p21 is one of the few PIP-box sequences to contain a tyrosine rather than a phenylalanine in the eighth conserved position, we probed the significance of the hydroxyl group at this position using a mutational approach. Here we present the cocrystal structure of PCNA bound to a mutant p21 PIP-box peptide, p21Tyr151Phe, with associated isothermal titration calorimetry data. The p21Tyr151Phe peptide showed a 3-fold difference in affinity, as well as differences in entropy and enthalpy of binding. These differences can be attributed to a loss of hydrogen bonding capacity, as well as structural plasticity in the PCNA interdomain connector loop and the hydrophobic cavity of PCNA to which p21 binds. Thus, the hydroxyl group of Tyr151 in p21 acts as a tethering point for ideal packing and surface recognition of the peptide interface, increasing the binding affinity of p21 for PCNA.
Publisher: Wiley
Date: 12-09-2021
Abstract: The serine 244 to aspartate (S244D) variant of the cytochrome P450 enzyme CYP199A4 was used to expand its substrate range beyond benzoic acids. Substrates, in which the carboxylate group of the benzoic acid moiety is replaced were oxidised with high activity by the S244D mutant (product formation rates nmol.(nmol‐CYP) −1 .min −1 ) and with total turnover numbers of up to 20,000. Ethyl α‐hydroxylation was more rapid than methyl oxidation, styrene epoxidation and S ‐oxidation. The S244D mutant catalysed the ethyl hydroxylation, epoxidation and sulfoxidation reactions with an excess of one stereoisomer (in some instances up to %). The crystal structure of 4‐methoxybenzoic acid‐bound CYP199A4 S244D showed that the active site architecture and the substrate orientation were similar to that of the WT enzyme. Overall, this work demonstrates that CYP199A4 can catalyse the stereoselective hydroxylation, epoxidation or sulfoxidation of substituted benzene substrates under mild conditions resulting in more sustainable transformations using this heme monooxygenase enzyme.
Publisher: Springer Science and Business Media LLC
Date: 13-10-2017
DOI: 10.1038/S41467-017-00978-7
Abstract: The vitamin D receptor/retinoid X receptor-α heterodimer (VDRRXRα) regulates bone mineralization via transcriptional control of osteocalcin ( BGLAP ) gene and is the receptor for 1α,25-dihydroxyvitamin D 3 (1,25D3). However, supra-physiological levels of 1,25D3 activates the calcium-regulating gene TRPV6 leading to hypercalcemia. An approach to attenuate this adverse effect is to develop selective VDR modulators (VDRMs) that differentially activate BGLAP but not TRPV6 . Here we present structural insight for the action of a VDRM compared with agonists by employing hydrogen/deuterium exchange. Agonist binding directs crosstalk between co-receptors upon DNA binding, stabilizing the activation function 2 (AF2) surfaces of both receptors driving steroid receptor co-activator-1 (SRC1) interaction. In contrast, AF2 of VDR within VDRM: BGLAP bound heterodimer is more vulnerable for large stabilization upon SRC1 interaction compared with VDRM: TRPV6 bound heterodimer. These results reveal that the combination of ligand structure and DNA sequence tailor the transcriptional activity of VDR toward specific target genes.
Publisher: Elsevier BV
Date: 04-2019
DOI: 10.1016/J.EJMECH.2019.02.034
Abstract: In search for effective multi-targeting drug ligands (MTDLs) to address low-grade inflammatory changes of metabolic disorders, we rationally designed some novel glitazones-like compounds. This was achieved by incorporating prominent pharmacophoric motifs from previously reported COX-2, 15-LOX and PPARγ ligands. Challenging our design with pre-synthetic docking experiments on PPARγ showed encouraging results. In vitro tests have identified 4 compounds as simultaneous partial PPARγ agonist, potent COX-2 antagonist (nanomolar IC
Publisher: Elsevier BV
Date: 12-2009
Publisher: International Union of Crystallography (IUCr)
Date: 10-2018
DOI: 10.1107/S2059798318010136
Abstract: Dethiobiotin synthetase from Mycobacterium tuberculosis ( Mt DTBS) is a promising antituberculosis drug target. Small-molecule inhibitors that target Mt DTBS provide a route towards new therapeutics for the treatment of antibiotic-resistant tuberculosis. Adenosine diphosphate (ADP) is an inhibitor of Mt DTBS however, structural studies into its mechanism of inhibition have been unsuccessful owing to competitive binding to the enzyme by crystallographic precipitants such as citrate and sulfate. Here, a crystallographic technique termed precipitant–ligand exchange has been developed to exchange protein-bound precipitants with ligands of interest. Proof of concept for the exchange method was demonstrated using cytidine triphosphate (CTP), which adopted the same binding mechanism as that obtained with traditional crystal-soaking techniques. Precipitant–ligand exchange also yielded the previously intractable structure of Mt DTBS in complex with ADP solved to 2.4 Å resolution. This result demonstrates the utility of precipitant–ligand exchange, which may be widely applicable to protein crystallography.
Publisher: Portland Press Ltd.
Date: 16-02-2018
DOI: 10.1042/BCJ20170946
Abstract: Members of the cytochrome P450 monooxygenase family CYP268 are found across a broad range of Mycobacterium species including the pathogens Mycobacterium avium, M. colombiense, M. kansasii, and M. marinum. CYP268A2, from M. marinum, which is the first member of this family to be studied, was purified and characterised. CYP268A2 was found to bind a variety of substrates with high affinity, including branched and straight chain fatty acids (C10–C12), acetate esters, and aromatic compounds. The enzyme was also found to bind phenylimidazole inhibitors but not larger azoles, such as ketoconazole. The monooxygenase activity of CYP268A2 was efficiently reconstituted using heterologous electron transfer partner proteins. CYP268A2 hydroxylated geranyl acetate and trans-pseudoionone at a terminal methyl group to yield (2E,6E)-8-hydroxy-3,7-dimethylocta-2,6-dien-1-yl acetate and (3E,5E,9E)-11-hydroxy-6,10-dimethylundeca-3,5,9-trien-2-one, respectively. The X-ray crystal structure of CYP268A2 was solved to a resolution of 2.0 Å with trans-pseudoionone bound in the active site. The overall structure was similar to that of the related phytanic acid monooxygenase CYP124A1 enzyme from Mycobacterium tuberculosis, which shares 41% sequence identity. The active site is predominantly hydrophobic, but includes the Ser99 and Gln209 residues which form hydrogen bonds with the terminal carbonyl group of the pseudoionone. The structure provided an explanation on why CYP268A2 shows a preference for shorter substrates over the longer chain fatty acids which bind to CYP124A1 and the selective nature of the catalysed monooxygenase activity.
Publisher: Springer New York
Date: 2019
DOI: 10.1007/978-1-4939-9195-2_21
Abstract: Obtaining high-throughput structural characterization of small molecule drug candidates bound to their protein target has been h ered by the challenges of cocrystallizing protein-ligand complexes. These challenges include poor ligand solubility, excess ligand molecules disrupting the homogeneity of the s le to be crystallized, and inefficiency in preparing in idual complexes and crystal screens for each drug candidate. Crystallizing apo protein followed by soaking with a solution containing the ligand of interest is a powerful tool for rapidly obtaining structural information of ligands. Here, we describe the process of purifying, crystallizing, and soaking PPARγ ligand binding domain as well as strategies for cocrystallizing ligands that are not amenable to soaking.
Publisher: International Union of Crystallography (IUCr)
Date: 28-04-2021
DOI: 10.1107/S2053230X21004052
Abstract: The phase problem is a persistent bottleneck that impedes the structure-determination pipeline and must be solved to obtain atomic resolution crystal structures of macromolecules. Although molecular replacement has become the predominant method of solving the phase problem, many scenarios still exist in which experimental phasing is needed. Here, a proof-of-concept study is presented that shows the efficacy of using tetrabromoterephthalic acid (B4C) as an experimental phasing compound. Incorporating B4C into the crystal lattice using co-crystallization, the crystal structure of hen egg-white lysozyme was solved using MAD phasing. The strong anomalous signal generated by its four Br atoms coupled with its compatibility with commonly used crystallization reagents render B4C an effective experimental phasing compound that can be used to overcome the phase problem.
Publisher: Cold Spring Harbor Laboratory
Date: 02-02-2022
DOI: 10.1101/2022.02.01.475276
Abstract: The basic-Helix-Loop-Helix Per-Arnt-Sim (PAS) homology domain (bHLH-PAS) transcription factor (TF) family comprises critical biological sensors of physiological (hypoxia, tryptophan metabolites, neuronal activity, and appetite) and environmental (diet derived metabolites and environmental pollutants) stimuli to regulate genes involved in signal adaptation and homeostasis 1 . bHLH TFs bind DNA as homo or heterodimers via E-box (CANNTG) response elements, however the DNA binding specificity of the PAS domain-containing bHLH subfamily remains unresolved 1 . We systematically analysed cognate DNA binding hierarchies of prototypical bHLH-PAS family members (ARNT, ARNT2, HIF1α, HIF2α, AhR, NPAS4, SIM1) and demonstrate distinct core (NNCGTG) specificities for different heterodimer classes. The results also show that bHLH-PAS TFs bind over a large footprint 12-15bp and recognise preferential DNA sequences flanking the core. For ex le, specificity beyond otherwise identical core binding by SIM1 and the HIFs is mediated through N-terminal HIFα-DNA interactions. We also reveal an intimate relationship between DNA shape and both core and flanking TF binding allowing motif sequence flexibility and underpinning TF binding specificity. Furthermore, DNA-shape affinity relationships revealed that novel downstream PAS-A-loop DNA interactions are associated with AT-rich sequences that lead to high-affinity binding, and that loss of this function underpins a monogenic cause of human hyperphagic obesity in a recapitulated SIM1.R171H knock-in mouse model. Importantly, models of protein-DNA binding accurately predict in vivo occupancy, while response element methylation blocks DNA binding and predicts cell type specific chromatin occupancy. These data provide a definitive and accurate map of bHLH-PAS TF specificity and target selectivity through novel flanking protein-DNA interactions that are crucial for in vivo biological function.
Publisher: Elsevier BV
Date: 08-2020
Publisher: Wiley
Date: 15-10-2020
Abstract: Proliferating cell nuclear antigen (PCNA) is an excellent inhibition target to shut down highly proliferative cells and thereby develop a broad-spectrum cancer therapeutic. It interacts with a wide variety of proteins through a conserved motif referred to as the PCNA-interacting protein (PIP) box. There is large sequence ersity between high-affinity PCNA binding partners, but with conservation of the binding structure-a well-defined 3
Publisher: Royal Society of Chemistry (RSC)
Date: 2015
DOI: 10.1039/C5RA08730A
Abstract: CYP199A4, a cytochrome P450 enzyme from Rhodopseudomonas palustris HaA2, is able to efficiently demethylate a range of benzoic acids at the para -position. It can also catalyse demethenylation reactions.
Publisher: Public Library of Science (PLoS)
Date: 17-01-2014
Publisher: American Society of Hematology
Date: 05-11-2020
DOI: 10.1182/BLOOD-2020-133491
Abstract: Introduction Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is a high-risk subtype of ALL associated with high relapse rates and poor survival. Rearrangements of Janus kinase 2 (JAK2r) are present in approximately 5% and 14% of pediatric and young adult Ph-like ALL cases respectively. The resultant JAK2 gene fusions drive leukemogenesis through constitutive activation of the JAK/STAT signaling pathway and are associated with very poor outcomes in patients with Ph-like ALL. All JAK inhibitors in clinical development are type I inhibitors, which bind in the ATP-binding site of JAK2. A phase II clinical trial is currently assessing the only FDA-approved JAK1/2 inhibitor, ruxolitinib in high-risk B-cell ALL cases harboring JAK2 alterations. The development of treatment resistance to targeted inhibitors in other diseases is well documented and often results in disease relapse. Elucidating mechanisms of ruxolitinib resistance in JAK2r ALL will inform approaches to monitor the emergence of resistance in ongoing clinical trials and enable the development of therapeutic strategies to overcome or avert resistance. Methods JAK2r B-ALL was modelled in the pro-B cell line, Ba/F3, by expressing the high-risk B-ALL fusion, ATF7IP-JAK2. Ruxolitinib resistance in three independent ATF7IP-JAK2 Ba/F3 cell lines was achieved following dose escalation to a clinically relevant dose of 1 μM ruxolitinib. Sanger sequencing of the RT-PCR lified JAK2 fusion revealed each resistant line had acquired a different mutation within the JAK2 kinase domain. Therapeutic sensitives were assessed by staining with Fixable Aqua Dead Cell Stain (Invitrogen) and Annexin V, and analysis by flow cytometry. Alterations in signaling pathways were determined using phosphoflow cytometry and western blot analysis. Computational modelling of acquired JAK2 mutations and subsequent influence on ruxolitinib binding was performed using ICM-Pro (Molsoft L.C.C.). Results In addition to the identification of two known ruxolitinib resistant mutations, JAK2 p.Y931C and p.L983F, a novel p.G993A mutation was identified. All mutations localized to the ATP/ruxolitinib binding site and conferred resistance to multiple type-I JAK inhibitors, including ruxolitinib, BMS-911543, and AZD-1480 (Table 1). JAK2 p.G993A ATF7IP-JAK2 Ba/F3 cells were also resistant to the type-II JAK inhibitor, CHZ-868, which binds in an allosteric site of JAK2 in addition to the ATP-binding site. Ruxolitinib resistance correlated with sustained downstream STAT5 activation in the presence of 1 μM ruxolitinib compared with non-mutant ATF7IP-JAK2 Ba/F3 cells. Intracellular phosphoflow cytometry of ruxolitinib-resistant ATF7IP-JAK2 Ba/F3 cells confirmed constitutive activation of JAK/STAT signaling in the presence of 50 nM ruxolitinib, in contrast to non-mutant ATF7IP-JAK2 Ba/F3 cells. Computational modelling suggested that JAK2 p.L983F (Fig. 1D) sterically hinders ruxolitinib binding, while JAK2 p.Y931C may reduce ruxolitinib binding affinity by disruption of a critical hydrogen-bond (Fig. 1B). The novel JAK2 p.G993A mutation is predicted to alter DFG-loop dynamics by stabilizing the JAK2 activation loop (Fig1C). Conclusions This study demonstrates that the JAK2 ATP-binding site is susceptible to JAK inhibitor resistant mutations following ruxolitinib exposure in the setting of JAK2r ALL, highlighting the importance of monitoring the emergence of mutations within this region. In addition to previously described mutations we identified a novel JAK2 p.G993A mutation that conferred resistance to both type-I and type-II JAK inhibitors. The JAK2 p.G993A mutation was postulated to modulate the stability of a conserved domain. Understanding mechanisms of ruxolitinib resistance, as modelled here, has the potential to inform future drug design and the development therapeutic strategies for this high-risk cohort. White: Amgen: Honoraria Bristol-Myers Squibb: Honoraria, Research Funding.
Publisher: American Chemical Society (ACS)
Date: 25-07-2023
Publisher: International Union of Crystallography (IUCr)
Date: 07-2019
DOI: 10.1107/S2059798319009008
Abstract: Two commonly encountered bottlenecks in the structure determination of a protein by X-ray crystallography are screening for conditions that give high-quality crystals and, in the case of novel structures, finding derivatization conditions for experimental phasing. In this study, the phasing molecule 5-amino-2,4,6-triiodoisophthalic acid (I3C) was added to a random microseed matrix screen to generate high-quality crystals derivatized with I3C in a single optimization experiment. I3C, often referred to as the magic triangle, contains an aromatic ring scaffold with three bound I atoms. This approach was applied to efficiently phase the structures of hen egg-white lysozyme and the N-terminal domain of the Orf11 protein from Staphylococcus phage P68 (Orf11 NTD) using SAD phasing. The structure of Orf11 NTD suggests that it may play a role as a virion-associated lysin or endolysin.
Publisher: Elsevier BV
Date: 02-2019
DOI: 10.1016/J.BMCL.2018.12.032
Abstract: A series of dipeptide aldehydes containing different N-terminal heterocycles was prepared and assayed in vitro against α-chymotrypsin to ascertain the importance of the heterocycle in maintaining a β-strand geometry while also providing a hydrogen bond donor equivalent to the backbone amide nitrogen of the surrogate amino acid. The dipeptide containing a pyrrole constraint (10) was the most potent inhibitor, with >30-fold improved activity over dipeptides which lacked a nitrogen hydrogen bond donor (namely thiophene 11, furan 12 and pyridine 13). Molecular docking studies of 10 bound to α-chymotrypsin demonstrates a hydrogen bond between the pyrrole nitrogen donor and the backbone carbonyl of Gly
Publisher: Elsevier BV
Date: 06-2020
Publisher: Elsevier BV
Date: 07-2022
DOI: 10.1016/J.JSBMB.2022.106097
Abstract: Members of the CYP51 family of cytochrome P450 enzymes are classified as sterol demethylases involved in the metabolic formation of cholesterol and related derivatives. The CYP51 enzyme from Mycobacterium marinum was studied and compared to its counterpart from Mycobacterium tuberculosis to determine the degree of functional conservation between them. Spectroscopic analyses of substrate and inhibitor binding of the purified CYP51 enzymes from M. marinum and M. tuberculosis were performed. The catalytic oxidation of lanosterol and related steroids was investigated. M. marinum CYP51 was structurally characterized by X-ray crystallography. The CYP51 enzyme of M. marinum is sequentially closely related to CYP51B1 from M. tuberculosis. However, differences in the heme spin state of each enzyme were observed upon the addition of steroids and other ligands. Both enzymes displayed different binding properties to those reported for the CYP51-Fdx fusion protein from the bacterium Methylococcus capsulatus. The enzymes were able to oxidatively demethylate lanosterol to generate 14-demethylanosterol, but no products were detected for the related species dihydrolanosterol and eburicol. The crystal structure of CYP51 from M. marinum in the absence of added substrate but with a Bis-Tris molecule within the active site was resolved. The CYP51 enzyme of M. marinum displays differences in how steroids and other ligands bind compared to the M. tuberculosis enzyme. This was related to structural differences between the two enzymes. Overall, both of these CYP51 enzymes from mycobacterial species displayed significant differences to the CYP51 enzymes of eukaryotic species and the bacterial CYP51-Fdx enzyme of Me. capsulatus.
Publisher: Wiley
Date: 27-02-2017
DOI: 10.1111/FEBS.14035
Abstract: The fungal pathogen Aspergillus fumigatus has been implicated in a drastic increase in life-threatening infections over the past decade. However, compared to other microbial pathogens, little is known about the essential molecular processes of this organism. One such fundamental process is DNA replication. The protein responsible for ensuring processive DNA replication is PCNA (proliferating cell nuclear antigen, also known as the sliding cl ), which cl s the replicative polymerase to DNA. Here we present the first crystal structure of a sliding cl from a pathogenic fungus (A. fumigatus), at 2.6Å. Surprisingly, the structure bears more similarity to the human sliding cl than other available fungal sliding cl s. Reflecting this, fluorescence polarization experiments demonstrated that AfumPCNA interacts with the PCNA-interacting protein (PIP-box) motif of human p21 with an affinity (K The atomic coordinates and structure factors for the Aspergillus fumigatus sliding cl can be found in the RCSB Protein Data Bank (www.rcsb.org) under the accession code 5TUP.
Publisher: Springer Science and Business Media LLC
Date: 10-08-2021
DOI: 10.1038/S41698-021-00215-X
Abstract: Ruxolitinib (rux) Phase II clinical trials are underway for the treatment of high-risk JAK2 -rearranged ( JAK2 r) B-cell acute lymphoblastic leukemia (B-ALL). Treatment resistance to targeted inhibitors in other settings is common elucidating potential mechanisms of rux resistance in JAK2 r B-ALL will enable development of therapeutic strategies to overcome or avert resistance. We generated a murine pro-B cell model of ATF7IP-JAK2 with acquired resistance to multiple type-I JAK inhibitors. Resistance was associated with mutations within the JAK2 ATP/rux binding site, including a JAK2 p.G993A mutation. Using in vitro models of JAK2 r B-ALL, JAK2 p.G993A conferred resistance to six type-I JAK inhibitors and the type-II JAK inhibitor, CHZ-868. Using computational modeling, we postulate that JAK2 p.G993A enabled JAK2 activation in the presence of drug binding through a unique resistance mechanism that modulates the mobility of the conserved JAK2 activation loop. This study highlights the importance of monitoring mutation emergence and may inform future drug design and the development of therapeutic strategies for this high-risk patient cohort.
Publisher: Elsevier BV
Date: 06-2021
Publisher: Springer Science and Business Media LLC
Date: 10-10-2010
DOI: 10.1038/NCHEMBIO.451
Publisher: Elsevier BV
Date: 10-2023
Publisher: American Chemical Society (ACS)
Date: 02-2021
Publisher: MyJove Corporation
Date: 16-01-2021
DOI: 10.3791/61894
Publisher: Wiley
Date: 06-10-2022
Abstract: The cytochrome P450 (CYP) family of heme monooxygenases catalyse the selective oxidation of C-H bonds under ambient conditions. The CYP199A4 enzyme from Rhodopseudomonas palustris catalyses aliphatic oxidation of 4-cyclohexylbenzoic acid but not the aromatic oxidation of 4-phenylbenzoic acid, due to the distinct mechanisms of aliphatic and aromatic oxidation. The aromatic substrates 4-benzyl-, 4-phenoxy- and 4-benzoyl-benzoic acid and methoxy-substituted phenylbenzoic acids were assessed to see if they could achieve an orientation more amenable to aromatic oxidation. CYP199A4 could catalyse the efficient benzylic oxidation of 4-benzylbenzoic acid. The methoxy-substituted phenylbenzoic acids were oxidatively demethylated with low activity. However, no aromatic oxidation was observed with any of these substrates. Crystal structures of CYP199A4 with 4-(3'-methoxyphenyl)benzoic acid demonstrated that the substrate binding mode was like that of 4-phenylbenzoic acid. 4-Phenoxy- and 4-benzoyl-benzoic acid bound with the ether or ketone oxygen atom hydrogen-bonded to the heme aqua ligand. We also investigated whether the substitution of phenylalanine residues in the active site could permit aromatic hydroxylation. Mutagenesis of the F298 residue to a valine did not significantly alter the substrate binding position or enable the aromatic oxidation of 4-phenylbenzoic acid however the F182L mutant was able to catalyse 4-phenylbenzoic acid oxidation generating 2'-hydroxy-, 3'-hydroxy- and 4'-hydroxy metabolites in a 83 : 9 : 8 ratio, respectively. Molecular dynamics simulations, in which the distance and angle of attack were considered, demonstrated that in the F182L variant, in contrast to the wild-type enzyme, the phenyl ring of 4-phenylbenzoic acid attained a productive geometry for aromatic oxidation to occur.
Publisher: Wiley
Date: 05-06-2014
Abstract: There is a real need for simple structures that define a β-strand conformation, a secondary structure that is central to peptide-protein interactions. For ex le, protease substrates and inhibitors almost universally adopt this geometry on active site binding. A planar pyrrole is used to replace two amino acids of a peptide backbone to generate a simple macrocycle that retains the required geometry for active site binding. The resulting β-strand templates have reduced peptide character and provide potent protease inhibitors with the attachment of an appropriate amino aldehyde to the C-terminus. Picomolar inhibitors of cathepsin L and S are reported and the mode of binding of one ex le to the model protease chymotrypsin is defined by X-ray crystallography.
Publisher: Wiley
Date: 23-07-2018
Publisher: Proceedings of the National Academy of Sciences
Date: 20-05-2008
Abstract: Here, we demonstrate that a single biochemical assay is able to predict the tissue-selective pharmacology of an array of selective estrogen receptor modulators (SERMs). We describe an approach to classify estrogen receptor (ER) modulators based on dynamics of the receptor-ligand complex as probed with hydrogen/deuterium exchange (HDX) mass spectrometry. Differential HDX mapping coupled with cluster and discriminate analysis effectively predicted tissue-selective function in most, but not all, cases tested. We demonstrate that analysis of dynamics of the receptor–ligand complex facilitates binning of ER modulators into distinct groups based on structural dynamics. Importantly, we were able to differentiate small structural changes within ER ligands of the same chemotype. In addition, HDX revealed differentially stabilized regions within the ligand-binding pocket that may contribute to the different pharmacology phenotypes of the compounds independent of helix 12 positioning. In summary, HDX provides a sensitive and rapid approach to classify modulators of the estrogen receptor that correlates with their pharmacological profile.
Publisher: Portland Press Ltd.
Date: 10-07-2014
DOI: 10.1042/BJ20131618
Abstract: The bHLH (basic helix–loop–helix) PAS (Per/Arnt/Sim) transcription factor SIM1 (single-minded 1) is important for development and function of regions of the hypothalamus that regulate energy homoeostasis and the feeding response. Low-activity SIM1 variants have been identified in in iduals with severe early-onset obesity, but the underlying molecular causes of impaired function are unknown. In the present study we assess a number of human SIM1 variants with reduced activity and determine that impaired function is frequently due to defects in dimerization with the essential partner protein ARNT2 (aryl hydrocarbon nuclear translocator 2). Equivalent variants generated in the highly related protein SIM2 (single-minded 2) produce near-identical impaired function and dimerization defects, indicating that these effects are not unique to the structure of SIM1. On the basis of these data, we predict that other select SIM1 and SIM2 variants reported in human genomic databases will also be deficient in activity, and identify two new low-activity SIM1 variants (V290E and V326F) present in the population. The cumulative data is used in homology modelling to make novel observations about the dimerization interface between the PAS domains of SIM1 and ARNT2, and to define a mutational ‘hot-spot’ in SIM1 that is critical for protein function.
Publisher: American Chemical Society (ACS)
Date: 12-04-2023
DOI: 10.1021/JACS.3C01456
Publisher: American Chemical Society (ACS)
Date: 06-02-2023
Publisher: Elsevier BV
Date: 08-2017
DOI: 10.1016/J.BBAGEN.2017.05.008
Abstract: Thiazolidinedione (TZD) compounds targeting the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) demonstrate unique benefits for the treatment of insulin resistance and type II diabetes. TZDs include rosiglitazone, pioglitazone and rivoglitazone, with the latter being the most potent. The TZDs are only marginally selective for the therapeutic target PPARγ as they also activate PPARα and PPARδ homologues to varying degrees, causing off-target effects. While crystal structures for TZD compounds in complex with PPARγ are available, minimal structural information is available for TZDs bound to PPARα and PPARδ. This paucity of structural information has h ered the determination of precise structural mechanisms involved in TZD selectivity between PPARs. To help address these questions molecular dynamic simulations were performed of rosiglitazone, pioglitazone and rivoglitazone in complex with PPARα, PPARδ, and PPARγ in order to better understand the mechanisms of PPAR selectivity. The simulations revealed that TZD interactions with residues Tyr314 and Phe318 of PPARα and residues Phe291 and Thr253 of PPARδ as well as the omega loop, are key determinants of TZD receptor selectivity. Notably, in this study, we solve the first X-ray crystal structure of rivoglitazone bound to any PPAR. Rivoglitazone forms a unique hydrogen bond network with the residues of the PPARγ co-activator binding surface (known as AF2) and makes more extensive contacts with helix 3 and the β-sheet as compared to model TZD compounds such as rosiglitazone.
Publisher: Wiley
Date: 11-11-2022
Abstract: The cytochrome P450 family of monooxygenase enzymes have essential biological roles involving the selective oxidation of carbon‐hydrogen bonds. They can also catalyze other important metabolic reactions including desaturation to form alkenes. Currently the factors that control the partition between P450 hydroxylation and desaturation pathways are poorly defined. The CYP199A4 enzyme from the bacterium Rhodopseudomonas palustris HaA2 catalyzes the oxidation of 4‐ethyl‐ and 4‐isopropyl‐ benzoic acids with hydroxylation and desaturation occurring in significant quantities. Here we demonstrate that 4‐cyclopropylbenzoic acid is regioselectively hydroxylated by CYP199A4 at the benzylic carbon. In contrast, the oxidation of 4‐ n ‐propylbenzoic acid by CYP199A4 results in three major metabolites: an alkene from desaturation and two hydroxylation products at the benzylic (Cα) and Cβ carbons in similar quantities. Extending the length of the alkyl substituent resulted in 4‐ n ‐butylbenzoic acid being oxidized at the benzylic position (45%) and desaturated (55%). In contrast, 4‐isobutylbenzoic generated very little alkene (5%) but was hydroxylated at the benzylic position (54%) and at the tertiary Cβ position (41%). The oxidation of 4‐ n ‐propylbenzoic acid by the F298 V mutant of CYP199A4 occurred with no hydroxylation at Cβ and a significant increase in metabolites arising from desaturation (73%). The X‐ray crystal structures of CYP199A4 with each substrate revealed that they bind in the active site with the alkyl substituent positioned over the heme. However, the longer alkylbenzoic acids were bound in a different conformation as was 4‐ n ‐propylbenzoic acid in the F298 V mutant. Overall, the changes in metabolite distribution could be ascribed to bond strength differences and the position of the alkyl group relative to the heme.
Publisher: Cold Spring Harbor Laboratory
Date: 11-04-2020
DOI: 10.1101/2020.04.09.034942
Abstract: Monitoring the mutation dynamics of SARS-CoV-2 is critical for the development of effective approaches to contain the pathogen. By analyzing 106 SARS-CoV-2 and 39 SARS genome sequences, we provided direct genetic evidence that SARS-CoV-2 has a much lower mutation rate than SARS. Minimum Evolution phylogeny analysis revealed the putative original status of SARS-CoV-2 and the early-stage spread history. The discrepant phylogenies for the spike protein and its receptor binding domain proved a previously reported structural rearrangement prior to the emergence of SARS-CoV-2. Despite that we found the spike glycoprotein of SARS-CoV-2 is particularly more conserved, we identified a receptor binding domain mutation that leads to weaker ACE2 binding capability based on in silico simulation, which concerns a SARS-CoV-2 s le collected on 27 th January 2020 from India. This represents the first report of a significant SARS-CoV-2 mutant, and requires attention from researchers working on vaccine development around the world. Based on the currently available genome sequence data, we provided direct genetic evidence that the SARS-COV-2 genome has a much lower mutation rate and genetic ersity than SARS during the 2002-2003 outbreak. The spike (S) protein encoding gene of SARS-COV-2 is found relatively more conserved than other protein-encoding genes, which is a good indication for the ongoing antiviral drug and vaccine development. Minimum Evolution phylogeny analysis revealed the putative original status of SARS-CoV-2 and the early-stage spread history. We confirmed a previously reported rearrangement in the S protein arrangement of SARS-COV-2, and propose that this rearrangement should have occurred between human SARS-CoV and a bat SARS-CoV, at a time point much earlier before SARS-COV-2 transmission to human. We provided first evidence that a mutated SARS-COV-2 with reduced human ACE2 receptor binding affinity have emerged in India based on a s le collected on 27th January 2020.
Publisher: American Chemical Society (ACS)
Date: 26-07-2022
DOI: 10.1021/ACSINFECDIS.2C00215
Abstract: The steroid binding CYP142 cytochrome P450 enzymes of
Publisher: Elsevier BV
Date: 05-2019
DOI: 10.1016/J.BBAGEN.2019.02.016
Abstract: Actinobacteria, including the Mycobacteria, have a large component of cytochrome P450 family monooxygenases. This includes Mycobacterium tuberculosis, M. ulcerans and M. marinum, and M. vanbaalenii. These enzymes can abstract CH bonds and have important roles in natural product biosynthesis. Two members of the bacterial CYP150 family, CYP150A5 and CYP150A6 from M. marinum, were produced, purified and characterised. The potential substrate ranges of both enzymes were analysed and the monooxygenase activity of CYP150A5 was reconstituted using a physiological electron transfer partner system. CYP150A6 was structurally characterised by X-ray crystallography. CYP150A5 was shown to bind various norisoprenoids and terpenoids. It could regioselectively hydroxylate β-ionol. The X-ray crystal structure of substrate-free CYP150A6 was solved to 1.5 Å. This displayed an open conformation with short F and G helices, an unresolved F-G loop region and exposed active site pocket. The active site residues could be identified and important variations were found among the CYP150A enzymes. Haem-binding azole inhibitors were identified for both enzymes. The structure of CYP150A6 will facilitate the identification of physiological substrates and the design of better inhibitors for members of this P450 family. Based on the observed differences in substrate binding preference and sequence variations among the active site residues, their roles are predicted to be different. Multiple CYP150 family members were found in many bacteria and are prevalent in the Mycobacteria including several human pathogens. Inhibition and structural data are reported here for these enzymes for the first time.
Publisher: American Chemical Society (ACS)
Date: 18-11-2022
Location: United States of America
No related grants have been discovered for John Bruning.