I03-Macromolecular Crystallography
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Yu
Liu
,
Ganka
Bineva-Todd
,
Richard W.
Meek
,
Laura
Mazo
,
Beatriz
Piniello
,
Olga
Moroz
,
Sean A.
Burnap
,
Nadima
Begum
,
André
Ohara
,
Chloe
Roustan
,
Sara
Tomita
,
Svend
Kjaer
,
Karen
Polizzi
,
Weston B.
Struwe
,
Carme
Rovira
,
Gideon J.
Davies
,
Benjamin
Schumann
Diamond Proposal Number(s):
[32736]
Open Access
Abstract: Correct elaboration of N-linked glycans in the secretory pathway of human cells is essential in physiology. Early N-glycan biosynthesis follows an assembly line principle before undergoing crucial elaboration points that feature the sequential incorporation of the sugar N-acetylglucosamine (GlcNAc). The activity of GlcNAc transferase V (MGAT5) primes the biosynthesis of an N-glycan antenna that is heavily upregulated in cancer. Still, the functional relevance and substrate choice of MGAT5 are ill-defined. Here, we employ protein engineering to develop a bioorthogonal substrate analog for the activity of MGAT5. Chemoenzymatic synthesis is used to produce a collection of nucleotide-sugar analogs with bulky, bioorthogonal acylamide side chains. We find that WT-MGAT5 displays considerable activity toward such substrate analogues. Protein engineering yields an MGAT5 variant that loses activity against the native nucleotide sugar and increases activity toward a 4-azidobutyramide-containing substrate analogue. By such restriction of substrate specificity, we show that the orthogonal enzyme–substrate pair is suitable to bioorthogonally tag glycoproteins. Through X-ray crystallography and molecular dynamics simulations, we establish the structural basis of MGAT5 engineering, informing the design rules for bioorthogonal precision chemical tools.
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Oct 2024
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Roi
Asor
,
Anna
Olerinyova
,
Sean A.
Burnap
,
Manish S.
Kushwah
,
Fabian
Soltermann
,
Lucas S. P.
Rudden
,
Mario
Hensen
,
Mario
Hensen
,
Snežana
Vasiljevic
,
Juliane
Brun
,
Michelle
Hill
,
Liu
Chang
,
Wanwisa
Dejnirattisai
,
Piyada
Supasa
,
Juthathip
Mongkolsapaya
,
Daming
Zhou
,
David I.
Stuart
,
Gavin R.
Screaton
,
Matteo T.
Degiacomi
,
Nicole
Zitzmann
,
Justin L. P.
Benesch
,
Weston B.
Struwe
,
Philipp
Kukura
Open Access
Abstract: Cellular processes are controlled by the thermodynamics of the underlying biomolecular interactions. Frequently, structural investigations use one monomeric binding partner, while ensemble measurements of binding affinities generally yield one affinity representative of a 1:1 interaction, despite the majority of the proteome consisting of oligomeric proteins. For example, viral entry and inhibition in SARS-CoV-2 involve a trimeric spike surface protein, a dimeric angiotensin-converting enzyme 2 (ACE2) cell-surface receptor and dimeric antibodies. Here, we reveal that cooperativity correlates with infectivity and inhibition as opposed to 1:1 binding strength. We show that ACE2 oligomerizes spike more strongly for more infectious variants, while exhibiting weaker 1:1 affinity. Furthermore, we find that antibodies use induced oligomerization both as a primary inhibition mechanism and to enhance the effects of receptor-site blocking. Our results suggest that naive affinity measurements are poor predictors of potency, and introduce an antibody-based inhibition mechanism for oligomeric targets. More generally, they point toward a much broader role of induced oligomerization in controlling biomolecular interactions.
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Oct 2024
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I04-Macromolecular Crystallography
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Lachlan P.
Deimel
,
Lucile
Moynie
,
Guoxuan
Sun
,
Viliyana
Lewis
,
Abigail
Turner
,
Charles J.
Buchanan
,
Sean A.
Burnap
,
Mikhail
Kutuzov
,
Carolin M.
Kobras
,
Yana
Demyaneko
,
Shabaz
Mohammed
,
Mathew
Stracy
,
Weston B.
Struwe
,
Andrew J.
Baldwin
,
James
Naismith
,
Benjamin G.
Davis
,
Quentin J.
Sattentau
Open Access
Abstract: Many archetypal and emerging classes of small-molecule therapeutics form covalent protein adducts. In vivo, both the resulting conjugates and their off-target side-conjugates have the potential to elicit antibodies, with implications for allergy and drug sequestration. Although β-lactam antibiotics are a drug class long associated with these immunological phenomena, the molecular underpinnings of off-target drug-protein conjugation and consequent drug-specific immune responses remain incomplete. Here, using the classical β-lactam penicillin G (PenG), we probe the B and T cell determinants of drug-specific IgG responses to such conjugates in mice. Deep B cell clonotyping reveals a dominant murine clonal antibody class encompassing phylogenetically-related IGHV1, IGHV5 and IGHV10 subgroup gene segments. Protein NMR and x-ray structural analyses reveal that these drive structurally convergent binding modes in adduct-specific antibody clones. Their common primary recognition mechanisms of the penicillin side-chain moiety (phenylacetamide in PenG)—regardless of CDRH3 length—limits cross-reactivity against other β-lactam antibiotics. This immunogenetics-guided discovery of the limited binding solutions available to antibodies against side products of an archetypal covalent inhibitor now suggests future potential strategies for the ‘germline-guided reverse engineering’ of such drugs away from unwanted immune responses.
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Aug 2024
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I04-Macromolecular Crystallography
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Suzana
Markolovic
,
Qinqin
Zhuang
,
Sarah E.
Wilkins
,
Charlotte D.
Eaton
,
Martine I.
Abboud
,
Maximiliano J.
Katz
,
Helen E.
Mcneil
,
Robert K.
Leśniak
,
Charlotte
Hall
,
Weston B.
Struwe
,
Rebecca
Konietzny
,
Simon
Davis
,
Ming
Yang
,
Wei
Ge
,
Justin L. P.
Benesch
,
Benedikt M.
Kessler
,
Peter J.
Ratcliffe
,
Matthew E.
Cockman
,
Roman
Fischer
,
Pablo
Wappner
,
Rasheduzzaman
Chowdhury
,
Mathew L.
Coleman
,
Christopher J.
Schofield
Abstract: Biochemical, structural and cellular studies reveal Jumonji-C (JmjC) domain-containing 7 (JMJD7) to be a 2-oxoglutarate (2OG)-dependent oxygenase that catalyzes (3S)-lysyl hydroxylation. Crystallographic analyses reveal JMJD7 to be more closely related to the JmjC hydroxylases than to the JmjC demethylases. Biophysical and mutation studies show that JMJD7 has a unique dimerization mode, with interactions between monomers involving both N- and C-terminal regions and disulfide bond formation. A proteomic approach identifies two related members of the translation factor (TRAFAC) family of GTPases, developmentally regulated GTP-binding proteins 1 and 2 (DRG1/2), as activity-dependent JMJD7 interactors. Mass spectrometric analyses demonstrate that JMJD7 catalyzes Fe(ii)- and 2OG-dependent hydroxylation of a highly conserved lysine residue in DRG1/2; amino-acid analyses reveal that JMJD7 catalyzes (3S)-lysyl hydroxylation. The functional assignment of JMJD7 will enable future studies to define the role of DRG hydroxylation in cell growth and disease.
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Jun 2018
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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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B21-High Throughput SAXS
I04-Macromolecular Crystallography
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Georg K. A.
Hochberg
,
Dale A.
Shepherd
,
Erik G.
Marklund
,
Indu
Santhanagoplan
,
Matteo T.
Degiacomi
,
Arthur
Laganowsky
,
Timothy M.
Allison
,
Eman
Basha
,
Michael T.
Marty
,
Martin R.
Galpin
,
Weston B.
Struwe
,
Andrew J.
Baldwin
,
Elizabeth
Vierling
,
Justin L. P.
Benesch
Diamond Proposal Number(s):
[9384]
Abstract: Oligomeric proteins assemble with exceptional selectivity, even in the presence of closely related proteins, to perform their cellular roles. We show that most proteins related by gene duplication of an oligomeric ancestor have evolved to avoid hetero-oligomerization and that this correlates with their acquisition of distinct functions. We report how coassembly is avoided by two oligomeric small heat-shock protein paralogs. A hierarchy of assembly, involving intermediates that are populated only fleetingly at equilibrium, ensures selective oligomerization. Conformational flexibility at noninterfacial regions in the monomers prevents coassembly, allowing interfaces to remain largely conserved. Homomeric oligomers must overcome the entropic benefit of coassembly and, accordingly, homomeric paralogs comprise fewer subunits than homomers that have no paralogs.
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Feb 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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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Alessandro T.
Caputo
,
Dominic S.
Alonzi
,
Lucia
Marti
,
Ida-Barbara
Reca
,
John
Kiappes
,
Weston B.
Struwe
,
Alice
Cross
,
Souradeep
Basu
,
Ed
Lowe
,
Benoit
Darlot
,
Angelo
Santino
,
Pietro
Roversi
,
Nicole
Zitzmann
Diamond Proposal Number(s):
[9306]
Abstract: The biosynthesis of enveloped viruses depends heavily on the host cell endoplasmic reticulum (ER) glycoprotein quality control (QC) machinery. This dependency exceeds the dependency of host glycoproteins, offering a window for the targeting of ERQC for the development of broad-spectrum antivirals. We determined small-angle X-ray scattering (SAXS) and crystal structures of the main ERQC enzyme, ER α-glucosidase II (α-GluII; from mouse), alone and in complex with key ligands of its catalytic cycle and antiviral iminosugars, including two that are in clinical trials for the treatment of dengue fever. The SAXS data capture the enzyme’s quaternary structure and suggest a conformational rearrangement is needed for the simultaneous binding of a monoglucosylated glycan to both subunits. The X-ray structures with key catalytic cycle intermediates highlight that an insertion between the +1 and +2 subsites contributes to the enzyme’s activity and substrate specificity, and reveal that the presence of d-mannose at the +1 subsite renders the acid catalyst less efficient during the cleavage of the monoglucosylated substrate. The complexes with iminosugar antivirals suggest that inhibitors targeting a conserved ring of aromatic residues between the α-GluII +1 and +2 subsites would have increased potency and selectivity, thus providing a template for further rational drug design.
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Aug 2016
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I02-Macromolecular Crystallography
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Jurgen
Brem
,
Weston B.
Struwe
,
Anna M.
Rydzik
,
Hanna
Tarhonskaya
,
Inga
Pfeffer
,
Emily
Flashman
,
Sander S.
Van Berkel
,
James
Spencer
,
Timothy D. W.
Claridge
,
Michael A.
Mcdonough
,
Justin L. P.
Benesch
,
Christopher J.
Schofield
Open Access
Abstract: Metallo-β-lactamases (MBLs) catalyse the hydrolysis of almost all β-lactam antibiotics. We report biophysical and kinetic studies on the São Paulo MBL (SPM-1), which reveal its Zn(II) ion usage and mechanism as characteristic of the clinically important di-Zn(II) dependent B1 MBL subfamily. Biophysical analyses employing crystallography, dynamic 19F NMR and ion mobility mass spectrometry, however, reveal that SPM-1 possesses loop and mobile element regions characteristic of the B2 MBLs. These include a mobile α3 region which is important in catalysis and determining inhibitor selectivity. SPM-1 thus appears to be a hybrid B1/B2 MBL. The results have implications for MBL evolution and inhibitor design.
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Oct 2014
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