I24-Microfocus Macromolecular Crystallography
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Sieglinde
De Cae
,
Inge
Van Molle
,
Loes
Van Schie
,
Sophie R.
Shoemaker
,
Julie
Deckers
,
Nincy
Debeuf
,
Sahine
Lameire
,
Wim
Nerinckx
,
Kenny
Roose
,
Daria
Fijalkowska
,
Simon
Devos
,
Anne-Sophie
De Smet
,
Jackeline Cecilia Zavala
Marchan
,
Toon
Venneman
,
Koen
Sedeyn
,
Lejla
Mujanovic
,
Marlies
Ballegeer
,
Manon
Vanheerswynghels
,
Caroline
De Wolf
,
Hans
Demol
,
Jasper
Zuallaert
,
Pieter
Vanhaverbeke
,
Gholamreza Hassanzadeh
Ghassabeh
,
Chiara
Lonigro
,
Viki
Bockstal
,
Manuela
Rinaldi
,
Rana
Abdelnabi
,
Johan
Neyts
,
Susan
Marqusee
,
Bart N.
Lambrecht
,
Nico
Callewaert
,
Han
Remaut
,
Xavier
Saelens
,
Bert
Schepens
Open Access
Abstract: Therapeutic monoclonal antibodies can prevent severe disease in SARS-CoV-2 exposed individuals. However, currently circulating virus variants have evolved to gain significant resistance to nearly all neutralizing human immune system-derived therapeutic monoclonal antibodies that had previously been emergency-authorized for use in the clinic. Here, we describe the discovery of a panel of single-domain antibodies (VHHs) directed against the spike protein S2 subunit that broadly neutralize SARS-CoV-1 and −2 with unusually high potency. One of these VHHs tightly clamps the spike’s monomers at a highly conserved, quaternary epitope in the membrane proximal part of the trimeric Heptad Repeat 2 (HR2) coiled-coil, thereby locking the HR2 in its prefusion conformation. Low dose systemic administration of a VHH-human IgG1 Fc fusion prevented SARS-CoV-2 infection in two animal models. Pseudovirus escape selection experiments demonstrate that the very rare escape variants are rendered almost non-infectious. This VHH-based antibody with a highly potent mechanism of antiviral action forms the basis for a new class of pan-sarbecovirus neutralizing biologics, which are currently under development. In addition, the unique quaternary binding mode of the VHHs to the prefusion HR2 could be exploited for other class I fusion proteins.
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May 2025
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[23248]
Abstract: Bacillus anthracis is a spore-forming gram-positive bacterium responsible for anthrax, an infectious disease with a high mortality rate and a target of concern due to bioterrorism and long-term site contamination. The entire surface of vegetative cells in exponential or stationary growth phase is covered in proteinaceous arrays called S-layers, composed of Sap or EA1 protein, respectively. The Sap S-layer represents an important virulence factor and cell envelope support structure whose paracrystalline nature is essential for its function. However, the spatial organization of Sap in its lattice state remains elusive. Here, we employed cryoelectron tomography and subtomogram averaging to obtain a map of the Sap S-layer from tubular polymers that revealed a conformational switch between the postassembly protomers and the previously available X-ray structure of the condensed monomers. To build and validate an atomic model of the lattice within this map, we used a combination of molecular dynamics simulations, X-ray crystallography, cross-linking mass spectrometry, and biophysics in an integrative structural biology approach. The Sap lattice model produced recapitulates a close-to-physiological arrangement, reveals high-resolution details of lattice contacts, and sheds light on the mechanisms underlying the stability of the Sap layer.
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Dec 2024
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[23248]
Open Access
Abstract: Nanobody-induced disassembly of Sap S-layers, a two-dimensional paracrystalline protein lattice from Bacillus anthracis, has been presented as a therapeutic intervention for lethal anthrax infections. However, only a subset of existing nanobodies with affinity to Sap exhibit depolymerization activity, suggesting that affinity and epitope recognition are not enough to explain inhibitory activity. In this study, we performed all-atom molecular dynamics simulations of each nanobody bound to the Sap binding site and trained a collection of machine learning classifiers to predict whether each nanobody induces depolymerization. We used feature importance analysis to filter out unnecessary features and engineered remaining features to regularize the feature landscape and encourage learning of the depolymerization mechanism. We find that, while not enforced in training, a gradient-boosting decision tree is able to reproduce the experimental activities of inhibitory nanobodies while maintaining high classification accuracy, whereas neural networks were only able to discriminate between classes. Further feature analysis revealed that inhibitory nanobodies restrain Sap motions toward an inhibitory conformational state described by domain-domain clamping and induced twisting of domains normal to the lattice plane. We believe these motions drive Sap lattice depolymerization and can be used as design targets for improved Sap-inhibitory nanobodies. Finally, we expect our method of study to apply to S-layers that serve as virulence factors in other pathogens, paving the way forward for nanobody therapeutics that target depolymerization mechanisms.
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Nov 2024
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I04-Macromolecular Crystallography
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Sibusiso B.
Maseko
,
Yasmine
Brammerloo
,
Inge
Van Molle
,
Adria
Sogues
,
Charlotte
Martin
,
Christoph
Gorgulla
,
Estelle
Plant
,
Julien
Olivet
,
Jeremy
Blavier
,
Thandokuhle
Ntombela
,
Frank
Delvigne
,
Haribabu
Arthanari
,
Hiba
El Hajj
,
Ali
Bazarbachi
,
Carine
Van Lint
,
Kourosh
Salehi-Ashtiani
,
Han
Remaut
,
Steven
Ballet
,
Alexander N.
Volkov
,
Jean-Claude
Twizere
Abstract: Human T-cell leukemia virus type-1 (HTLV-1) is the first pathogenic retrovirus discovered in human. Although HTLV-1-induced diseases are well-characterized and linked to the encoded Tax-1 oncoprotein, there is currently no strategy to target Tax-1 functions with small molecules. Here, we analyzed the binding of Tax-1 to the human homolog of the drosophila discs large tumor suppressor (hDLG1/SAP97), a multi-domain scaffolding protein involved in Tax-1-transformation ability. We have solved the structures of the PDZ binding motif (PBM) of Tax-1 in complex with the PDZ1 and PDZ2 domains of hDLG1 and assessed the binding of 10 million molecules by virtual screening. Among the 19 experimentally confirmed compounds, one systematically inhibited the Tax-1-hDLG1 interaction in different biophysical and cellular assays, as well as HTLV-1 cell-to-cell transmission in a T-cell model. Thus, our work demonstrates that interactions involving Tax-1 PDZ-domains are amenable to small-molecule inhibition, which provides a framework for the design of targeted therapies for HTLV-1-induced diseases.
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Sep 2023
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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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I03-Macromolecular Crystallography
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Vincent
Debruycker
,
Andrew
Hutchin
,
Matthieu
Masureel
,
Emel
Ficici
,
Chloé
Martens
,
Pierre
Legrand
,
Richard A.
Stein
,
Hassane S.
Mchaourab
,
José D.
Faraldo-Gómez
,
Han
Remaut
,
Cedric
Govaerts
Diamond Proposal Number(s):
[12718]
Abstract: Multidrug efflux pumps present a challenge to the treatment of bacterial infections, making it vitally important to understand their mechanism of action. Here, we investigate the nature of substrate binding within Lactococcus lactis LmrP, a prototypical multidrug transporter of the major facilitator superfamily. We determined the crystal structure of LmrP in a ligand-bound outward-open state and observed an embedded lipid in the binding cavity of LmrP, an observation supported by native mass spectrometry analyses. Molecular dynamics simulations suggest that the anionic lipid stabilizes the observed ligand-bound structure. Mutants engineered to disrupt binding of the embedded lipid display reduced transport of some, but not all, antibiotic substrates. Our results suggest that a lipid within the binding cavity could provide a malleable hydrophobic component that allows adaptation to the presence of different substrates, helping to explain the broad specificity of this protein and possibly other multidrug transporters.
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Jul 2020
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[17150]
Abstract: The β-barrel assembly machinery (BAM) inserts outer membrane β-barrel proteins (OMPs) in the outer membrane of Gram-negative bacteria. In Enterobacteriacea, BAM also mediates export of the stress sensor lipoprotein RcsF to the cell surface by assembling RcsF–OMP complexes. Here, we report the crystal structure of the key BAM component BamA in complex with RcsF. BamA adopts an inward-open conformation, with the lateral gate to the membrane closed. RcsF is lodged deep within the lumen of the BamA barrel, binding regions proposed to undergo outward and lateral opening during OMP insertion. On the basis of our structural and biochemical data, we propose a push-and-pull model for RcsF export following conformational cycling of BamA, and provide a mechanistic explanation for how RcsF uses its interaction with BamA to detect envelope stress. Our data also suggest that the flux of incoming OMP substrates is involved in the control of BAM activity.
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Jun 2020
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I03-Macromolecular Crystallography
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Dorien
De Vlieger
,
Katja
Hoffmann
,
Inge
Van Molle
,
Wim
Nerinckx
,
Lien
Van Hoecke
,
Marlies
Ballegeer
,
Sarah
Creytens
,
Han
Remaut
,
Hartmut
Hengel
,
Bert
Schepens
,
Xavier
Saelens
Open Access
Abstract: Lower respiratory tract infections, such as infections caused by influenza A viruses, are a constant threat for public health. Antivirals are indispensable to control disease caused by epidemic as well as pandemic influenza A. We developed a novel anti-influenza A virus approach based on an engineered single-domain antibody (VHH) construct that can selectively recruit innate immune cells to the sites of virus replication. This protective construct comprises two VHHs. One VHH binds with nanomolar affinity to the conserved influenza A matrix protein 2 (M2) ectodomain (M2e). Co-crystal structure analysis revealed that the complementarity determining regions 2 and 3 of this VHH embrace M2e. The second selected VHH specifically binds to the mouse Fcγ Receptor IV (FcγRIV) and was genetically fused to the M2e-specific VHH, which resulted in a bi-specific VHH-based construct that could be efficiently expressed in Pichia pastoris. In the presence of M2 expressing or influenza A virus-infected target cells, this single domain antibody construct selectively activated the mouse FcγRIV. Moreover, intranasal delivery of this bispecific FcγRIV-engaging VHH construct protected wild type but not FcγRIV−/− mice against challenge with an H3N2 influenza virus. These results provide proof of concept that VHHs directed against a surface exposed viral antigen can be readily armed with effector functions that trigger protective antiviral activity beyond direct virus neutralization.
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Dec 2019
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I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[12718, 17150]
Abstract: Anthrax is an ancient and deadly disease caused by the spore-forming bacterial pathogen Bacillus anthracis. At present, anthrax mostly affects wildlife and livestock, although it remains a concern for human public health—primarily for people who handle contaminated animal products and as a bioterrorism threat due to the high resilience of spores, a high fatality rate of cases and the lack of a civilian vaccination programme. The cell surface of B. anthracis is covered by a protective paracrystalline monolayer—known as surface layer or S-layer—that is composed of the S-layer proteins Sap or EA1. Here, we generate nanobodies to inhibit the self-assembly of Sap, determine the structure of the Sap S-layer assembly domain (SapAD) and show that the disintegration of the S-layer attenuates the growth of B. anthracis and the pathology of anthrax in vivo. SapAD comprises six β-sandwich domains that fold and support the formation of S-layers independently of calcium. Sap-inhibitory nanobodies prevented the assembly of Sap and depolymerized existing Sap S-layers in vitro. In vivo, nanobody-mediated disruption of the Sap S-layer resulted in severe morphological defects and attenuated bacterial growth. Subcutaneous delivery of Sap inhibitory nanobodies cleared B. anthracis infection and prevented lethality in a mouse model of anthrax disease. These findings highlight disruption of S-layer integrity as a mechanism that has therapeutic potential in S-layer-carrying pathogens.
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Jul 2019
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I02-Macromolecular Crystallography
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Kristof
Moonens
,
Youssef
Hamway
,
Matthias
Neddermann
,
Marc
Reschke
,
Nicole
Tegtmeyer
,
Tobias
Kruse
,
Robert
Kammerer
,
Raquel
Mejías‐luque
,
Bernhard B
Singer
,
Steffen
Backert
,
Markus
Gerhard
,
Han
Remaut
Diamond Proposal Number(s):
[12718]
Abstract: The human gastric pathogen Helicobacter pylori is a major causative agent of gastritis, peptic ulcer disease, and gastric cancer. As part of its adhesive lifestyle, the bacterium targets members of the carcinoembryonic antigen‐related cell adhesion molecule (CEACAM) family by the conserved outer membrane adhesin HopQ. The HopQ–CEACAM1 interaction is associated with inflammatory responses and enables the intracellular delivery and phosphorylation of the CagA oncoprotein via a yet unknown mechanism. Here, we generated crystal structures of HopQ isotypes I and II bound to the N‐terminal domain of human CEACAM1 (C1ND) and elucidated the structural basis of H. pylori specificity toward human CEACAM receptors. Both HopQ alleles target the β‐strands G, F, and C of C1ND, which form the trans dimerization interface in homo‐ and heterophilic CEACAM interactions. Using SAXS, we show that the HopQ ectodomain is sufficient to induce C1ND monomerization and thus providing H. pylori a route to influence CEACAM‐mediated cell adherence and signaling events.
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Jun 2018
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