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33 items found (0 items not approved), displaying 1 to 10.[First/Prev] 1, 2, 3, 4 [Next/Last]

Instruments / Technical Area	Publication Details	Published
I03-Macromolecular Crystallography I04-Macromolecular Crystallography	Structures of SALSA/DMBT1 SRCR domains reveal the conserved ligand-binding mechanism of the ancient SRCR fold Martin P. Reichhardt , Vuokko Loimaranta , Susan M. Lea , Steven Johnson Life Science Alliance 3 DOI: 10.26508/lsa.201900502 Diamond Proposal Number(s): [18069] Open Access Show Abstract ▼ Abstract: The scavenger receptor cysteine-rich (SRCR) family of proteins comprises more than 20 membrane-associated and secreted molecules. Characterised by the presence of one or more copies of the ∼110 amino-acid SRCR domain, this class of proteins have widespread functions as antimicrobial molecules, scavenger receptors, and signalling receptors. Despite the high level of structural conservation of SRCR domains, no unifying mechanism for ligand interaction has been described. The SRCR protein SALSA, also known as DMBT1/gp340, is a key player in mucosal immunology. Based on detailed structural data of SALSA SRCR domains 1 and 8, we here reveal a novel universal ligand-binding mechanism for SALSA ligands. The binding interface incorporates a dual cation-binding site, which is highly conserved across the SRCR superfamily. Along with the well-described cation dependency on most SRCR domain–ligand interactions, our data suggest that the binding mechanism described for the SALSA SRCR domains is applicable to all SRCR domains. We thus propose to have identified in SALSA a conserved functional mechanism for the SRCR class of proteins.	Feb 2020
I03-Macromolecular Crystallography	An inhibitor of complement C5 provides structural insights into activation Martin P. Reichhardt , Steven Johnson , Terence Tang , Thomas Morgan , Nchimunya Tebeka , Niko Popitsch , Justin C. Deme , Matthijs M. Jore , Susan Lea Proceedings Of The National Academy Of Sciences 117 DOI: 10.1073/pnas.1909973116 Diamond Proposal Number(s): [18069] Open Access Show Abstract ▼ Abstract: The complement system is a crucial part of innate immune defenses against invading pathogens. The blood-meal of the tick Rhipicephalus pulchellus lasts for days, and the tick must therefore rely on inhibitors to counter complement activation. We have identified a class of inhibitors from tick saliva, the CirpT family, and generated detailed structural data revealing their mechanism of action. We show direct binding of a CirpT to complement C5 and have determined the structure of the C5–CirpT complex by cryoelectron microscopy. This reveals an interaction with the peripheral macro globulin domain 4 (C5_MG4) of C5. To achieve higher resolution detail, the structure of the C5_MG4–CirpT complex was solved by X-ray crystallography (at 2.7 Å). We thus present the fold of the CirpT protein family, and provide detailed mechanistic insights into its inhibitory function. Analysis of the binding interface reveals a mechanism of C5 inhibition, and provides information to expand our biological understanding of the activation of C5, and thus the terminal complement pathway.	Dec 2019
I02-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength)	Structure-based design of chimeric antigens for multivalent protein vaccines S. Hollingshead , I. Jongerius , R. M. Exley , S. Johnson , S. Lea , C. M. Tang Nature Communications 9 DOI: 10.1038/s41467-018-03146-7 Open Access Show Abstract ▼ Abstract: There is an urgent need to develop vaccines against pathogenic bacteria. However, this is often hindered by antigenic diversity and difficulties encountered manufacturing membrane proteins. Here we show how to use structure-based design to develop chimeric antigens (ChAs) for subunit vaccines. ChAs are generated against serogroup B Neisseria meningitidis (MenB), the predominant cause of meningococcal disease in wealthy countries. MenB ChAs exploit factor H binding protein (fHbp) as a molecular scaffold to display the immunogenic VR2 epitope from the integral membrane protein PorA. Structural analyses demonstrate fHbp is correctly folded and the PorA VR2 epitope adopts an immunogenic conformation. In mice, immunisation with ChAs generates fHbp and PorA antibodies that recognise the antigens expressed by clinical MenB isolates; these antibody responses correlate with protection against meningococcal disease. Application of ChAs is therefore a potentially powerful approach to develop multivalent subunit vaccines, which can be tailored to circumvent pathogen diversity.	Mar 2018
I03-Macromolecular Crystallography I04-Macromolecular Crystallography	Structural Basis for Mitotic Centrosome Assembly in Flies Zhe Feng , Anna Caballe , Alan Wainman , Steven Johnson , Andreas F. M. Haensele , Matthew A. Cottee , Paul T. Conduit , Susan M. Lea , Jordan W. Raff Cell 169, 1078 - 1089.e13 DOI: 10.1016/j.cell.2017.05.030 Diamond Proposal Number(s): [9306, 12346] Open Access Show Abstract ▼ Abstract: In flies, Centrosomin (Cnn) forms a phosphorylation-dependent scaffold that recruits proteins to the mitotic centrosome, but how Cnn assembles into a scaffold is unclear. We show that scaffold assembly requires conserved leucine zipper (LZ) and Cnn-motif 2 (CM2) domains that co-assemble into a 2:2 complex in vitro. We solve the crystal structure of the LZ:CM2 complex, revealing that both proteins form helical dimers that assemble into an unusual tetramer. A slightly longer version of the LZ can form micron-scale structures with CM2, whose assembly is stimulated by Plk1 phosphorylation in vitro. Mutating individual residues that perturb LZ:CM2 tetramer assembly perturbs the formation of these micron-scale assemblies in vitro and Cnn-scaffold assembly in vivo. Thus, Cnn molecules have an intrinsic ability to form large, LZ:CM2-interaction-dependent assemblies that are critical for mitotic centrosome assembly. These studies provide the first atomic insight into a molecular interaction required for mitotic centrosome assembly.	Jun 2017
I02-Macromolecular Crystallography I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography	Structural and functional dissection of the interplay between lipid and Notch binding by human Notch ligands Richard J. Suckling , Boguslawa Korona , Pat Whiteman , Chandramouli Chillakuri , Laurie Holt , Penny A. Handford , Susan M. Lea The Embo Journal DOI: 10.15252/embj.201796632 Diamond Proposal Number(s): [12346] Open Access Show Abstract ▼ Abstract: Recent data have expanded our understanding of Notch signalling by identifying a C2 domain at the N‐terminus of Notch ligands, which has both lipid‐ and receptor‐binding properties. We present novel structures of human ligands Jagged2 and Delta‐like4 and human Notch2, together with functional assays, which suggest that ligand‐mediated coupling of membrane recognition and Notch binding is likely to be critical in establishing the optimal context for Notch signalling. Comparisons between the Jagged and Delta family show a huge diversity in the structures of the loops at the apex of the C2 domain implicated in membrane recognition and Jagged1 missense mutations, which affect these loops and are associated with extrahepatic biliary atresia, lead to a loss of membrane recognition, but do not alter Notch binding. Taken together, these data suggest that C2 domain binding to membranes is an important element in tuning ligand‐dependent Notch signalling in different physiological contexts.	Jun 2017
I02-Macromolecular Crystallography I03-Macromolecular Crystallography	A key centriole assembly interaction interface between human Plk4 and STIL appears to not be conserved in flies Matthew A. Cottee , Steven Johnson , Jordan W. Raff , Susan M. Lea Biology Open DOI: 10.1242/bio.024661 Diamond Proposal Number(s): [9306] Open Access Show Abstract ▼ Abstract: A small number of proteins form a conserved pathway of centriole duplication. In humans and flies, the binding of Plk4/Sak to STIL/Ana2 initiates daughter centriole assembly. In humans, this interaction is mediated by an interaction between the Polo-Box-3 (PB3) domain of Plk4 and the coiled-coil domain of STIL (HsCCD). We showed previously that the Drosophila Ana2 coiled-coil domain (DmCCD) is essential for centriole assembly, but it forms a tight parallel tetramer in vitro that likely precludes an interaction with PB3. Here we show that the isolated HsCCD and HsPB3 domains form a mixture of homo-multimers in vitro, but these readily dissociate when mixed to form the previously described 1:1 HsCCD:HsPB3 complex. In contrast, although Drosophila PB3 (DmPB3) adopts a canonical polo-box fold, it does not detectably interact with DmCCD in vitro. Thus, surprisingly, a key centriole assembly interaction interface appears to differ between humans and flies.	Feb 2017
I02-Macromolecular Crystallography Data acquisition Detectors Diagnostics Health Physics	A Comparative Structure/Function Analysis of Two Type IV Pilin DNA Receptors Defines a Novel Mode of DNA Binding Jamie Berry , Yingqi Xu , Philip n. Ward , Susan Lea , Steve Matthews , Vladimir Pelicic Structure 24, 926 - 934 DOI: 10.1016/j.str.2016.04.001 Show Abstract ▼ Abstract: DNA transformation is a widespread process allowing bacteria to capture free DNA by using filamentous nano-machines composed of type IV pilins. These proteins can act as DNA receptors as demonstrated by the finding that Neisseria meningitidis ComP minor pilin has intrinsic DNA-binding ability. ComP binds DNA better when it contains the DNA-uptake sequence (DUS) motif abundant in this species genome, playing a role in its trademark ability to selectively take up its own DNA. Here, we report high-resolution structures for meningococcal ComP and Neisseria subflava ComPsub, which recognize different DUS motifs. We show that they are structurally identical type IV pilins that pack readily into filament models and display a unique DD region delimited by two disulfide bonds. Functional analysis of ComPsub defines a new mode of DNA binding involving the DD region, adapted for exported DNA receptors.	Jun 2016
I02-Macromolecular Crystallography	Structural basis for therapeutic inhibition of complement C5 Matthijs Jore , Steven Johnson , Devon Sheppard , Natalie Barber , Yang I Li , Miles A Nunn , Hans Elmlund , Susan Lea Nature Structural & Molecular Biology DOI: 10.1038/nsmb.3196 Diamond Proposal Number(s): [9306] Show Abstract ▼ Abstract: Activation of complement C5 generates the potent anaphylatoxin C5a and leads to pathogen lysis, inflammation and cell damage. The therapeutic potential of C5 inhibition has been demonstrated by eculizumab, one of the world's most expensive drugs. However, the mechanism of C5 activation by C5 convertases remains elusive, thus limiting development of therapeutics. Here we identify and characterize a new protein family of tick-derived C5 inhibitors. Structures of C5 in complex with the new inhibitors, the phase I and phase II inhibitor OmCI, or an eculizumab Fab reveal three distinct binding sites on C5 that all prevent activation of C5. The positions of the inhibitor-binding sites and the ability of all three C5–inhibitor complexes to competitively inhibit the C5 convertase conflict with earlier steric-inhibition models, thus suggesting that a priming event is needed for activation.	Mar 2016
I03-Macromolecular Crystallography	Non-Linear and Flexible Regions of the Human Notch1 Extracellular Domain Revealed by High-Resolution Structural Studies Philip C. Weisshuhn , Devon Sheppard , Paul Taylor , Pat Whiteman , Susan M. Lea , Penny A. Handford , Christina Redfield Structure 24, 1-12 DOI: 10.1016/j.str.2016.02.010 Diamond Proposal Number(s): [12346] Open Access Show Abstract ▼ Abstract: The Notch receptor is a key component of a core metazoan signaling pathway activated by Delta/Serrate/Lag-2 ligands expressed on an adjacent cell. This results in a short-range signal with profound effects on cell-fate determination, cell proliferation, and cell death. Key to understanding receptor function is structural knowledge of the large extracellular portion of Notch which contains multiple repeats of epidermal growth factor (EGF)-like domains. Here we investigate the EGF4-13 region of human Notch1 (hN1) using a multidisciplinary approach. Ca2+-binding measurements, X-ray crystallography, {1H}-15N heteronuclear nuclear Overhauser effects, and residual dipolar couplings support a non-linear organization for the EGF4-13 region with a rigid, bent conformation for EGF4-7 and a single flexible linkage between EGF9 and EGF10. These data allow us to construct an informed model for EGF10-13 which, in conjunction with comparative binding studies, demonstrates that EGF10 has an important role in determining Notch receptor sensitivity to Dll-4.	Mar 2016
I04-Macromolecular Crystallography	Structural basis for specificity and promiscuity in a carrier protein/enzyme system from the sulfur cycle Daniel B. Grabarczyk , Paul Chappell , Steven Johnson , Lukas S. Stelzl , Susan Lea , Ben C. Berks Proceedings Of The National Academy Of Sciences DOI: 10.1073/pnas.1506386112 PMID: 26655737 Diamond Proposal Number(s): [9306] Show Abstract ▼ Abstract: The bacterial Sox (sulfur oxidation) pathway is an important route for the oxidation of inorganic sulfur compounds. Intermediates in the Sox pathway are covalently attached to the heterodimeric carrier protein SoxYZ through conjugation to a cysteine on a protein swinging arm. We have investigated how the carrier protein shuttles intermediates between the enzymes of the Sox pathway using the interaction between SoxYZ and the enzyme SoxB as our model. The carrier protein and enzyme interact only weakly, but we have trapped their complex by using a “suicide enzyme” strategy in which an engineered cysteine in the SoxB active site forms a disulfide bond with the incoming carrier arm cysteine. The structure of this trapped complex, together with calorimetric data, identifies sites of protein–protein interaction both at the entrance to the enzyme active site tunnel and at a second, distal, site. We find that the enzyme distinguishes between the substrate and product forms of the carrier protein through differences in their interaction kinetics and deduce that this behavior arises from substrate-specific stabilization of a conformational change in the enzyme active site. Our analysis also suggests how the carrier arm-bound substrate group is able to outcompete the adjacent C-terminal carboxylate of the carrier arm for binding to the active site metal ions. We infer that similar principles underlie carrier protein interactions with other enzymes of the Sox pathway.	Dec 2015

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