I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[15304]
Abstract: Glycolipids play a central role in a variety of important biological processes in all living organisms. PatA is a membrane acyltransferase involved in the biosynthesis of phosphatidyl-myo-inositol mannosides (PIMs), key structural elements and virulence factors of Mycobacterium tuberculosis. PatA catalyzes the transfer of a palmitoyl moiety from palmitoyl-CoA to the 6-position of the mannose ring linked to the 2-position of inositol in PIM1/PIM2. We report here the crystal structure of PatA in the presence of 6-O-palmitoyl-α-D-mannopyranoside, unraveling the acceptor binding mechanism. The acceptor mannose ring localizes in a cavity at the end of a surface-exposed, long groove where the active site is located, whereas the palmitate moiety accommodates into a hydrophobic pocket deeply buried in the α/β core of the protein. Both fatty acyl chains of the PIM2 acceptor are essential for the reaction to take place, highlighting their critical role in the generation of a competent active site. By the use of combined structural and quantum-mechanics/molecular-mechanics (QM/MM) metadynamics we unravel the catalytic mechanism of PatA at the atomic-electronic level. Our study provides a detailed structural rationale for a stepwise reaction, with the generation of a tetrahedral transition state for the rate-determining step. Finally, the crystal structure of PatA in the presence of β-D-mannopyranose and palmitate suggest an inhibitory mechanism for the enzyme, providing exciting possibilities for inhibitor design and the discovery of chemotherapeutic agents against this major human pathogen.
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Nov 2017
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I03-Macromolecular Crystallography
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Matthew K.
Bilyard
,
Henry J.
Bailey
,
Lluís
Raich
,
Maria A.
Gafitescu
,
Takuya
Machida
,
Javier
Iglésias-fernández
,
Seung Seo
Lee
,
Christopher D.
Spicer
,
Carme
Rovira
,
Wyatt W.
Yue
,
Benjamin G.
Davis
Abstract: Biosynthesis of glycogen, the essential glucose (and hence energy) storage molecule in humans, animals and fungi1, is initiated by the glycosyltransferase enzyme, glycogenin (GYG). Deficiencies in glycogen formation cause neurodegenerative and metabolic disease2,3,4, and mouse knockout5 and inherited human mutations6 of GYG impair glycogen synthesis. GYG acts as a ‘seed core’ for the formation of the glycogen particle by catalysing its own stepwise autoglucosylation to form a covalently bound gluco-oligosaccharide chain at initiation site Tyr 195. Precise mechanistic studies have so far been prevented by an inability to access homogeneous glycoforms of this protein, which unusually acts as both catalyst and substrate. Here we show that unprecedented direct access to different, homogeneously glucosylated states of GYG can be accomplished through a palladium-mediated enzyme activation ‘shunt’ process using on-protein C–C bond formation. Careful mimicry of GYG intermediates recapitulates catalytic activity at distinct stages, which in turn allows discovery of triphasic kinetics and substrate plasticity in GYG’s use of sugar substrates. This reveals a tolerant but ‘proof-read’ mechanism that underlies the precision of this metabolic process. The present demonstration of direct, chemically controlled access to intermediate states of active enzymes suggests that such ligation-dependent activation could be a powerful tool in the study of mechanism.
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Oct 2018
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I03-Macromolecular Crystallography
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Matilde
De Las Rivas
,
Earnest James
Paul Daniel
,
Helena
Coelho
,
Erandi
Lira-navarrete
,
Lluis
Raich
,
Ismael
Compañón
,
Ana
Diniz
,
Laura
Lagartera
,
Jesús
Jiménez-barbero
,
Henrik
Clausen
,
Carme
Rovira
,
Filipa
Marcelo
,
Francisco
Corzana
,
Thomas A.
Gerken
,
Ramon
Hurtado-guerrero
Diamond Proposal Number(s):
[10121]
Open Access
Abstract: Mucin-type O-glycosylation is initiated by a family of polypeptide GalNAc-transferases (GalNAc-Ts) which are type-II transmembrane proteins that contain Golgi luminal catalytic and lectin domains that are connected by a flexible linker. Several GalNAc-Ts, including GalNAc-T4, show both long-range and short-range prior glycosylation specificity, governed by their lectin and catalytic domains, respectively. While the mechanism of the lectin-domain-dependent glycosylation is well-known, the molecular basis for the catalytic-domain-dependent glycosylation of glycopeptides is unclear. Herein, we report the crystal structure of GalNAc-T4 bound to the diglycopeptide GAT*GAGAGAGT*TPGPG (containing two α-GalNAc glycosylated Thr (T*), the PXP motif and a “naked” Thr acceptor site) that describes its catalytic domain glycopeptide GalNAc binding site. Kinetic studies of wild-type and GalNAc binding site mutant enzymes show the lectin domain GalNAc binding activity dominates over the catalytic domain GalNAc binding activity and that these activities can be independently eliminated. Surprisingly, a flexible loop protruding from the lectin domain was found essential for the optimal activity of the catalytic domain. This work provides the first structural basis for the short-range glycosylation preferences of a GalNAc-T.
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Sep 2018
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Andrew J.
Thompson
,
Gaetano
Speciale
,
Javier
Iglesias-fernández
,
Zalihe
Hakki
,
Tyson
Belz
,
Alan
Cartmell
,
Richard J.
Spears
,
Emily
Chandler
,
Max J.
Temple
,
Judith
Stepper
,
Harry J.
Gilbert
,
Carme
Rovira
,
Spencer J.
Williams
,
Gideon J.
Davies
Diamond Proposal Number(s):
[7864, 9948]
Abstract: carbohydrates;computational chemistry;conformational analysis;enzymatic mechanisms;glycosidase inhibitors
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Mar 2015
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Erandi
Lira-navarrete
,
Matilde
De Las Rivas
,
Ismael
Compañón
,
María Carmen
Pallarés
,
Yun
Kong
,
Javier
Iglesias-fernández
,
Gonçalo J. L.
Bernardes
,
Jesús M.
Peregrina
,
Carme
Rovira
,
Pau
Bernadó
,
Pierpaolo
Bruscolini
,
Henrik
Clausen
,
Anabel
Lostao
,
Francisco
Corzana
,
Ramon
Hurtado-guerrero
Diamond Proposal Number(s):
[8035, 10121]
Open Access
Abstract: Protein O-glycosylation is controlled by polypeptide GalNAc-transferases (GalNAc-Ts) that uniquely feature both a catalytic and lectin domain. The underlying molecular basis of how the lectin domains of GalNAc-Ts contribute to glycopeptide specificity and catalysis remains unclear. Here we present the first crystal structures of complexes of GalNAc-T2 with glycopeptides that together with enhanced sampling molecular dynamics simulations demonstrate a cooperative mechanism by which the lectin domain enables free acceptor sites binding of glycopeptides into the catalytic domain. Atomic force microscopy and small-angle X-ray scattering experiments further reveal a dynamic conformational landscape of GalNAc-T2 and a prominent role of compact structures that are both required for efficient catalysis. Our model indicates that the activity profile of GalNAc-T2 is dictated by conformational heterogeneity and relies on a flexible linker located between the catalytic and the lectin domains. Our results also shed light on how GalNAc-Ts generate dense decoration of proteins with O-glycans.
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May 2015
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Andrew
Thompson
,
Jerome
Dabin
,
Javier
Iglesias-fernández
,
Albert
Ardèvol
,
Zoran
Dinev
,
Spencer J.
Williams
,
Omprakash
Bande
,
Aloysius
Siriwardena
,
Carl
Moreland
,
Ting-chou
Hu
,
David K.
Smith
,
Harry J.
Gilbert
,
Carme
Rovira
,
Gideon J.
Davies
Abstract: Mannosidases are glycoside hydrolases that face special stereoelectronic challenges in effecting the hydrolysis of the glycosidic bond. In their Communication on page?10997?ff., C. Rovira, G.?J. Davies, and co-workers use QM/MM calculations, supported by X-ray structures of the enzyme with ligands mimicking the substrate, transition state, and product, to show that the free-energy landscape of an isolated alpha-mannoside is shaped on-enzyme into a single conformational itinerary along the reaction coordinate.
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Oct 2012
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Andrew J.
Thompson
,
Jerome
Dabin
,
Javier
Iglesias-fernández
,
Albert
Ardèvol
,
Zoran
Dinev
,
Spencer J.
Williams
,
Omprakash
Bande
,
Aloysius
Siriwardena
,
Carl
Moreland
,
Ting-chou
Hu
,
David K.
Smith
,
Harry J.
Gilbert
,
Carme
Rovira
,
Gideon J.
Davies
Open Access
Abstract: Mannosides in the southern hemisphere: Conformational analysis of enzymatic mannoside hydrolysis informs strategies for enzyme inhibition and inspires solutions to mannoside synthesis. Atomic resolution structures along the reaction coordinate of an inverting α-mannosidase show how the enzyme distorts the substrate and transition state. QM/MM calculations reveal how the free energy landscape of isolated α-D-mannose is molded on enzyme to only allow one conformationally accessible reaction coordinate.
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Oct 2012
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Andrew J.
Thompson
,
Gaetano
Speciale
,
Javier
Iglesias-fernández
,
Zalihe
Hakki
,
Tyson
Belz
,
Alan
Cartmell
,
Richard J.
Spears
,
Emily
Chandler
,
Max J.
Temple
,
Judith
Stepper
,
Harry J.
Gilbert
,
Carme
Rovira
,
Spencer J.
Williams
,
Gideon J.
Davies
Abstract: α-Mannosidases and α-mannanases have attracted attention for the insight they provide into nucleophilic substitution at the hindered anomeric center of α-mannosides, and the potential of mannosidase inhibitors as cellular probes and therapeutic agents. We report the conformational itinerary of the family GH76 α-mannanases studied through structural analysis of the Michaelis complex and synthesis and evaluation of novel aza/imino sugar inhibitors. A Michaelis complex in an OS2 conformation, coupled with distortion of an azasugar in an inhibitor complex to a high energy B2,5 conformation are rationalized through ab initio QM/MM metadynamics that show how the enzyme surface restricts the conformational landscape of the substrate, rendering the B2,5 conformation the most energetically stable on-enzyme. We conclude that GH76 enzymes perform catalysis using an itinerary that passes through OS2 and B2,5≠ conformations, information that should inspire the development of new antifungal agents.
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Apr 2015
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Sybrin P.
Schröder
,
Casper
De Boer
,
Nicholas G. S.
Mcgregor
,
Rhianna J.
Rowland
,
Olga
Moroz
,
Elena
Blagova
,
Jos
Reijngoud
,
Mark
Arentshorst
,
David
Osborn
,
Marc D.
Morant
,
Eric
Abbate
,
Mary A.
Stringer
,
Kristian B. R. M.
Krogh
,
Lluís
Raich
,
Carme
Rovira
,
Jean-guy
Berrin
,
Gilles P.
Van Wezel
,
Arthur F. J.
Ram
,
Bogdan I.
Florea
,
Gijsbert A.
Van Der Marel
,
Jeroen D. C.
Codée
,
Keith S.
Wilson
,
Liang
Wu
,
Gideon J.
Davies
,
Herman S.
Overkleeft
Diamond Proposal Number(s):
[13587]
Abstract: Plant polysaccharides represent a virtually unlimited feedstock for the generation of biofuels and other commodities. However, the extraordinary recalcitrance of plant polysaccharides toward breakdown necessitates a continued search for enzymes that degrade these materials efficiently under defined conditions. Activity-based protein profiling provides a route for the functional discovery of such enzymes in complex mixtures and under industrially relevant conditions. Here, we show the detection and identification of β-xylosidases and endo-β-1,4-xylanases in the secretomes of Aspergillus niger, by the use of chemical probes inspired by the β-glucosidase inhibitor cyclophellitol. Furthermore, we demonstrate the use of these activity-based probes (ABPs) to assess enzyme–substrate specificities, thermal stabilities, and other biotechnologically relevant parameters. Our experiments highlight the utility of ABPs as promising tools for the discovery of relevant enzymes useful for biomass breakdown.
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May 2019
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[13587]
Abstract: The varied yet family-specific conformational pathways utilized by individual glycoside hydrolases (GHs) offer a tantalising prospect for the design of tight binding and specific enzyme inhibitors. A cardinal example of a GH family specific inhibitor, and one that finds widespread practical use, is the natural product kifunensine, which is a low nanomolar inhibitor selective for GH family 47 inverting a-mannosidases. Here we show, through quantum mechanical approaches, that kifunensine is restrained to a 'ring-flipped' 1C4 conformation with another accessible, but higher-energy, region around the 1,4B conformation. The conformations of kifunensine in complex with a range of GH47 enzymes including an atomic level (1 Å) resolution structure of kifunensine with Caulobacter sp. CkGH47 reported herein, and on GH family 38 and 92 a-mannosidases, were mapped onto the kifunensine free energy landscape. These studies revealed that kifunensine has the ability to mimic the product state of GH47 enzymes but cannot mimic any conformational states relevant to the reaction coordinate of mannosidases from other families.
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May 2017
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