I03-Macromolecular Crystallography
Krios II-Titan Krios II at Diamond
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James
Hillier
,
Yuguang
Zhao
,
Loic
Carrique
,
Tomas
Malinauskas
,
Reinis R.
Ruza
,
Tao-Hsin
Chang
,
Gangshun
Yi
,
Helen M. E.
Duyvesteyn
,
Jing
Yu
,
Weixian
Lu
,
Els
Pardon
,
Jan
Steyaert
,
Yanan
Zhu
,
Tao
Ni
,
E. Yvonne
Jones
Diamond Proposal Number(s):
[19946, 28713]
Open Access
Abstract: The Wnt receptor Frizzled3 (FZD3) is important for brain axonal development and cancer progression. We report structures of FZD3 in complex with extracellular and intracellular binding nanobodies (Nb). The crystal structure of Nb8 in complex with the FZD3 cysteine-rich domain (CRD) reveals that the nanobody binds at the base of the lipid-binding groove and can compete with Wnt5a. Nb8 fused with the Dickkopf-1 C-terminal domain behaves as a FZD3-specific Wnt surrogate, activating β-catenin signalling. The cryo-EM structure of FZD3 in complex with Nb9 reveals partially resolved density for the CRD, which exhibits positional flexibility, and a transmembrane conformation that resembles active GPCRs. Nb9 binds to the cytoplasmic region of FZD3 at the putative Dishevelled (DVL) or G protein-binding site, competes with DVL binding, and inhibits GαS coupling. In combination, our FZD3 structures with nanobody modulators map extracellular and intracellular interaction surfaces of functional, and potentially therapeutic, relevance.
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Aug 2024
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I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Baptiste
Fischer
,
Tomasz
Uchanski
,
Aidana
Sheryazdanova
,
Simon
Gonzalez
,
Alexander N.
Volkov
,
Elke
Brosens
,
Thomas
Zogg
,
Valentina
Kalichuk
,
Steven
Ballet
,
Wim
Versees
,
Anna A.
Sablina
,
Els
Pardon
,
Alexandre
Wohlkonig
,
Jan
Steyaert
Diamond Proposal Number(s):
[17150]
Open Access
Abstract: Protein-protein interactions (PPIs) are central in cell metabolism but research tools for the structural and functional characterization of these PPIs are often missing. Here we introduce broadly applicable immunization (Cross-link PPIs and immunize llamas, ChILL) and selection strategies (Display and co-selection, DisCO) for the discovery of diverse nanobodies that either stabilize or disrupt PPIs in a single experiment. We apply ChILL and DisCO to identify competitive, connective, or fully allosteric nanobodies that inhibit or facilitate the formation of the SOS1•RAS complex and modulate the nucleotide exchange rate on this pivotal GTPase in vitro as well as RAS signalling in cellulo. One of these connective nanobodies fills a cavity that was previously identified as the binding pocket for a series of therapeutic lead compounds. The long complementarity-determining region (CDR3) that penetrates this binding pocket serves as pharmacophore for extending the repertoire of potential leads.
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Jul 2024
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I24-Microfocus Macromolecular Crystallography
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Karin E. J.
Roedstroem
,
Alexander
Cloake
,
Janina
Sörmann
,
Agnese
Baronina
,
Kathryn H. M.
Smith
,
Ashley C. W.
Pike
,
Jackie
Ang
,
Peter
Proks
,
Marcus
Schewe
,
Ingelise
Holland-Kaye
,
Simon R.
Bushell
,
Jenna
Elliott
,
Els
Pardon
,
Thomas
Baukrowitz
,
Raymond J.
Owens
,
Simon
Newstead
,
Jan
Steyaert
,
Elisabeth P.
Carpenter
,
Stephen J.
Tucker
Diamond Proposal Number(s):
[15433, 19301]
Open Access
Abstract: Potassium channels of the Two-Pore Domain (K2P) subfamily, KCNK1-KCNK18, play crucial roles in controlling the electrical activity of many different cell types and represent attractive therapeutic targets. However, the identification of highly selective small molecule drugs against these channels has been challenging due to the high degree of structural and functional conservation that exists not only between K2P channels, but across the whole K+ channel superfamily. To address the issue of selectivity, here we generate camelid antibody fragments (nanobodies) against the TREK-2 (KCNK10) K2P K+ channel and identify selective binders including several that directly modulate channel activity. X-ray crystallography and CryoEM data of these nanobodies in complex with TREK-2 also reveal insights into their mechanisms of activation and inhibition via binding to the extracellular loops and Cap domain, as well as their suitability for immunodetection. These structures facilitate design of a biparatropic inhibitory nanobody with markedly improved sensitivity. Together, these results provide important insights into TREK channel gating and provide an alternative, more selective approach to modulation of K2P channel activity via their extracellular domains.
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May 2024
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I04-Macromolecular Crystallography
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Lorenzo
Maso
,
Filippo
Vascon
,
Monica
Chinellato
,
Frédéric
Goormaghtigh
,
Pierangelo
Bellio
,
Enrica
Campagnaro
,
Laurence
Van Melderen
,
Maria
Ruzzene
,
Els
Pardon
,
Alessandro
Angelini
,
Giuseppe
Celenza
,
Jan
Steyaert
,
Donatella
Tondi
,
Laura
Cendron
Diamond Proposal Number(s):
[21741]
Abstract: Antimicrobial resistance threatens the eradication of infectious diseases and impairs the efficacy of available therapeutics. The bacterial SOS pathway is a conserved response triggered by genotoxic stresses and represents one of the principal mechanisms that lead to resistance. The RecA recombinase acts as a DNA-damage sensor inducing the autoproteolysis of the transcriptional repressor LexA, thereby derepressing SOS genes that mediate DNA repair, survival to chemotherapy, and hypermutation. The inhibition of such pathway represents a promising strategy for delaying the evolution of antimicrobial resistance. We report the identification, via llama immunization and phage display, of nanobodies that bind LexA with sub-micromolar affinity and block autoproteolysis, repressing SOS response in Escherichia coli. Biophysical characterization of nanobody-LexA complexes revealed that they act by trapping LexA in an inactive conformation and interfering with RecA engagement. Our studies pave the way to the development of new-generation antibiotic adjuvants for the treatment of bacterial infections.
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Oct 2022
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Krios I-Titan Krios I at Diamond
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John J.
Kelly
,
Dale
Tranter
,
Els
Pardon
,
Gamma
Chi
,
Holger
Kramer
,
Lotta
Happonen
,
Kelly M.
Knee
,
Jay M.
Janz
,
Jan
Steyaert
,
Christine
Bulawa
,
Ville O.
Paavilainen
,
Juha T.
Huiskonen
,
Wyatt W.
Yue
Diamond Proposal Number(s):
[20223]
Open Access
Abstract: The integrity of a cell’s proteome depends on correct folding of polypeptides by chaperonins. The chaperonin TCP-1 ring complex (TRiC) acts as obligate folder for >10% of cytosolic proteins, including he cytoskeletal proteins actin and tubulin. Although its architecture and how it recognizes folding substrates are emerging from structural studies, the subsequent fate of substrates inside the TRiC chamber is not defined. We trapped endogenous human TRiC with substrates (actin, tubulin) and cochaperone (PhLP2A) at different folding stages, for structure determination by cryo-EM. The already-folded regions of client proteins are anchored at the chamber wall, positioning unstructured regions toward the central space to achieve their native fold. Substrates engage with different sections of the chamber during the folding cycle, coupled to TRiC open-and-close transitions. Further, the cochaperone PhLP2A modulates folding, acting as a molecular strut between substrate and TRiC chamber. Our structural snapshots piece together an emerging model of client protein folding within TRiC.
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Apr 2022
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[19946]
Open Access
Abstract: Influenza A viruses cause seasonal epidemics and global pandemics, representing a considerable burden to healthcare systems. Central to the replication cycle of influenza viruses is the viral RNA-dependent RNA polymerase which transcribes and replicates the viral RNA genome. The polymerase undergoes conformational rearrangements and interacts with viral and host proteins to perform these functions. Here we determine the structure of the 1918 influenza virus polymerase in transcriptase and replicase conformations using cryo-electron microscopy (cryo-EM). We then structurally and functionally characterise the binding of single-domain nanobodies to the polymerase of the 1918 pandemic influenza virus. Combining these functional and structural data we identify five sites on the polymerase which are sensitive to inhibition by nanobodies. We propose that the binding of nanobodies at these sites either prevents the polymerase from assuming particular functional conformations or interactions with viral or host factors. The polymerase is highly conserved across the influenza A subtypes, suggesting these sites as effective targets for potential influenza antiviral development.
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Jan 2022
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Krios I-Titan Krios I at Diamond
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Claire E.
Coupland
,
Sebastian A.
Andrei
,
T. Bertie
Ansell
,
Loic
Carrique
,
Pramod
Kumar
,
Lea
Sefer
,
Rebekka A.
Schwab
,
Eamon F. X.
Byrne
,
Els
Pardon
,
Jan
Steyaert
,
Anthony I.
Magee
,
Thomas
Lanyon-Hogg
,
Mark S. P.
Sansom
,
Edward W.
Tate
,
Christian
Siebold
Diamond Proposal Number(s):
[20223]
Open Access
Abstract: The Sonic Hedgehog (SHH) morphogen pathway is fundamental for embryonic development and stem cell maintenance and is implicated in various cancers. A key step in signaling is transfer of a palmitate group to the SHH N terminus, catalyzed by the multi-pass transmembrane enzyme Hedgehog acyltransferase (HHAT). We present the high-resolution cryo-EM structure of HHAT bound to substrate analog palmityl-coenzyme A and a SHH-mimetic megabody, revealing a heme group bound to HHAT that is essential for HHAT function. A structure of HHAT bound to potent small-molecule inhibitor IMP-1575 revealed conformational changes in the active site that occlude substrate binding. Our multidisciplinary analysis provides a detailed view of the mechanism by which HHAT adapts the membrane environment to transfer an acyl chain across the endoplasmic reticulum membrane. This structure of a membrane-bound O-acyltransferase (MBOAT) superfamily member provides a blueprint for other protein-substrate MBOATs and a template for future drug discovery.
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Dec 2021
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I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Daniel
Scholl
,
Maud
Sigoillot
,
Marie
Overtus
,
Rafael Colomer
Martinez
,
Chloé
Martens
,
Yiting
Wang
,
Els
Pardon
,
Toon
Laeremans
,
Abel
Garcia-Pino
,
Jan
Steyaert
,
David N.
Sheppard
,
Jelle
Hendrix
,
Cedric
Govaerts
Diamond Proposal Number(s):
[12718, 9426]
Abstract: The cystic fibrosis transmembrane conductance regulator (CFTR) anion channel is essential to maintain fluid homeostasis in key organs. Functional impairment of CFTR due to mutations in the cftr gene leads to cystic fibrosis. Here, we show that the first nucleotide-binding domain (NBD1) of CFTR can spontaneously adopt an alternate conformation that departs from the canonical NBD fold previously observed. Crystallography reveals that this conformation involves a topological reorganization of NBD1. Single-molecule fluorescence resonance energy transfer microscopy shows that the equilibrium between the conformations is regulated by adenosine triphosphate binding. However, under destabilizing conditions, such as the disease-causing mutation F508del, this conformational flexibility enables unfolding of the β-subdomain. Our data indicate that, in wild-type CFTR, this conformational transition of NBD1 regulates channel function, but, in the presence of the F508del mutation, it allows domain misfolding and subsequent protein degradation. Our work provides a framework to design conformation-specific therapeutics to prevent noxious transitions.
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Aug 2021
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B21-High Throughput SAXS
I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Tomasz
Uchański
,
Simonas
Masiulis
,
Baptiste
Fischer
,
Valentina
Kalichuk
,
Uriel
López-Sánchez
,
Eleftherios
Zarkadas
,
Miriam
Weckener
,
Andrija
Sente
,
Philip
Ward
,
Alexandre
Wohlkonig
,
Thomas
Zogg
,
Han
Remaut
,
James
Naismith
,
Hugues
Nury
,
Wim
Vranken
,
A. Radu
Aricescu
,
Els
Pardon
,
Jan
Steyaert
Abstract: Nanobodies are popular and versatile tools for structural biology. They have a compact single immunoglobulin domain organization, bind target proteins with high affinities while reducing their conformational heterogeneity and stabilize multi-protein complexes. Here we demonstrate that engineered nanobodies can also help overcome two major obstacles that limit the resolution of single-particle cryo-electron microscopy reconstructions: particle size and preferential orientation at the water–air interfaces. We have developed and characterized constructs, termed megabodies, by grafting nanobodies onto selected protein scaffolds to increase their molecular weight while retaining the full antigen-binding specificity and affinity. We show that the megabody design principles are applicable to different scaffold proteins and recognition domains of compatible geometries and are amenable for efficient selection from yeast display libraries. Moreover, we demonstrate that megabodies can be used to obtain three-dimensional reconstructions for membrane proteins that suffer from severe preferential orientation or are otherwise too small to allow accurate particle alignment.
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Jan 2021
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[19800]
Open Access
Abstract: Energy-coupling factor (ECF) transporters mediate import of micronutrients in prokaryotes. They consist of an integral membrane S-component (that binds substrate) and ECF module (that powers transport by ATP hydrolysis). It has been proposed that different S-components compete for docking onto the same ECF module, but a minimal liposome-reconstituted system, required to substantiate this idea, is lacking. Here, we co-reconstituted ECF transporters for folate (ECF-FolT2) and pantothenate (ECF-PanT) into proteoliposomes, and assayed for crosstalk during active transport. The kinetics of transport showed that exchange of S-components is part of the transport mechanism. Competition experiments suggest much slower substrate association with FolT2 than with PanT. Comparison of a crystal structure of ECF-PanT with previously determined structures of ECF-FolT2 revealed larger conformational changes upon binding of folate than pantothenate, which could explain the kinetic differences. Our work shows that a minimal in vitro system with two reconstituted transporters recapitulates intricate kinetics behaviour observed in vivo.
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Dec 2020
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