I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[29835]
Open Access
Abstract: The IgG-specific endoglycosidases EndoS and EndoS2 from Streptococcus pyogenes can remove conserved N-linked glycans present on the Fc region of host antibodies to inhibit Fc-mediated effector functions. These enzymes are therefore being investigated as therapeutics for suppressing unwanted immune activation, and have additional application as tools for antibody glycan remodelling. EndoS and EndoS2 differ in Fc glycan substrate specificity due to structural differences within their catalytic glycosyl hydrolase domains. However, a chimeric EndoS enzyme with a substituted glycosyl hydrolase from EndoS2 loses catalytic activity, despite high structural homology between the two enzymes, indicating either mechanistic divergence of EndoS and EndoS2, or improperly-formed domain interfaces in the chimeric enzyme. Here, we present the crystal structure of the EndoS2-IgG1 Fc complex determined to 3.0 Å resolution. Comparison of complexed and unliganded EndoS2 reveals relative reorientation of the glycosyl hydrolase, leucine-rich repeat and hybrid immunoglobulin domains. The conformation of the complexed EndoS2 enzyme is also different when compared to the earlier EndoS-IgG1 Fc complex, and results in distinct contact surfaces between the two enzymes and their Fc substrate. These findings indicate mechanistic divergence of EndoS2 and EndoS. It will be important to consider these differences in the design of IgG-specific endoglycosidases, developed to enable customisable antibody glycosylation.
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Apr 2024
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[29835]
Open Access
Abstract: Enzymatic cleavage of IgG antibodies is a common strategy used by pathogenic bacteria to ablate immune effector function. The Streptococcus pyogenes bacterium secretes the protease IdeS and the glycosidase EndoS, which specifically catalyse cleavage and deglycosylation of human IgG, respectively. IdeS has received clinical approval for kidney transplantation in hypersensitised individuals, while EndoS has found application in engineering antibody glycosylation. We present crystal structures of both enzymes in complex with their IgG1 Fc substrate, which was achieved using Fc engineering to disfavour preferential Fc crystallisation. The IdeS protease displays extensive Fc recognition and encases the antibody hinge. Conversely, the glycan hydrolase domain in EndoS traps the Fc glycan in a “flipped-out” conformation, while additional recognition of the Fc peptide is driven by the so-called carbohydrate binding module. In this work, we reveal the molecular basis of antibody recognition by bacterial enzymes, providing a template for the development of next-generation enzymes.
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Dec 2022
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I04-Macromolecular Crystallography
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Giorgia
Chiodin
,
Joel D.
Allen
,
Dean J.
Bryant
,
Philip
Rock
,
Enrica A.
Martino
,
Beatriz
Valle-Argos
,
Patrick J.
Duriez
,
Yasunori
Watanabe
,
Isla
Henderson
,
James S.
Blachly
,
Katy J.
Mccann
,
Jonathan C.
Strefford
,
Graham
Packham
,
Teunis B. H.
Geijtenbeek
,
Carl G.
Figdor
,
George W.
Wright
,
Louis M.
Staudt
,
Richard
Burack
,
Thomas A.
Bowden
,
Max
Crispin
,
Freda K.
Stevenson
,
Francesco
Forconi
Diamond Proposal Number(s):
[14744]
Abstract: Glycosylation of the surface immunoglobulin (Ig) variable region is a remarkable follicular lymphoma–associated feature rarely seen in normal B cells. Here, we define a subset of diffuse large B-cell lymphomas (DLBCLs) that acquire N-glycosylation sites selectively in the Ig complementarity-determining regions (CDRs) of the antigen-binding sites. Mass spectrometry and X-ray crystallography demonstrate how the inserted glycans are stalled at oligomannose-type structures because they are buried in the CDR loops. Acquisition of sites occurs in ∼50% of germinal-center B-cell–like DLBCL (GCB-DLBCL), mainly of the genetic EZB subtype, irrespective of IGHV-D-J use. This markedly contrasts with the activated B-cell–like DLBCL Ig, which rarely has sites in the CDR and does not seem to acquire oligomannose-type structures. Acquisition of CDR-located acceptor sites associates with mutations of epigenetic regulators and BCL2 translocations, indicating an origin shared with follicular lymphoma. Within the EZB subtype, these sites are associated with more rapid disease progression and with significant gene set enrichment of the B-cell receptor, PI3K/AKT/MTORC1 pathway, glucose metabolism, and MYC signaling pathways, particularly in the fraction devoid of MYC translocations. The oligomannose-type glycans on the lymphoma cells interact with the candidate lectin dendritic cell–specific intercellular adhesion molecule 3 grabbing non-integrin (DC-SIGN), mediating low-level signals, and lectin-expressing cells form clusters with lymphoma cells. Both clustering and signaling are inhibited by antibodies specifically targeting the DC-SIGN carbohydrate recognition domain. Oligomannosylation of the tumor Ig is a posttranslational modification that readily identifies a distinct GCB-DLBCL category with more aggressive clinical behavior, and it could be a potential precise therapeutic target via antibody-mediated inhibition of the tumor Ig interaction with DC-SIGN–expressing M2-polarized macrophages.
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Oct 2021
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Joel D.
Allen
,
Himanshi
Chawla
,
Firdaus
Samsudin
,
Lorena
Zuzic
,
Aishwary Tukaram
Shivgan
,
Yasunori
Watanabe
,
Wan-Ting
He
,
Sean
Callaghan
,
Ge
Song
,
Peter
Yong
,
Philip J. M.
Brouwer
,
Yutong
Song
,
Yongfei
Cai
,
Helen M. E.
Duyvesteyn
,
Tomas
Malinauskas
,
Joeri
Kint
,
Paco
Pino
,
Maria J.
Wurm
,
Martin
Frank
,
Bing
Chen
,
David I.
Stuart
,
Rogier W.
Sanders
,
Raiees
Andrabi
,
Dennis R.
Burton
,
Sai
Li
,
Peter J.
Bond
,
Max
Crispin
Open Access
Abstract: A central tenet in the design of vaccines is the display of native-like antigens in the elicitation of protective immunity. The abundance of N-linked glycans across the SARS-CoV-2 spike protein is a potential source of heterogeneity among the many different vaccine candidates under investigation. Here, we investigate the glycosylation of recombinant SARS-CoV-2 spike proteins from five different laboratories and compare them against S protein from infectious virus, cultured in Vero cells. We find patterns that are conserved across all samples, and this can be associated with site-specific stalling of glycan maturation that acts as a highly sensitive reporter of protein structure. Molecular dynamics simulations of a fully glycosylated spike support a model of steric restrictions that shape enzymatic processing of the glycans. These results suggest that recombinant spike-based SARS-CoV-2 immunogen glycosylation reproducibly recapitulates signatures of viral glycosylation.
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Jul 2021
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Krios II-Titan Krios II at Diamond
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Yasunori
Watanabe
,
Luiza
Mendonca
,
Elizabeth R.
Allen
,
Andrew
Howe
,
Mercede
Lee
,
Joel D.
Allen
,
Himanshi
Chawla
,
David
Pulido
,
Francesca
Donnellan
,
Hannah
Davies
,
Marta
Ulaszewska
,
Sandra
Belij-Rammerstorfer
,
Susan
Morris
,
Anna-Sophia
Krebs
,
Wanwisa
Dejnirattisai
,
Juthathip
Mongkolsapaya
,
Piyada
Supasa
,
Gavin R.
Screaton
,
Catherine M.
Green
,
Teresa
Lambe
,
Peijun
Zhang
,
Sarah C.
Gilbert
,
Max
Crispin
Diamond Proposal Number(s):
[18477, 21005, 21004]
Open Access
Abstract: Vaccine development against the SARS-CoV-2 virus focuses on the principal target of the neutralizing immune response, the spike (S) glycoprotein. Adenovirus-vectored vaccines offer an effective platform for the delivery of viral antigen, but it is important for the generation of neutralizing antibodies that they produce appropriately processed and assembled viral antigen that mimics that observed on the SARS-CoV-2 virus. Here, we describe the structure, conformation, and glycosylation of the S protein derived from the adenovirus-vectored ChAdOx1 nCoV-19/AZD1222 vaccine. We demonstrate native-like post-translational processing and assembly, and reveal the expression of S proteins on the surface of cells adopting the trimeric prefusion conformation. The data presented here confirm the use of ChAdOx1 adenovirus vectors as a leading platform technology for SARS-CoV-2 vaccines.
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Apr 2021
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Abstract: The development of domain-exchanged antibodies offers a route to high-affinity targeting to clustered multivalent epitopes, such as those associated with viral infections and many cancers. One strategy to generate these antibodies is to introduce mutations into target antibodies to drive domain exchange using the only known naturally occurring domain-exchanged anti-HIV (anti-human immunodeficiency virus) IgG1 antibody, 2G12, as a template. Here, we show that domain exchange can be sensitively monitored by ion-mobility mass spectrometry and gas-phase collision-induced unfolding. Using native 2G12 and a mutated form that disrupts domain exchange such that it has a canonical IgG1 architecture (2G12 I19R), we show that the two forms can be readily distinguished by their unfolding profiles. Importantly, the same signature of domain exchange is observed for both intact antibody and isolated Fab fragments. The development of a mass spectrometric method to detect antibody domain exchange will enable rapid screening and selection of candidate antibodies engineered to exhibit this and other unusual quaternary antibody architectures.
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May 2018
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I02-Macromolecular Crystallography
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Diamond Proposal Number(s):
[10627]
Abstract: Transmission of hemorrhagic fever New World arenaviruses from their rodent reservoirs to human populations poses substantial public health and economic dangers. These zoonotic events are enabled by the specific interaction between the New World arenaviral attachment glycoprotein, GP1, and cell surface human transferrin receptor (hTfR1). Here, we present the structural basis for how a mouse-derived neutralizing antibody (nAb), OD01, disrupts this interaction by targeting the receptor-binding surface of the GP1 glycoprotein from Junín virus (JUNV), a hemorrhagic fever arenavirus endemic in central Argentina. Comparison of our structure with that of a previously reported nAb complex (JUNV GP1–GD01) reveals largely overlapping epitopes but highly distinct antibody-binding modes. Despite differences in GP1 recognition, we find that both antibodies present a key tyrosine residue, albeit on different chains, that inserts into a central pocket on JUNV GP1 and effectively mimics the contacts made by the host TfR1. These data provide a molecular-level description of how antibodies derived from different germline origins arrive at equivalent immunological solutions to virus neutralization.
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Jun 2017
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B21-High Throughput SAXS
I04-1-Macromolecular Crystallography (fixed wavelength)
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Jonathan T. S.
Hopper
,
Stephen
Ambrose
,
Oliver C.
Grant
,
Stefanie A.
Krumm
,
Timothy
Allison
,
Matteo T.
Degiacomi
,
Mark D.
Tully
,
Laura K.
Pritchard
,
Gabriel
Ozorowski
,
Andrew B.
Ward
,
Max
Crispin
,
Katie J.
Doores
,
Robert J.
Woods
,
Justin L. P.
Benesch
,
Carol V.
Robinson
,
Weston B.
Struwe
Diamond Proposal Number(s):
[9384]
Abstract: Select lectins have powerful anti-viral properties that effectively neutralize HIV-1 by targeting the dense glycan shield on the virus. Here, we reveal the mechanism by which one of the most potent lectins, BanLec, achieves its inhibition. We identify that BanLec recognizes a subset of high-mannose glycans via bidentate interactions spanning the two binding sites present on each BanLec monomer that were previously considered separate carbohydrate recognition domains. We show that both sites are required for high-affinity glycan binding and virus neutralization. Unexpectedly we find that BanLec adopts a tetrameric stoichiometry in solution whereby the glycan-binding sites are positioned to optimally target glycosylated viral spikes. The tetrameric architecture, together with bidentate binding to individual glycans, leads to layers of multivalency that drive viral neutralization through enhanced avidity effects. These structural insights will prove useful in engineering successful lectin therapeutics targeting the dense glycan shield of HIV.
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Apr 2017
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[8423]
Abstract: An emergent viral pathogen termed severe fever with thrombocytopenia syndrome virus (SFTSV) is responsible for thousands of clinical cases and associated fatalities in China, Japan, and South Korea. Akin to other phleboviruses, SFTSV relies on a viral glycoprotein, Gc, to catalyze the merger of endosomal host and viral membranes during cell entry. Here, we describe the postfusion structure of SFTSV Gc, revealing that the molecular transformations the phleboviral Gc undergoes upon host cell entry are conserved with otherwise unrelated alpha- and flaviviruses. By comparison of SFTSV Gc with that of the prefusion structure of the related Rift Valley fever virus, we show that these changes involve refolding of the protein into a trimeric state. Reverse genetics and rescue of site-directed histidine mutants enabled localization of histidines likely to be important for triggering this pH-dependent process. These data provide structural and functional evidence that the mechanism of phlebovirus–host cell fusion is conserved among genetically and patho-physiologically distinct viral pathogens.
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Jun 2016
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[8423]
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Sep 2013
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