I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Andrew
Chancellor
,
Robert A.
Simmons
,
Rahul C.
Khanolkar
,
Vladimir
Nosi
,
Aisha
Beshirova
,
Giuliano
Berloffa
,
Rodrigo
Colombo
,
Vijaykumar
Karuppiah
,
Johanne M.
Pentier
,
Vanessa
Tubb
,
Hemza
Ghadbane
,
Richard J.
Suckling
,
Keith
Page
,
Rory M.
Crean
,
Alessandro
Vacchini
,
Corinne
De Gregorio
,
Verena
Schaefer
,
Daniel
Constantin
,
Thomas
Gligoris
,
Angharad
Lloyd
,
Miriam
Hock
,
Velupillai
Srikannathasan
,
Ross A.
Robinson
,
Gurdyal S.
Besra
,
Marc W.
Van Der Kamp
,
Lucia
Mori
,
Raffaele
Calogero
,
David K.
Cole
,
Gennaro
De Libero
,
Marco
Lepore
Diamond Proposal Number(s):
[22870, 28224]
Abstract: Mucosal-associated invariant T (MAIT) cells use canonical semi-invariant T cell receptors (TCR) to recognize microbial riboflavin precursors displayed by the antigen-presenting molecule MR1. The extent of MAIT TCR crossreactivity toward physiological, microbially unrelated antigens remains underexplored. We describe MAIT TCRs endowed with MR1-dependent reactivity to tumor and healthy cells in the absence of microbial metabolites. MAIT cells bearing TCRs crossreactive toward self are rare but commonly found within healthy donors and display T-helper-like functions in vitro. Experiments with MR1-tetramers loaded with distinct ligands revealed significant crossreactivity among MAIT TCRs both ex vivo and upon in vitro expansion. A canonical MAIT TCR was selected on the basis of extremely promiscuous MR1 recognition. Structural and molecular dynamic analyses associated promiscuity to unique TCRβ-chain features that were enriched within self-reactive MAIT cells of healthy individuals. Thus, self-reactive recognition of MR1 represents a functionally relevant indication of MAIT TCR crossreactivity, suggesting a potentially broader role of MAIT cells in immune homeostasis and diseases, beyond microbial immunosurveillance.
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Sep 2023
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Lu
Zhang
,
Yao
Zhao
,
Yan
Gao
,
Lijie
Wu
,
Ruogu
Gao
,
Qi
Zhang
,
Yinan
Wang
,
Chengyao
Wu
,
Fangyu
Wu
,
Sudagar S.
Gurcha
,
Natacha
Veerapen
,
Sarah M.
Batt
,
Wei
Zhao
,
Ling
Qin
,
Xiuna
Yang
,
Manfu
Wang
,
Yan
Zhu
,
Bing
Zhang
,
Lijun
Bi
,
Xian’en
Zhang
,
Haitao
Yang
,
Luke W.
Guddat
,
Wenqing
Xu
,
Quan
Wang
,
Jun
Li
,
Gurdyal S.
Besra
,
Zihe
Rao
Abstract: The arabinosyltransferases EmbA, EmbB, and EmbC are involved in Mycobacterium tuberculosis cell wall synthesis and are recognized as the targets for the anti-tuberculosis drug ethambutol. We have determined cryo-electron microscopy and x-ray crystal structures of mycobacterial EmbA-EmbB and EmbC-EmbC complexes, in the presence of their glycosyl donor and acceptor substrates and with ethambutol. These structures show how the donor and acceptor substrates bind in the active site and how ethambutol inhibits by binding to the same site as both substrates in EmbB and EmbC. The majority of drug-resistant mutations are located nearby to the ethambutol-binding site. Collectively, our work provides a structural basis for understanding the biochemical function and inhibition of arabinosyltransferases and development of new anti-tuberculosis agents.
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Apr 2020
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[14692]
Open Access
Abstract: Growth and division by most bacteria requires remodelling and cleavage of their cell wall. A byproduct of this process is the generation of free peptidoglycan (PG) fragments known as muropeptides, which are recycled in many model organisms. Bacteria and hosts can harness the unique nature of muropeptides as a signal for cell wall damage and infection, respectively. Despite this critical role for muropeptides, it has long been thought that pathogenic mycobacteria such as Mycobacterium tuberculosis do not recycle their PG. Herein we show that M. tuberculosis and Mycobacterium bovis BCG are able to recycle components of their PG. We demonstrate that the core mycobacterial gene lpqI, encodes an authentic NagZ β-N-acetylglucosaminidase and that it is essential for PG-derived amino sugar recycling via an unusual pathway. Together these data provide a critical first step in understanding how mycobacteria recycle their peptidoglycan.
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Jun 2019
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I02-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[6388, 8359, 10369]
Open Access
Abstract: A growing body of evidence implicates the mycobacterial capsule - the outermost layer of the mycobacterial cell envelope - in modulation of the host immune response and virulence of mycobacteria. Mycobacteria synthesize the dominant capsule component, α(1→4)-linked glucan, via three interconnected, and potentially redundant metabolic pathways. Here, we report the crystal structure of the Mycobacterium smegmatis TreS-Pep2 complex, containing trehalose synthase (TreS) and maltokinase (Pep2), which convert trehalose to maltose-1-phosphate as part of the TreS-Pep2-GlgE pathway. The structure, at 3.6 Å resolution, revealed that a diamond-shaped TreS tetramer forms the core of the complex, and that pairs of Pep2 monomers bind to opposite apices of the tetramer in a 4+4 configuration. However, for the M. smegmatis orthologues, results from isothermal titration calorimetry and analytical ultracentrifugation experiments indicated that the prevalent stoichiometry in solution is 4 TreS + 2 Pep2 protomers. The observed discrepancy between the crystallised complex and the behaviour in the solution state may be explained by the relatively weak affinity of Pep2 for TreS (Kd 3.5 μM at mildly acidic pH) and crystal packing favouring the 4+4 complex. Proximity of the ATP binding site in Pep2 to the complex interface provides a rational basis for rate enhancement of Pep2 upon binding to TreS, but the complex structure appears to rule out substrate channeling between the active sites of TreS and Pep2. Our findings provide a structural model for the trehalose synthase-maltokinase complex in M. smegmatis that offers critical insights into capsule assembly.
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Mar 2019
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[14692]
Open Access
Abstract: The intracellular pathogen Mycobacterium tuberculosis is the causative agent of tuberculosis, which is a leading cause of mortality worldwide. The survival of M. tuberculosis in host macrophages through long-lasting periods of persistence depends, in part, on breaking down host cell lipids as a carbon source. The critical role of fatty-acid catabolism in this organism is underscored by the extensive redundancy of the genes implicated in β-oxidation (∼100 genes). In a previous study, the enzymology of the M. tuberculosis L-3-hydroxyacyl-CoA dehydrogenase FadB2 was characterized. Here, the crystal structure of this enzyme in a ligand-free form is reported at 2.1 Å resolution. FadB2 crystallized as a dimer with three unique dimer copies per asymmetric unit. The structure of the monomer reveals a dual Rossmann-fold motif in the N-terminal domain, while the helical C-terminal domain mediates dimer formation. Comparison with the CoA- and NAD+-bound human orthologue mitochondrial hydroxyacyl-CoA dehydrogenase shows extensive conservation of the residues that mediate substrate and cofactor binding. Superposition with the multi-catalytic homologue M. tuberculosis FadB, which forms a trifunctional complex with the thiolase FadA, indicates that FadB has developed structural features that prevent its self-association as a dimer. Conversely, FadB2 is unable to substitute for FadB in the tetrameric FadA–FadB complex as it lacks the N-terminal hydratase domain of FadB. Instead, FadB2 may functionally (or physically) associate with the enoyl-CoA hydratase EchA8 and the thiolases FadA2, FadA3, FadA4 or FadA6 as suggested by interrogation of the STRING protein-network database.
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Jan 2019
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I03-Macromolecular Crystallography
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Adrian
Richter
,
Ines
Rudolph
,
Ute
Möllmann
,
Kerstin
Voigt
,
Chun-Wa
Chung
,
Onkar M. P.
Singh
,
Michael
Rees
,
Alfonso
Mendoza-Losana
,
Robert
Bates
,
Lluís
Ballell
,
Sarah
Batt
,
Natacha
Veerapen
,
Klaus
Fütterer
,
Gurdyal
Besra
,
Peter
Imming
,
Argyrides
Argyrou
Diamond Proposal Number(s):
[12279]
Open Access
Abstract: Nitro-substituted 1,3-benzothiazinones (nitro-BTZs) are mechanism-based covalent inhibitors of Mycobacterium tuberculosis decaprenylphosphoryl-β-D-ribose-2′-oxidase (DprE1) with strong antimycobacterial properties. We prepared a number of oxidized and reduced forms of nitro-BTZs to probe the mechanism of inactivation of the enzyme and to identify opportunities for further chemistry. The kinetics of inactivation of DprE1 was examined using an enzymatic assay that monitored reaction progress up to 100 min, permitting compound ranking according to kinact/Ki values. The side-chain at the 2-position and heteroatom identity at the 1-position of the BTZs were found to be important for inhibitory activity. We obtained crystal structures with several compounds covalently bound. The data suggest that steps upstream from the covalent end-points are likely the key determinants of potency and reactivity. The results of protein mass spectrometry using a 7-chloro-nitro-BTZ suggest that nucleophilic reactions at the 7-position do not operate and support a previously proposed mechanism in which BTZ activation by a reduced flavin intermediate is required. Unexpectedly, a hydroxylamino-BTZ showed time-dependent inhibition and mass spectrometry corroborated that this hydroxylamino-BTZ is a mechanism-based suicide inhibitor of DprE1. With this BTZ derivative, we propose a new covalent mechanism of inhibition of DprE1 that takes advantage of the oxidation cycle of the enzyme.
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Sep 2018
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[8359]
Open Access
Abstract: Mycobacterium tuberculosis (Mtb) DprE1, an essential isomerase for the biosynthesis of the mycobacterial cell wall, is a validated target for tuberculosis (TB) drug development. Here we report the X-ray crystal structures of DprE1 and the DprE1 resistant mutant (Y314C) in complexes with TCA1 derivatives to elucidate the molecular basis of their inhibitory activities and an unconventional resistance mechanism, which enabled us to optimize the potency of the analogs. The selected lead compound showed excellent in vitro and in vivo activities, and low risk of toxicity profile except for the inhibition of CYP2C9. A crystal structure of CYP2C9 in complex with a TCA1 analog revealed the similar interaction patterns to the DprE1-TCA1 complex. Guided by the structures, an optimized molecule was generated with differential inhibitory activities against DprE1 and CYP2C9, which provides insights for development of a clinical candidate to treat TB.
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Aug 2017
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I04-Macromolecular Crystallography
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Katherine A.
Abrahams
,
Jonathan A. G.
Cox
,
Klaus
Futterer
,
Joaquín
Rullas
,
Fátima
Ortega-Muro
,
Nicholas J.
Loman
,
Patrick J.
Moynihan
,
Esther
Pérez-Herrán
,
Elena
Jiménez
,
Jorge
Esquivias
,
David
Barros
,
Lluís
Ballell
,
Carlos
Alemparte
,
Gurdyal S.
Besra
Open Access
Abstract: Drug discovery efforts against the pathogen Mycobacterium tuberculosis (Mtb) have been advanced through phenotypic screens of extensive compound libraries. Such a screen revealed sulfolane 1 and indoline-5-sulfonamides 2 and 3 as potent inhibitors of mycobacterial growth. Optimization in the sulfolane series led to compound 4, which has proven activity in an in vivo murine model of Mtb infection. Here we identify the target and mode of inhibition of these compounds based on whole genome sequencing of spontaneous resistant mutants, which identified mutations locating to the essential α- and β-subunits of tryptophan synthase. Over-expression studies confirmed tryptophan synthase as the biological target. Biochemical techniques probed the mechanism of inhibition, revealing the mutant enzyme complex incurs a fitness cost but does not prevent inhibitor binding. Mapping of the resistance conferring mutations onto a low-resolution crystal structure of Mtb tryptophan synthase showed they locate to the interface between the α- and β-subunits. The discovery of anti-tubercular agents inhibiting tryptophan synthase highlights the therapeutic potential of this enzyme and draws attention to the prospect of other amino acid biosynthetic pathways as future Mtb drug targets.
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Aug 2017
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I04-Macromolecular Crystallography
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Katherine A.
Abrahams
,
Chun-Wa
Chung
,
Sonja
Ghidelli-Disse
,
Joaquín
Rullas
,
María José
Rebollo-López
,
Sudagar S.
Gurcha
,
Jonathan A. G.
Cox
,
Alfonso
Mendoza
,
Elena
Jiménez-Navarro
,
María Santos
Martínez-Martínez
,
Margarete
Neu
,
Anthony
Shillings
,
Paul
Homes
,
Argyrides
Argyrou
,
Ruth
Casanueva
,
Nicholas J.
Loman
,
Patrick J.
Moynihan
,
Joël
Lelièvre
,
Carolyn
Selenski
,
Matthew
Axtman
,
Laurent
Kremer
,
Marcus
Bantscheff
,
Iñigo
Angulo-Barturen
,
Mónica Cacho
Izquierdo
,
Nicholas C.
Cammack
,
Gerard
Drewes
,
Lluis
Ballell
,
David
Barros
,
Gurdyal S.
Besra
,
Robert H.
Bates
Diamond Proposal Number(s):
[12279]
Open Access
Abstract: Phenotypic screens for bactericidal compounds are starting to yield promising hits against tuberculosis. In this regard, whole-genome sequencing of spontaneous resistant mutants generated against an indazole sulfonamide (GSK3011724A) identifies several specific single-nucleotide polymorphisms in the essential Mycobacterium tuberculosis β-ketoacyl synthase (kas) A gene. Here, this genomic-based target assignment is confirmed by biochemical assays, chemical proteomics and structural resolution of a KasA-GSK3011724A complex by X-ray crystallography. Finally, M. tuberculosis GSK3011724A-resistant mutants increase the in vitro minimum inhibitory concentration and the in vivo 99% effective dose in mice, establishing in vitro and in vivo target engagement. Surprisingly, the lack of target engagement of the related β-ketoacyl synthases (FabH and KasB) suggests a different mode of inhibition when compared with other Kas inhibitors of fatty acid biosynthesis in bacteria. These results clearly identify KasA as the biological target of GSK3011724A and validate this enzyme for further drug discovery efforts against tuberculosis.
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Sep 2016
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[6388]
Open Access
Abstract: Mycobacterium tuberculosis (Mtb), the aetiological agent of tuberculosis, has evolved to scavenge nutrients from the confined environment of host macrophages with mycobacterial ATP-binding cassette (ABC) transporters playing a key role in nutrient acquisition. Mtb-UspC (Rv2318) is the solute-binding protein of the essential transporter UspABC, one of four Mtb ABC transporters implicated by homology in sugar acquisition. Herein, we report the structural and functional characterization of Mtb-UspC. The 1.5 Å resolution structure of UspC reveals a two subdomain architecture that forms a highly acidic carbohydrate-substrate binding cleft. This has allowed a distinct preference of Mtb-UspC for amino sugars as determined by thermal shift analysis and solution saturation transfer difference-NMR. Taken together our data support the functional assignment of UspABC as an amino-sugar transporter. Given the limited availability of carbohydrates within the phagosomal environmental niche during Mtb intracellular infection, our studies suggest that UspABC enables Mtb to optimize the use of scarce nutrients during intracellular infection, linking essentiality of this protein to a potential role in recycling components of cell-wall peptidoglycan.
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Jun 2016
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