I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Robert M.
Cleverley
,
Zoe J.
Rutter
,
Jeanine
Rismondo
,
Federico
Corona
,
Ho-Ching Tiffany
Tsui
,
Fuad A.
Alatawi
,
Richard A.
Daniel
,
Sven
Halbedel
,
Orietta
Massidda
,
Malcolm E.
Winkler
,
Richard J.
Lewis
Diamond Proposal Number(s):
[9948, 13587]
Open Access
Abstract: Bacterial growth and cell division requires precise spatiotemporal regulation of the synthesis and remodelling of the peptidoglycan layer that surrounds the cytoplasmic membrane. GpsB is a cytosolic protein that affects cell wall synthesis by binding cytoplasmic mini-domains of peptidoglycan synthases to ensure their correct subcellular localisation. Here, we describe critical structural features for the interaction of GpsB with peptidoglycan synthases from three bacterial species (Bacillus subtilis, Listeria monocytogenes and Streptococcus pneumoniae) and suggest their importance for cell wall growth and viability in L. monocytogenes and S. pneumoniae. We use these structural motifs to identify novel partners of GpsB in B. subtilis and extend the members of the GpsB interactome in all three bacterial species. Our results support that GpsB functions as an adaptor protein that mediates the interaction between membrane proteins, scaffolding proteins, signalling proteins and enzymes to generate larger protein complexes at specific sites in a bacterial cell cycle-dependent manner.
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Jan 2019
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I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[7864]
Open Access
Abstract: Bacterial biofilms are a complex architecture of cells that grow on moist interfaces, and are held together by a molecular glue of extracellular proteins, sugars and nucleic acids. Biofilms are particularly problematic in human healthcare as they can coat medical implants and are thus a potential source of disease. The enzymatic dispersal of biofilms is increasingly being developed as a new strategy to treat this problem. Here, we have characterized NucB, a biofilm-dispersing nuclease from a marine strain of Bacillus licheniformis, and present its crystal structure together with the biochemistry and a mutational analysis required to confirm its active site. Taken together, these data support the categorization of NucB into a unique subfamily of the ββα metal-dependent non-specific endonucleases. Understanding the structure and function of NucB will facilitate its future development into an anti-biofilm therapeutic agent.
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Nov 2017
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Richard A
Ward
,
Paul A.
Bethel
,
Calum
Cook
,
Emma
Davies
,
Judit E.
Debreczeni
,
Gary
Fairley
,
Lyman
Feron
,
Vikki
Flemington
,
Mark A.
Graham
,
Ryan
Greenwood
,
Nicola
Griffin
,
Lyndsey
Hanson
,
Philip
Hopcroft
,
Tina D.
Howard
,
Julian
Hudson
,
Michael
James
,
Clifford D.
Jones
,
Christopher R
Jones
,
Scott
Lamont
,
Richard James
Lewis
,
Nicola
Lindsay
,
Karen
Roberts
,
Iain
Simpson
,
Steve
St-Gallay
,
Steve
Swallow
,
Jia
Tang
,
Michael
Tonge
,
Zhenhua
Wang
,
Baochang
Zhai
Abstract: There are a number of small molecule inhibitors targeting the RAS/RAF/MEK/ERK signaling pathway either approved or in clinical development for oncology across a range of disease indications. The inhibition of ERK1/2 is of significant current interest as cell lines with acquired resistance to BRAF and MEK inhibitors have been shown to maintain sensitivity to ERK1/2 inhibition in pre-clinical models. This manuscript reports on our recent work to identify novel, potent and selective reversible ERK1/2 inhibitors from a low molecular weight, modestly active and highly promiscuous chemical starting point 4. To guide and inform the evolution of this series, inhibitor binding mode information from x-ray crystal structures was critical in the rapid exploration of this template to compound 35, which was active when tested in in vivo anti tumour efficacy experiments.
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Apr 2017
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[1221]
Open Access
Abstract: GpsB, a key regulator of cell division in Gram-positive bacteria, interacts with a key peptidoglycan synthase at the cell division septum, the penicillin binding protein PBP1 (a.k.a. PonA). Bacillus subtilis GpsB has been reported to interact with other components of the cell division machinery, including EzrA, MreC, and PrkC. In this study, we report an analysis of the arrangement of subunits in Listeria monocytogenes GpsB by small-angle X-ray scattering. The resulting model has an elongated shape with residues critical for interaction with PBP1 and the cell membrane clustered at one end of the molecule. Mutations that destabilize the hexameric assembly of the wild-type protein have a gpsB null phenotype, indicating that oligomerization is critical for the correct function of GpsB. We suggest a model in which a single GpsB hexamer can interact with multiple PBP1 molecules and can therefore influence the arrangement of PBP1 molecules within the cell division machinery, a dynamic multiprotein complex called the divisome, consistent with a role for GpsB in modulating the synthesis of the cell wall.
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Sep 2016
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I02-Macromolecular Crystallography
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Diamond Proposal Number(s):
[9948]
Open Access
Abstract: Peptidoglycan (PG) is an essential structural component of the bacterial cell wall and maintains the integrity and shape of the cell by forming a continuous layer around the cytoplasmic membrane. The thin PG layer of Escherichia coli resides in the periplasm, a unique compartment whose composition and pH can vary depending on the local environment of the cell. Hence, the growth of the PG layer must be sufficiently robust to allow cell growth and division under different conditions. We have analyzed the PG composition of 28 mutants lacking multiple PG enzymes (penicillin-binding proteins [PBPs]) after growth in acidic or near-neutral-pH media. Statistical analysis of the muropeptide profiles identified dd-carboxypeptidases (DD-CPases) that were more active in cells grown at acidic pH. In particular, the absence of the DD-CPase PBP6b caused a significant increase in the pentapeptide content of PG as well as morphological defects when the cells were grown at acidic pH. Other DD-CPases (PBP4, PBP4b, PBP5, PBP6a, PBP7, and AmpH) and the PG synthase PBP1B made a smaller or null contribution to the pentapeptide-trimming activity at acidic pH. We solved the crystal structure of PBP6b and also demonstrated that the enzyme is more stable and has a lower Km at acidic pH, explaining why PBP6b is more active at low pH. Hence, PBP6b is a specialized DD-CPase that contributes to cell shape maintenance at low pH, and E. coli appears to utilize redundant DD-CPases for normal growth under different conditions.
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Jun 2016
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I04-Macromolecular Crystallography
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Abstract: Each bacterium has to co-ordinate its growth with division to ensure genetic stability of the population. Consequently, cell division and growth are tightly-regulated phenomena, albeit different bacteria utilise one of several alternative regulatory mechanisms to maintain control. Here we consider GpsB, which is linked to cell growth and division in Gram-positive bacteria. ΔgpsB mutants of the human pathogen Listeria monocytogenes show severe lysis, division and growth defects due to distortions of cell wall biosynthesis. Consistent with this premise, GpsB interacts both in vitro and in vivo with the major bi-functional penicillin binding protein. We solved the crystal structure of GpsB and the interaction interfaces in both proteins are identified and validated. The inactivation of gpsB results in strongly attenuated virulence in animal experiments, comparable in degree to classical listerial virulence factor mutants. Therefore, GpsB is essential for in vitro and in vivo growth of a highly virulent food-borne pathogen, suggesting that GpsB could be a target for the future design of novel antibacterials.
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Nov 2015
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I03-Macromolecular Crystallography
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Andriy
Kryshtafovych
,
John
Moult
,
Arnaud
Basle
,
Alex
Burgin
,
Timothy K.
Craig
,
Robert A.
Edwards
,
Deborah
Fass
,
Marcus
Hartmann
,
Mateusz
Korycinski
,
Rick
Lewis
,
Donald
Lorimer
,
Andrei N.
Lupas
,
Janet
Newman
,
Thomas S.
Peat
,
Kurt H.
Piepenbrink
,
Janani
Prahlad
,
Mark
Van Raaij
,
Forest
Rohwer
,
Anca M.
Segall
,
Victor
Seguritan
,
Eric J.
Sundberg
,
Abhimanyu
Singh
,
Mark A.
Wilson
,
Torsten
Schwede
Diamond Proposal Number(s):
[9948]
Open Access
Abstract: The Critical Assessment of protein Structure Prediction (CASP) experiment would not have been possible without the prediction targets provided by the experimental structural biology community. In this article, selected crystallographers providing targets for the CASP11 experiment discuss the functional and biological significance of the target proteins, highlight their most interesting structural features and assess whether these features were correctly reproduced in the predictions submitted to CASP11.
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Oct 2015
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Robert
Cleverley
,
Jeffrey R.
Barrett
,
Arnaud
Basle
,
Nhat Khai
Bui
,
Lorraine
Hewitt
,
Alexandra
Solovyova
,
Zhi-Qiang
Xu
,
Richard A.
Daniel
,
Nicholas E.
Dixon
,
Elizabeth J.
Harry
,
Aaron J.
Oakley
,
Waldemar
Vollmer
,
Richard J/
Lewis
Diamond Proposal Number(s):
[9948]
Open Access
Abstract: Bacterial cell division is facilitated by a molecular machinethe divisomethat assembles at mid-cell in dividing cells. The formation of the cytokinetic Z-ring by the tubulin homologue FtsZ is regulated by several factors, including the divisome component EzrA. Here we describe the structure of the 60-kDa cytoplasmic domain of EzrA, which comprises five linear repeats of an unusual triple helical bundle. The EzrA structure is bent into a semicircle, providing the protein with the potential to interact at both N- and C-termini with adjacent membrane-bound divisome components. We also identify at least two binding sites for FtsZ on EzrA and map regions of EzrA that are responsible for regulating FtsZ assembly. The individual repeats, and their linear organization, are homologous to the spectrin proteins that connect actin filaments to the membrane in eukaryotes, and we thus propose that EzrA is the founding member of the bacterial spectrin family.
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Nov 2014
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I02-Macromolecular Crystallography
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Christopher
Hoyland
,
Christine
Aldridge
,
Robert
Cleverley
,
Marie-Clémence
Duchêne
,
George
Minasov
,
Olena
Onopriyenko
,
Karzan
Sidiq
,
Peter J.
Stogios
,
Wayne F.
Anderson
,
Richard A.
Daniel
,
Alexei
Savchenko
,
Waldemar
Vollmer
,
Richard J.
Lewis
Open Access
Abstract: Peptidoglycan surrounds the bacterial cytoplasmic membrane to protect the cell against osmolysis. The biosynthesis of peptidoglycan, made of glycan strands crosslinked by short peptides, is the target of antibiotics like β-lactams and glycopeptides. Nascent peptidoglycan contains pentapeptides that are trimmed by carboxypeptidases to tetra- and tripeptides. The well-characterized DD-carboxypeptidases hydrolyze the terminal D-alanine from the stem pentapeptide to produce a tetrapeptide. However, few LD-carboxypeptidases that produce tripeptides have been identified, and nothing is known about substrate specificity in these enzymes. We report biochemical properties and crystal structures of the LD-carboxypeptidases LdcB from Streptococcus pneumoniae, Bacillus anthracis, and Bacillus subtilis. The enzymes are active against bacterial cell wall tetrapeptides and adopt a zinc-carboxypeptidase fold characteristic of the LAS superfamily. We have also solved the structure of S. pneumoniae LdcB with a product mimic, elucidating the residues essential for peptidoglycan recognition and the conformational changes that occur on ligand binding.
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Jul 2014
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I02-Macromolecular Crystallography
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Abstract: Bacillus subtilis mutants lacking ymdB are unable to form biofilms, exhibit a strong overexpression of the flagellin gene hag, and are deficient in SlrR, a SinR antagonist. Here, we report the functional and structural characterization of YmdB, and we find that YmdB is a phosphodiesterase with activity against 2' 3' and 3' 5 'cyclic nucleotide monophosphates. The structure of YmdB reveals that the enzyme adopts a conserved phosphodiesterase fold with a binuclear metal center. Mutagenesis of a catalytically crucial residue demonstrates that the enzymatic activity of YmdB is essential for biofilm formation. The deletion of ymdB affects the expression of more than 800 genes; the levels of the sigma D-dependent motility regulon and several sporulation genes are increased, and the levels of the SinR-repressed biofilm genes are decreased, confirming the role of YmdB in regulating late adaptive responses of B. subtilis.
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Dec 2013
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