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1,301 items found (0 items not approved), displaying 51 to 60.[First/Prev] 2, 3, 4, 5, 6, 7, 8, 9 [Next/Last]

Instruments / Technical Area	Publication Details	Published
I03-Macromolecular Crystallography I04-Macromolecular Crystallography	The structure of a polygamous repressor reveals how phage-inducible chromosomal islands spread in nature J. Rafael Ciges-tomas , Christian Alite , Suzanne Humphrey , Jorge Donderis , Janine Bowring , Xavier Salvatella , José R. Penadés , Alberto Marina Nature Communications 10 DOI: 10.1038/s41467-019-11504-2 Diamond Proposal Number(s): [14739, 16258] Open Access Show Abstract ▼ Abstract: Stl is a master repressor encoded by Staphylococcus aureus pathogenicity islands (SaPIs) that maintains integration of these elements in the bacterial chromosome. After infection or induction of a resident helper phage, SaPIs are de-repressed by specific interactions of phage proteins with Stl. SaPIs have evolved a fascinating mechanism to ensure their promiscuous transfer by targeting structurally unrelated proteins performing identically conserved functions for the phage. Here we decipher the molecular mechanism of this elegant strategy by determining the structure of SaPIbov1 Stl alone and in complex with two structurally unrelated dUTPases from different S. aureus phages. Remarkably, SaPIbov1 Stl has evolved different domains implicated in DNA and partner recognition specificity. This work presents the solved structure of a SaPI repressor protein and the discovery of a modular repressor that acquires multispecificity through domain recruiting. Our results establish the mechanism that allows widespread dissemination of SaPIs in nature.	Aug 2019
I03-Macromolecular Crystallography	Mapping the hydrophobic substrate binding site of phenylalanine ammonia lyase from petroselinum crispum Emma Zsófia Aletta Nagy , Souad Diana Tork , Pauline A. Lang , Alina Filip , Florin Dan Irimie , László Poppe , Monica Ioana Toşa , Christopher J. Schofield , Jurgen Brem , Csaba Paizs , Laszlo Csaba Bencze Acs Catalysis DOI: 10.1021/acscatal.9b02108 Diamond Proposal Number(s): [19458] Show Abstract ▼ Abstract: Modification of the hydrophobic binding pocket of phenylalanine ammonia-lyase from Petroselinum crispum (PcPAL) enables increased activity and selectivity towards phenylalanines and cinnamic acids mono-substituted with both electron donating (-CH3, -OCH3) and electron withdrawing (-CF3, -Br) groups at all positions (o-, m-, p-) of their aromatic ring. The results reveal specific residues involved in accommodating substituents at o-, m-, p-positions of the substrate’s phenyl ring. The predicted interactions were validated by crystallographic analysis of the binding mode of para-methoxy cinnamic acid complexed at the active site of PcPAL. The biocatalytic utility of the tailored PcPAL mutants was demonstrated by the efficient preparative scale synthesis of (S)-m-bromo-phenylalanine (ee: > 99%, yield: 60%) and (R)-p-methyl-phenylalanine (ee: 97%, yield: 49%), using the corresponding ammonia addition and ammonia elimination reactions catalyzed by the L134A and I460V PcPAL variants, respectively. Overall, the results reveal the potential for structure based protein engineering of PALs to provide enzymes with enhanced catalytic properties and which are specifically tailored for differently substituted phenylalanine analogues of high synthetic value.	Aug 2019
I02-Macromolecular Crystallography I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography	Molecular features of lipoprotein CD0873 – a potential vaccine against the human pathogen Clostridioides difficile William J. Bradshaw , Jean-françois Bruxelle , Andrea Kovacs-simon , Nicholas J. Harmer , Claire Janoir , Severine Péchiné , K. Ravi Acharya , Stephen L. Michell Journal Of Biological Chemistry DOI: 10.1074/jbc.RA119.010120 Diamond Proposal Number(s): [12342, 17212] Show Abstract ▼ Abstract: Clostridioides difficile is the primary cause of antibiotic-associated diarrhoea and colitis, a healthcare-associated intestinal disease resulting in a significant fatality rate. Colonization of the gut is critical for C. difficile pathogenesis, and the bacterial molecules essential for efficient colonization therefore offer great potential as vaccine candidates. Here we present findings demonstrating that the C. difficile immunogenic lipoprotein CD0873 plays a critical role in pathogen success in vivo. We found that in a dixenic colonization model, a CD0873-positive strain of C. difficile significantly outcompeted a CD0873-negative strain. Immunization of mice with recombinant CD0873 prevented long-term gut colonization and was correlated with a strong secretory IgA immune response. We further present high-resolution crystal structures of CD0873, at 1.80-2.50 Å resolutions, offering a first view of the ligand-binding pocket of CD0873 and provide evidence that this lipoprotein adhesin is part of a tyrosine import system, an amino acid key in C. difficile infection. These findings suggest that CD0873 could serve as a effective component in a vaccine against C. difficile.	Aug 2019
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	The complex of ferric-enterobactin with its transporter from Pseudomonas aeruginosa suggests a two-site model Lucile Moynie , Stefan Milenkovic , Gaëtan L. A. Mislin , Véronique Gasser , Giuliano Malloci , Etienne Baco , Rory P. Mccaughan , Malcolm G. P. Page , Isabelle J. Schalk , Matteo Ceccarelli , James H. Naismith Nature Communications 10 DOI: 10.1038/s41467-019-11508-y Open Access Show Abstract ▼ Abstract: Bacteria use small molecules called siderophores to scavenge iron. Siderophore-Fe3+ complexes are recognised by outer-membrane transporters and imported into the periplasm in a process dependent on the inner-membrane protein TonB. The siderophore enterobactin is secreted by members of the family Enterobacteriaceae, but many other bacteria including Pseudomonas species can use it. Here, we show that the Pseudomonas transporter PfeA recognises enterobactin using extracellular loops distant from the pore. The relevance of this site is supported by in vivo and in vitro analyses. We suggest there is a second binding site deeper inside the structure and propose that correlated changes in hydrogen bonds link binding-induced structural re-arrangements to the structural adjustment of the periplasmic TonB-binding motif.	Aug 2019
I03-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Diversity of oligomerization in Drosophila semaphorins suggests a mechanism of functional fine-tuning Daniel Rozbesky , Ross A. Robinson , Vitul Jain , Max Renner , Tomas Malinauskas , Karl Harlos , Christian Siebold , E. Yvonne Jones Nature Communications 10 DOI: 10.1038/s41467-019-11683-y Diamond Proposal Number(s): [14744, 10627] Open Access Show Abstract ▼ Abstract: Semaphorin ligands and their plexin receptors are one of the major cell guidance factors that trigger localised changes in the cytoskeleton. Binding of semaphorin homodimer to plexin brings two plexins in close proximity which is a prerequisite for plexin signalling. This model appears to be too simplistic to explain the complexity and functional versatility of these molecules. Here, we determine crystal structures for all members of Drosophila class 1 and 2 semaphorins. Unlike previously reported semaphorin structures, Sema1a, Sema2a and Sema2b show stabilisation of sema domain dimer formation via a disulfide bond. Unexpectedly, our structural and biophysical data show Sema1b is a monomer suggesting that semaphorin function may not be restricted to dimers. We demonstrate that semaphorins can form heterodimers with members of the same semaphorin class. This heterodimerization provides a potential mechanism for cross-talk between different plexins and co-receptors to allow fine-tuning of cell signalling.	Aug 2019
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography	The lipid transfer protein Saposin B does not directly bind CD1d for lipid antigen loading Maria Shamin , Tomasz H. Benedyk , Stephen C. Graham , Janet E. Deane Wellcome Open Research 4 DOI: 10.12688/wellcomeopenres.15368.2 Diamond Proposal Number(s): [15916] Open Access Show Abstract ▼ Abstract: Background: Lipid antigens are presented on the surface of cells by the CD1 family of glycoproteins, which have structural and functional similarity to MHC class I molecules. The hydrophobic lipid antigens are embedded in membranes and inaccessible to the lumenal lipid-binding domain of CD1 molecules. Therefore, CD1 molecules require lipid transfer proteins for lipid loading and editing. CD1d is loaded with lipids in late endocytic compartments, and lipid transfer proteins of the saposin family have been shown to play a crucial role in this process. However, the mechanism by which saposins facilitate lipid binding to CD1 molecules is not known and is thought to involve transient interactions between protein components to ensure CD1-lipid complexes can be efficiently trafficked to the plasma membrane for antigen presentation. Of the four saposin proteins, the importance of Saposin B (SapB) for loading of CD1d is the most well-characterised. However, a direct interaction between CD1d and SapB has yet to be described. Methods: In order to determine how SapB might load lipids onto CD1d, we used purified, recombinant CD1d and SapB and carried out a series of highly sensitive binding assays to monitor direct interactions. We performed equilibrium binding analysis, chemical cross-linking and co-crystallisation experiments, under a range of different conditions. Results: We could not demonstrate a direct interaction between SapB and CD1d using any of these binding assays. Conclusions: This work establishes comprehensively that the role of SapB in lipid loading does not involve direct binding to CD1d. We discuss the implication of this for our understanding of lipid loading of CD1d and propose several factors that may influence this process.	Aug 2019
I03-Macromolecular Crystallography	Heparan sulfates are critical regulators of the inhibitory megakaryocyte-platelet receptor G6b-B Timo Vögtle , Sumana Sharma , Jun Mori , Zoltan Nagy , Daniela Semeniak , Cyril Scandola , Mitchell J. Geer , Christopher W. Smith , Jordan Lane , Scott Pollack , Riitta Lassila , Annukka Jouppila , Alastair J. Barr , Derek J. Ogg , Tina D. Howard , Helen J. Mcmiken , Juli Warwicker , Catherine Geh , Rachel Rowlinson , W. Mark Abbott , Anita Eckly , Harald Schulze , Gavin J. Wright , Alexandra Mazharian , Klaus Futterer , Sundaresan Rajesh , Michael R. Douglas , Yotis A. Senis Elife 8 DOI: 10.7554/eLife.46840 Diamond Proposal Number(s): [20026] Open Access Show Abstract ▼ Abstract: The immunoreceptor tyrosine-based inhibition motif (ITIM)-containing receptor G6b-B is critical for platelet production and activation. Loss of G6b-B results in severe macrothrombocytopenia, myelofibrosis and aberrant platelet function in mice and humans. Using a combination of immunohistochemistry, affinity chromatography and proteomics, we identified the extracellular matrix heparan sulfate (HS) proteoglycan perlecan as a G6b-B binding partner. Subsequent in vitro biochemical studies and a cell-based genetic screen demonstrated that the interaction is specifically mediated by the HS chains of perlecan. Biophysical analysis revealed that heparin forms a high-affinity complex with G6b-B and mediates dimerization. Using platelets from humans and genetically-modified mice, we demonstrate that binding of G6b-B to HS and multivalent heparin inhibits platelet and megakaryocyte function by inducing downstream signaling via the tyrosine phosphatases Shp1 and Shp2. Our findings provide novel insights into how G6b-B is regulated and contribute to our understanding of the interaction of megakaryocytes and platelets with glycans.	Aug 2019
I02-Macromolecular Crystallography I03-Macromolecular Crystallography I04-Macromolecular Crystallography	A natural Diels‐Alder biocatalyst enables efficient [4 + 2] cycloaddition under harsh reaction conditions Carl Marsh , Nicholas Lees , Li-chen Han , Matthew Byrne , Sbusisiwe Mbatha , Laurence Maschio , Sebastian Pagden-ratcliffe , Phillip Duke , James Stach , Paul Curnow , Christine Willis , Paul Race Chemcatchem DOI: 10.1002/cctc.201901285 Diamond Proposal Number(s): [17212] Open Access Show Abstract ▼ Abstract: Carbon‐carbon bond formation is a fundamental transformation in both synthetic chemistry and biosynthesis. Enzymes catalyze such reactions with exquisite selectivity which often cannot be achieved using non‐biological methods but may suffer from an intolerance of high temperature and the presence of organic solvents limiting their applications. Here we report the thermodynamic and kinetic stability of the β‐barrel natural Diels‐Alderase AbyU, which catalyzes formation of the spirotetronate core of the antimicrobial natural product abyssomicin C, with creation of 3 new asymmetric centers. This enzyme is shown to catalyze [4 + 2] cycloadditions at elevated temperature (up to 65 oC), and in the presence of organic solvents (MeOH, CH3CN and DMSO) and the chemical denaturant guanidinium hydrochloride, revealing that AbyU has potential widespread value as a biocatalyst.	Aug 2019
I03-Macromolecular Crystallography	Tool inhibitors and assays to interrogate the biology of the TRAF2 and NCK interacting kinase Jon Read , Iain T. Collie , Michelle Nguyen-mccarty , Christopher Lucaj , James Robinson , Leslie Conway , Jayanta Mukherjee , Eileen Mccall , Gerard Donohoe , Elizabeth Flavell , Karolina Peciak , Juli Warwicker , Carly Dix , Bernard G. Van Den Hoven , Andrew Madin , Dean G. Brown , Stephen Moss , Stephen J. Haggarty , Nicholas J. Brandon , Roland W. Bürli Bioorganic & Medicinal Chemistry Letters 29, 1962 - 1967 DOI: 10.1016/j.bmcl.2019.05.032 Show Abstract ▼ Abstract: The TRAF2 and NCK interacting kinase (TNIK) has been proposed to play a role in cytoskeletal organization and synaptic plasticity and has been linked, among others, to neurological disorders. However, target validation efforts for TNIK have been hampered by the limited kinase selectivity of small molecule probes and possible functional compensation in mouse models. Both issues are at least in part due to its close homology to the kinases MINK1 (or MAP4K6) and MAP4K4 (or HGK). As part of our interest in validating TNIK as a therapeutic target for neurological diseases, we set up a panel of biochemical and cellular assays, which are described herein. We then examined the activity of known amino-pyridine-based TNIK inhibitors (1, 3) and prepared structurally very close analogs that lack the ability to inhibit the target. We also developed a structurally orthogonal, naphthyridine-based TNIK inhibitor (9) and an inactive control molecule of the same chemical series. These validated small-molecule probes will enable dissection of the function of TNIK family in the context of human disease biology.	Aug 2019
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I24-Microfocus Macromolecular Crystallography	The role of the light chain in the structure and binding activity of two cattle antibodies that neutralize bovine respiratory syncytial virus Jingshan Ren , Joanne E. Nettleship , Gemma Harris , William Mwangi , Nahid Rhaman , Clare Grant , Abhay Kotecha , Elizabeth Fry , Bryan Charleston , David I. Stuart , John Hammond , Raymond J. Owens Molecular Immunology 112, 123 - 130 DOI: 10.1016/j.molimm.2019.04.026 Diamond Proposal Number(s): [10627, 14744] Open Access Show Abstract ▼ Abstract: Cattle antibodies have unusually long CDR3 loops in their heavy chains (HCs), and limited light chain (LC) diversity, raising the question of whether these mask the effect of LC variation on antigen recognition. We have investigated the role of the LC in the structure and activity of two neutralizing cattle antibodies (B4 and B13) that bind the F protein of bovine respiratory syncytial virus (bRSV). Recombinant Fab fragments of B4 and B13 bound bRSV infected cells and showed similar affinities for purified bRSV F protein. Exchanging the LCs between the Fab fragments produced hybrid Fabs: B13* (B13 HC/B4 LC) and B4* (B4 HC/B13 LC). The affinity of B13* to the F protein was found to be two-fold lower than B13 whilst the binding affinity of B4* was reduced at least a hundred-fold compared to B4 such that it no longer bound to bRSV infected cells. Comparison of the structures of B4 and B13 with their LC exchanged counterparts B4* and B13* showed that paratope of the HC variable domain (VH) of B4 was disrupted on pairing with the B13 LC, consistent with the loss of binding activity. By contrast, B13 H3 adopts a similar conformation when paired with either B13 or B4 LCs. These observations confirm the expected key role of the extended H3 loop in antigen-binding by cattle antibodies but also show that the quaternary LC/HC subunit interaction can be crucial for its presentation and thus the LC variable domain (VL) is also important for antigen recognition.	Aug 2019

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