I04-Macromolecular Crystallography
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Rebecca J.
Parr
,
Yoann G.
Santin
,
Giedrė
Ratkevičiūte
,
Simon G.
Caulton
,
Paul
Radford
,
Dominik
Gurvič
,
Matthew
Jenkins
,
Matthew T.
Doyle
,
Liam
Mead
,
Augustinas
Silale
,
Bert
Van Den Berg
,
Timothy J.
Knowles
,
R. Elizabeth
Sockett
,
Phillip J.
Stansfeld
,
Géraldine
Laloux
,
Andrew L.
Lovering
Diamond Proposal Number(s):
[19880]
Open Access
Abstract: Outer membrane proteins (OMPs) define the surface biology of Gram-negative bacteria, with roles in adhesion, transport, catalysis and signalling. Specifically, porin beta-barrels are common diffusion channels, predominantly monomeric/trimeric in nature. Here we show that the major OMP of the bacterial predator Bdellovibrio bacteriovorus, PopA, differs from this architecture, forming a pentameric porin-like superstructure. Our X-ray and cryo-EM structures reveal a bowl-shape composite outer β-wall, which houses a central chamber that encloses a section of the lipid bilayer. We demonstrate that PopA, reported to insert into prey inner membrane, causes defects when directed into Escherichia coli membranes. We discover widespread PopA homologues, including likely tetramers and hexamers, that retain the lipid chamber; a similar chamber is formed by an unrelated smaller closed-barrel family, implicating this as a general feature. Our work thus defines oligomeric OMP superfamilies, whose deviation from prior structures requires us to revisit existing membrane-interaction motifs and folding models.
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Jul 2025
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Krios I-Titan Krios I at Diamond
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Diamond Proposal Number(s):
[33797]
Open Access
Abstract: Model membranes allow for structural and biophysical studies on membrane biochemistry at the molecular level, albeit on systems of reduced complexity which can limit biological accuracy. Floating supported bilayers offer a means of producing planar lipid membrane models not adhered to a surface, which allows for improved accuracy compared to other model membranes. Here we communicate the incorporation of an integral membrane protein complex, the multidomain β-barrel assembly machinery (Bam), into our recently developed in situ self-assembled floating supported bilayers. Using neutron reflectometry and quartz crystal microbalance measurements we show this sample system can be fabricated using a two-step self-assembly process. We then demonstrate the complexity of the model membrane and tuneability of the membrane-to-surface distance using changes in the salt concentration of the bulk solution. Results demonstrate an easily fabricated, biologically accurate and tuneable membrane assay system which can be utilized for studies on integral membrane proteins within their native lipid matrix.
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Jun 2024
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I04-Macromolecular Crystallography
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Benjamin F.
Cooper
,
Giedrė
Ratkevičiūtė
,
Luke A.
Clifton
,
Hannah
Johnston
,
Rachel
Holyfield
,
David J.
Hardy
,
Simon G.
Caulton
,
William
Chatterton
,
Pooja
Sridhar
,
Peter
Wotherspoon
,
Gareth W.
Hughes
,
Stephen C. L.
Hall
,
Andrew L.
Lovering
,
Timothy J.
Knowles
Diamond Proposal Number(s):
[26803]
Open Access
Abstract: The E. coli Paraquat Inducible (Pqi) Pathway is a putative Gram-negative phospholipid transport system. The pathway comprises three components: an integral inner membrane protein (PqiA), a periplasmic spanning MCE family protein (PqiB) and an outer membrane lipoprotein (PqiC). Interactions between all complex components, including stoichiometry, remain uncharacterised; nevertheless, once assembled into their quaternary complex, the trio of Pqi proteins are anticipated to provide a continuous channel between the inner and outer membranes of diderms. Here, we present X-ray structures of both the native and a truncated, soluble construct of the PqiC lipoprotein, providing insight into its biological assembly, and utilise neutron reflectometry to characterise the nature of the PqiB-PqiC-membrane interaction. Finally, we employ phenotypic complementation assays to probe specific PqiC residues, which imply the interaction between PqiB and PqiC is less intimate than previously anticipated.
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Jan 2024
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Stephen C. I.
Hall
,
Luke
Clifton
,
Pooja
Sridhar
,
David J.
Hardy
,
Peter
Wotherspoon
,
Jack
Wright
,
James
Whitehouse
,
Nadisha
Gamage
,
Claire S.
Laxton
,
Caitlin
Hatton
,
Gareth W.
Hughes
,
Mark
Jeeves
,
Timothy J.
Knowles
Abstract: The outer membrane of Gram-negative bacteria presents a robust physicochemical barrier protecting the cell from both the natural environment and acting as the first line of defense against antimicrobial materials. The proteins situated within the outer membrane are responsible for a range of biological functions including controlling influx and efflux. These outer membrane proteins (OMPs) are ultimately inserted and folded within the membrane by the β-barrel assembly machine (Bam) complex. The precise mechanism by which the Bam complex folds and inserts OMPs remains unclear. Here, we have developed a platform for investigating Bam-mediated OMP insertion. By derivatizing a gold surface with a copper-chelating self-assembled monolayer, we were able to assemble a planar system containing the complete Bam complex reconstituted within a phospholipid bilayer. Structural characterization of this interfacial protein-tethered bilayer by polarized neutron reflectometry (PNR) revealed distinct regions consistent with known high-resolution models of the Bam complex. Additionally, by monitoring changes of mass associated with OMP insertion by quartz crystal microbalance with dissipation monitoring (QCM-D), we were able to demonstrate the functionality of this system by inserting two diverse OMPs within the membrane, pertactin and OmpT. This platform has promising application in investigating the mechanism of Bam-mediated OMP insertion, in addition to OMP function and activity within a phospholipid bilayer environment.
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Oct 2021
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Open Access
Abstract: Over recent years, there has been a rapid development of membrane-mimetic systems to encapsulate and stabilize planar segments of phospholipid bilayers in solution. One such system has been the use of amphipathic copolymers to solubilize lipid bilayers into nanodiscs. The attractiveness of this system, in part, stems from the capability of these polymers to solubilize membrane proteins directly from the host cell membrane. The assumption has been that the native lipid annulus remains intact, with nanodiscs providing a snapshot of the lipid environment. Recent studies have provided evidence that phospholipids can exchange from the nanodiscs with either lipids at interfaces, or with other nanodiscs in bulk solution. Here we investigate kinetics of lipid exchange between three recently studied polymer-stabilized nanodiscs and supported lipid bilayers at the silicon-water interface. We show that lipid and polymer exchange occurs in all nanodiscs tested, although the rate and extent differs between different nanodisc types. Furthermore, we observe adsorption of nanodiscs to the supported lipid bilayer for one nanodisc system which used a polymer made using reversible addition-fragmentation chain transfer polymerization. These results have important implications in applications of polymer-stabilized nanodiscs, such as in the fabrication of solid-supported films containing membrane proteins.
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Apr 2020
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I04-Macromolecular Crystallography
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Gareth W.
Hughes
,
Stephen C. L.
Hall
,
Claire S.
Laxton
,
Pooja
Sridhar
,
Amirul H.
Mahadi
,
Caitlin
Hatton
,
Thomas J.
Piggot
,
Peter J.
Wotherspoon
,
Aneika C.
Leney
,
Douglas G.
Ward
,
Mohammed
Jamshad
,
Vaclav
Spana
,
Ian T.
Cadby
,
Christopher
Harding
,
Georgia L.
Isom
,
Jack A.
Bryant
,
Rebecca J.
Parr
,
Yasin
Yakub
,
Mark
Jeeves
,
Damon
Huber
,
Ian R.
Henderson
,
Luke A.
Clifton
,
Andrew L.
Lovering
,
Timothy J.
Knowles
Diamond Proposal Number(s):
[14692]
Abstract: The Mla pathway is believed to be involved in maintaining the asymmetrical Gram-negative outer membrane via retrograde phospholipid transport. The pathway is composed of three components: the outer membrane MlaA–OmpC/F complex, a soluble periplasmic protein, MlaC, and the inner membrane ATPase, MlaFEDB complex. Here, we solve the crystal structure of MlaC in its phospholipid-free closed apo conformation, revealing a pivoting β-sheet mechanism that functions to open and close the phospholipid-binding pocket. Using the apo form of MlaC, we provide evidence that the inner-membrane MlaFEDB machinery exports phospholipids to MlaC in the periplasm. Furthermore, we confirm that the phospholipid export process occurs through the MlaD component of the MlaFEDB complex and that this process is independent of ATP. Our data provide evidence of an apparatus for lipid export away from the inner membrane and suggest that the Mla pathway may have a role in anterograde phospholipid transport.
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Jun 2019
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I02-Macromolecular Crystallography
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Mohammed
Jamshad
,
Timothy J.
Knowles
,
Scott A.
White
,
Douglas G
Ward
,
Fiyaz
Mohammed
,
Kazi Fahmida
Rahman
,
Max
Wynne
,
Gareth W.
Hughes
,
Günter
Kramer
,
Bernd
Bukau
,
Damon
Huber
Diamond Proposal Number(s):
[10369]
Open Access
Abstract: In bacteria, the translocation of proteins across the cytoplasmic membrane by the Sec machinery requires the ATPase SecA. SecA binds ribosomes and recognises nascent substrate proteins, but the molecular mechanism of nascent substrate recognition is unknown. We investigated the role of the C-terminal tail (CTT) of SecA in nascent polypeptide recognition. The CTT consists of a flexible linker (FLD) and a small metal-binding domain (MBD). Phylogenetic analysis and ribosome binding experiments indicated that the MBD interacts with 70S ribosomes. Disruption of the MBD only or the entire CTT had opposing effects on ribosome binding, substrate-protein binding, ATPase activity and in vivo function, suggesting that the CTT influences the conformation of SecA. Site-specific crosslinking indicated that F399 in SecA contacts ribosomal protein uL29, and binding to nascent chains disrupts this interaction. Structural studies provided insight into the CTT-mediated conformational changes in SecA. Our results suggest a mechanism for nascent substrate protein recognition.
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Jun 2019
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B21-High Throughput SAXS
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Stephen C. L.
Hall
,
Cecilia
Tognoloni
,
Jack
Charlton
,
Eilis C.
Bragginton
,
Alice J.
Rothnie
,
Pooja
Sridhar
,
Mark
Wheatley
,
Timothy J.
Knowles
,
Thomas
Arnold
,
Karen J.
Edler
,
Tim R.
Dafforn
Diamond Proposal Number(s):
[9727]
Open Access
Abstract: The fundamental importance of membrane proteins in drug discovery has meant that membrane mimetic systems for studying membrane proteins is of increasing interest. One such system has been the amphipathic, negatively charged poly(styrene-co-maleic acid) (SMA) polymer to form “SMA Lipid Particles” (SMALPs) which have been widely adopted to solubilize membrane proteins directly from the cell membrane. However, SMALPs are only soluble under basic conditions and precipitate in the presence of divalent cations required for many downstream applications. Here, we show that the positively charged poly(styrene-co-maleimide) (SMI) forms similar nanoparticles with comparable efficiency to SMA, whilst remaining functional at acidic pH and compatible with high concentrations of divalent cations. We have performed a detailed characterization of the performance of SMI that enables a direct comparison with similar data published for SMA. We also demonstrate that SMI is capable of extracting proteins directly from the cell membrane and can solubilize functional human G-protein coupled receptors (GPCRs) expressed in cultured HEK 293T cells. “SMILPs” thus provide an alternative membrane solubilization method that successfully overcomes some of the limitations of the SMALP method.
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May 2018
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I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Mahboob
Salim
,
Timothy J.
Knowles
,
Alfie T.
Baker
,
Martin S.
Davey
,
Mark
Jeeves
,
Pooja
Sridhar
,
John
Wilkie
,
Carrie R.
Willcox
,
Hachemi
Kadri
,
Taher E.
Taher
,
Pierre
Vantourout
,
Adrian
Hayday
,
Youcef
Mehellou
,
Fiyaz
Mohammed
,
Benjamin E.
Willcox
Diamond Proposal Number(s):
[14692]
Abstract: Human Vγ9/Vδ2 T-cells detect tumour cells and microbial infections by recognising small phosphorylated prenyl metabolites termed phosphoantigens (P-Ag). The type-1 transmembrane protein Butyrophilin 3A1 (BTN3A1) is critical to the P-Ag-mediated activation of Vγ9/Vδ2 T-cells, however, the molecular mechanisms involved in BTN3A1-mediated metabolite sensing are unclear, including how P-Ag are discriminated from non-antigenic small molecules. Here, we utilised NMR and X-ray crystallography to probe P-Ag sensing by BTN3A1. Whereas the BTN3A1 Immunoglobulin Variable domain failed to bind P-Ag, the intracellular B30.2 domain bound a range of negatively-charged small molecules, including P-Ag, in a positively-charged surface pocket. However, NMR chemical shift perturbations indicated BTN3A1 discriminated P-Ag from non-antigenic small molecules by their ability to induce a specific conformational change in the B30.2 domain that propagated from the P-Ag binding site to distal parts of the domain. These results suggest BTN3A1 selectively detects P-Ag intracellularly via a conformational antigenic sensor in its B30.2 domain, and have implications for rational design of antigens for Vγ9/Vδ2 -based T-cell immunotherapies.
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Sep 2017
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I22-Small angle scattering & Diffraction
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Abstract: Membranes of Gram-negative bacteria, mitochondria and chloroplasts receive and fold beta-barrel transmembrane proteins through the action of polypeptide transport-associated (POTRA) domains. In Escherichia coli, folding substrates are inserted into the outer membrane by the essential protein YaeT, a prototypic Omp85 protein. Here, the articulation between tandem POTRA domains in solution is defined by nuclear magnetic resonance (NMR) spectroscopy, indicating an unprecedented juxtaposition. The novel solution orientations of all five POTRA domains are revealed by small-angle X-ray scattering of the entire 46 kDa periplasmic region. NMR titration studies show that strands from YaeT's canonical folding substrate, PhoE, bind non-specifically along alternating sides of its mixed beta sheets, thus providing an ideal platform for helping to fold nascent outer-membrane proteins. Together, this provides the first structural model of how multiple POTRA domains recruit substrates from the periplasmic solution into the outer membrane.
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Jun 2008
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