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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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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[22717, 15433, 19301]
Open Access
Abstract: SHIP1, an inositol 5-phosphatase, plays a central role in cellular signaling. As such, it has been implicated in many conditions. Exploiting SHIP1 as a drug target will require structural knowledge and the design of selective small molecules. We have determined apo, and magnesium and phosphate-bound structures of the phosphatase and C2 domains of SHIP1. The C2 domains of SHIP1 and the related SHIP2 modulate the activity of the phosphatase domain. To understand the mechanism, we performed activity assays, hydrogen-deuterium exchange mass spectrometry, and molecular dynamics on SHIP1 and SHIP2. Our findings demonstrate that the influence of the C2 domain is more pronounced for SHIP2 than SHIP1. We determined 91 structures of SHIP1 with fragments bound, with some near the interface between the two domains. We performed a mass spectrometry screen and determined four structures with covalent fragments. These structures could act as starting points for the development of potent, selective probes.
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Feb 2024
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B23-Circular Dichroism
I24-Microfocus Macromolecular Crystallography
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Ryan M.
Lithgo
,
Marko
Hanževački
,
Gemma
Harris
,
Jos J. A. G.
Kamps
,
Ellie
Holden
,
Tiberiu-Marius
Gianga
,
Justin L. P.
Benesch
,
Christof M.
Jäger
,
Anna K.
Croft
,
Rohanah
Hussain
,
Jon L.
Hobman
,
Allen M.
Orville
,
Andrew
Quigley
,
Stephen B.
Carr
,
David J.
Scott
Open Access
Abstract: The periplasmic chaperone SilF has been identified as part of an Ag(I) detoxification system in Gram negative bacteria. Sil proteins also bind Cu(I), but with reported weaker affinity, therefore leading to the designation of a specific detoxification system for Ag(I). Using isothermal titration calorimetry we show that binding of both ions is not only tighter than previously thought, but of very similar affinities. We investigated the structural origins of ion binding using molecular dynamics and QM/MM simulations underpinned by structural and biophysical experiments. The results of this analysis showed that the binding site adapts to accommodate either ion, with key interactions with the solvent in the case of Cu(I). The implications of this are that Gram negative bacteria do not appear to have evolved a specific Ag(I) efflux system but take advantage of the existing Cu(I) detoxification system. Therefore, there are consequences for how we define a particular metal resistance mechanism and understand its evolution in the environment.
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Oct 2023
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Miranda P.
Collier
,
T. Reid
Alderson
,
Carin P.
De Villiers
,
Daisy
Nicholls
,
Heidi Y.
Gastall
,
Timothy
Allison
,
Matteo T.
Degiacomi
,
He
Jiang
,
Georg
Mlynek
,
Dieter O.
Fürst
,
Peter F. M.
Van Der Ven
,
Kristina
Djinovic-Carugo
,
Andrew J.
Baldwin
,
Hugh
Watkins
,
Katja
Gehmlich
,
Justin L. P.
Benesch
Diamond Proposal Number(s):
[12346]
Open Access
Abstract: Mechanical force–induced conformational changes in proteins underpin a variety of physiological functions, typified in muscle contractile machinery. Mutations in the actin-binding protein filamin C (FLNC) are linked to musculoskeletal pathologies characterized by altered biomechanical properties and sometimes aggregates. HspB1, an abundant molecular chaperone, is prevalent in striated muscle where it is phosphorylated in response to cues including mechanical stress. We report the interaction and up-regulation of both proteins in three mouse models of biomechanical stress, with HspB1 being phosphorylated and FLNC being localized to load-bearing sites. We show how phosphorylation leads to increased exposure of the residues surrounding the HspB1 phosphosite, facilitating their binding to a compact multidomain region of FLNC proposed to have mechanosensing functions. Steered unfolding of FLNC reveals that its extension trajectory is modulated by the phosphorylated region of HspB1. This may represent a posttranslationally regulated chaperone-client protection mechanism targeting over-extension during mechanical stress.
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May 2019
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[9384]
Open Access
Abstract: Small heat-shock proteins (sHsps) are ubiquitous molecular chaperones, and sHsp mutations or altered expression are linked to multiple human disease states. sHsp monomers assemble into large oligomers with dimeric substructure, and the dynamics of sHsp oligomers has led to major questions about the form that captures substrate, a critical aspect of their mechanism of action. We show here that sub-structural dimers of two plant dodecameric sHsps, Ta16.9 and homologous Ps18.1, are functional units in the initial encounter with unfolding substrate. We introduced inter-polypeptide disulfide bonds at the two dodecameric interfaces, dimeric and nondimeric, to restrict how their assemblies can dissociate. When disulfide bonded at the non-dimeric interface, mutants of Ta16.9 and Ps18.1 (TaCT-ACD and PsCT-ACD) were inactive, but when reduced, had wildtype-like chaperone activity, demonstrating that dissociation at non-dimeric interfaces is essential for sHsp activity. Moreover, the size of the TaCT-ACD and PsCT-ACD covalent unit defined a new tetrahedral geometry for these sHsps, different from that observed in the Ta16.9 X-ray structure. Importantly, oxidized Tadimer (disulfide bonded at the dimeric interface) exhibited greatly enhanced ability to protect substrate, indicating that strengthening the dimeric interface increases chaperone efficiency. Temperature-induced size and secondary structure changes revealed that folded sHsp dimers interact with substrate and that dimer stability affects chaperone efficiency. These results yield a model in which sHsp dimers capture substrate before assembly into larger, heterogeneous sHsp–substrate complexes for substrate refolding or degradation and suggest that tuning the strength of the dimer interface can be used to engineer sHsp chaperone efficiency.
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Oct 2018
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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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I02-Macromolecular Crystallography
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Alice
Clark
,
Wilma Vree
Egberts
,
Frances D. L.
Kondrat
,
Gillian R.
Hilton
,
Nicholas J.
Ray
,
Ambrose R.
Cole
,
John A.
Carver
,
Justin L. P.
Benesch
,
Nicholas
Keep
,
Wilbert C.
Boelens
,
Christine
Slingsby
Diamond Proposal Number(s):
[7197]
Open Access
Abstract: Heterogeneity in small heat shock proteins (sHsps) spans multiple spatiotemporal regimes – from fast fluctuations of part of the protein, to conformational variability of tertiary structure, plasticity of the interfaces, and polydispersity of the inter-converting, and co-assembling oligomers. This heterogeneity and dynamic nature of sHsps has significantly hindered their structural characterisation. Atomic-coordinates are particularly lacking for vertebrate sHsps, where most available structures are of extensively truncated homomers. sHsps play important roles in maintaining protein levels in the cell and therefore in organismal health and disease. HspB2 and HspB3 are vertebrate sHsps that are found co-assembled in neuromuscular cells, and variants thereof are associated with disease. Here, we present the structure of human HspB2/B3, which crystallised as a hetero-tetramer in a 3:1 ratio. In the HspB2/B3 tetramer, the four α-crystallin domains (ACDs) assemble into a flattened tetrahedron which is pierced by two non-intersecting approximate dyads. Assembly is mediated by flexible “nuts and bolts” involving IXI/V motifs from terminal regions filling ACD pockets. Parts of the N-terminal region bind in an unfolded conformation into the anti-parallel shared ACD dimer grooves. Tracts of the terminal regions are not resolved, most likely due to their disorder in the crystal lattice. This first structure of a full-length human sHsp heteromer reveals the heterogeneous interactions of the terminal regions and suggests a plasticity that is important for the cytoprotective functions of sHsps.
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Jun 2018
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B21-High Throughput SAXS
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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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I24-Microfocus Macromolecular Crystallography
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Christos
Pliotas
,
Samuel C.
Grayer
,
Silvia
Ekkerman
,
Anthony K. N.
Chan
,
Jess
Healy
,
Phedra
Marius
,
Wendy
Bartlett
,
Amjad
Khan
,
Wilian A.
Cortopassi
,
Shane A.
Chandler
,
Tim
Rasmussen
,
Justin L. P.
Benesch
,
Robert S.
Paton
,
Timothy D. W.
Claridge
,
Samantha
Miller
,
Ian R.
Booth
,
James
Naismith
,
Stuart J.
Conway
Open Access
Abstract: Ligand binding is one of the most fundamental properties of proteins. Ligand functions fall into three basic types: substrates, regulatory molecules, and cofactors essential to protein stability, reactivity, or enzyme–substrate complex formation. The regulation of potassium ion movement in bacteria is predominantly under the control of regulatory ligands that gate the relevant channels and transporters, which possess subunits or domains that contain Rossmann folds (RFs). Here we demonstrate that adenosine monophosphate (AMP) is bound to both RFs of the dimeric bacterial Kef potassium efflux system (Kef), where it plays a structural role. We conclude that AMP binds with high affinity, ensuring that the site is fully occupied at all times in the cell. Loss of the ability to bind AMP, we demonstrate, causes protein, and likely dimer, instability and consequent loss of function. Kef system function is regulated via the reversible binding of comparatively low-affinity glutathione-based ligands at the interface between the dimer subunits. We propose this interfacial binding site is itself stabilized, at least in part, by AMP binding.
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Aug 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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