I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[13587]
Abstract: Peptides play an important signalling role in Bacillus subtilis, where their uptake by one of two ABC-type oligopeptide transporters, Opp and App, is required for efficient sporulation. Homologues of these transporters in Clostridium difficile have been characterized, but their role, and hence that of peptides, in regulating sporulation in this organism is less clear. Here, the oligopeptide-binding receptor proteins for these transporters, CdAppA and CdOppA, have been purified and partially characterized, and the crystal structure of CdAppA has been determined in an open unliganded form. Peptide binding to either protein could not be observed in Thermofluor assays with a set of ten peptides of varying lengths and compositions. Re-examination of the protein sequences together with structure comparisons prompts the proposal that CdAppA is not a versatile peptide-binding protein but instead may bind a restricted set of peptides. Meanwhile, CdOppA is likely to be the receptor protein for a nickel-uptake system.
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Apr 2019
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I02-Macromolecular Crystallography
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Nadine
Daou
,
Yuanguo
Wang
,
Vladimir M.
Levdikov
,
Madhumitha
Nandakumar
,
Jonathan
Livny
,
Laurent
Bouillaut
,
Elena
Blagova
,
Keshan
Zhang
,
Boris R.
Belitsky
,
Kyu
Rhee
,
Anthony J.
Wilkinson
,
Xingmin
Sun
,
Abraham L.
Sonenshein
Diamond Proposal Number(s):
[9948]
Open Access
Abstract: Toxin synthesis and endospore formation are two of the most critical factors that determine the outcome of infection by Clostridioides difficile. The two major toxins, TcdA and TcdB, are the principal factors causing damage to the host. Spores are the infectious form of C. difficile, permit survival of the bacterium during antibiotic treatment and are the predominant cell form that leads to recurrent infection. Toxin production and sporulation have their own specific mechanisms of regulation, but they share negative regulation by the global regulatory protein CodY. Determining the extent of such regulation and its detailed mechanism is important for understanding the linkage between two apparently independent biological phenomena and raises the possibility of creating new ways of limiting infection. The work described here shows that a codY null mutant of a hypervirulent (ribotype 027) strain is even more virulent than its parent in a mouse model of infection and that the mutant expresses most sporulation genes prematurely during exponential growth phase. Moreover, examining the expression patterns of mutants producing CodY proteins with different levels of residual activity revealed that expression of the toxin genes is dependent on total CodY inactivation, whereas most sporulation genes are turned on when CodY activity is only partially diminished. These results suggest that, in wild-type cells undergoing nutrient limitation, sporulation genes can be turned on before the toxin genes.
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Jan 2019
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I12-JEEP: Joint Engineering, Environmental and Processing
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Diamond Proposal Number(s):
[9333]
Abstract: The influence of statistics on calculated lattice strains has been studied by comparing crystal plasticity finite element (CPFE) calculations with strains measured experimentally. Experimentally, when Bragg's law is obeyed, a plane normal must lie within a narrow orientation range (∼ 0.02° for synchrotron diffraction), or Bragg tolerance. However, CPFE models consider only a small number of grains compared to experiments, necessitating a justification of the statistically representative volume. It also becomes necessary to assess the threshold of Bragg tolerance allowable for the determined statistically representative volume. In this study, an 8 × 8 × 8 model was deemed as statistically representative such that only small benefits are obtained in terms of lattice strain calculations by adopting larger models such as 10 × 10 × 10. Based on the selected model, an allowable Bragg tolerance of approximately 5° was calculated. Also highlighted was the coupling between lattice strain, texture, hardening and applied boundary condition which are discriminators that will affect the choice of model size and Bragg tolerance threshold.
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May 2018
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Aurélie
Mousnier
,
Andrew S.
Bell
,
Dawid P.
Swieboda
,
Julia
Morales-sanfrutos
,
Inmaculada
Perez-dorado
,
James A.
Brannigan
,
Joseph
Newman
,
Markus
Ritzefeld
,
Jennie A.
Hutton
,
Anabel
Guedán
,
Amin S.
Asfor
,
Sean W.
Robinson
,
Iva
Hopkins-navratilova
,
Anthony J.
Wilkinson
,
Sebastian L.
Johnston
,
Robin J.
Leatherbarrow
,
Tobias J.
Tuthill
,
Roberto
Solari
,
Edward W.
Tate
Diamond Proposal Number(s):
[12579, 7864, 9948]
Abstract: Rhinoviruses (RVs) are the pathogens most often responsible for the common cold, and are a frequent cause of exacerbations in asthma, chronic obstructive pulmonary disease and cystic fibrosis. Here we report the discovery of IMP-1088, a picomolar dual inhibitor of the human N-myristoyltransferases NMT1 and NMT2, and use it to demonstrate that pharmacological inhibition of host-cell N-myristoylation rapidly and completely prevents rhinoviral replication without inducing cytotoxicity. The identification of cooperative binding between weak-binding fragments led to rapid inhibitor optimization through fragment reconstruction, structure-guided fragment linking and conformational control over linker geometry. We show that inhibition of the co-translational myristoylation of a specific virus-encoded protein (VP0) by IMP-1088 potently blocks a key step in viral capsid assembly, to deliver a low nanomolar antiviral activity against multiple RV strains, poliovirus and foot and-mouth disease virus, and protection of cells against virus-induced killing, highlighting the potential of host myristoylation as a drug target in picornaviral infections.
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May 2018
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I02-Macromolecular Crystallography
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Diamond Proposal Number(s):
[7864]
Abstract: PP2C phosphatases control biological processes including stress responses, development, and cell division in all kingdoms of life. Diverse regulatory domains adapt PP2C phosphatases to specific functions, but how these domains control phosphatase activity was unknown. We present structures representing active and inactive states of the PP2C phosphatase SpoIIE from Bacillus subtilis. Based on structural analyses and genetic and biochemical experiments, we identify an α-helical switch that shifts a carbonyl oxygen into the active site to coordinate a metal cofactor. Our analysis indicates that this switch is widely conserved among PP2C family members, serving as a platform to control phosphatase activity in response to diverse inputs. Remarkably, the switch is shared with proteasomal proteases, which we identify as evolutionary and structural relatives of PP2C phosphatases. Although these proteases use an unrelated catalytic mechanism, rotation of equivalent helices controls protease activity by movement of the equivalent carbonyl oxygen into the active site.
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May 2017
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I12-JEEP: Joint Engineering, Environmental and Processing
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Diamond Proposal Number(s):
[9333]
Open Access
Abstract: Common alloys used in sheet form can display a significant ductility benefit when they are subjected to
certain multiaxial strain paths. This effect has been studied here for a polycrystalline ferritic steel using a
combination of Nakajima bulge testing, X-ray diffraction during biaxial testing of cruciform samples and
crystal plasticity finite element (CPFE) modelling. Greatest gains in strain to failure were found when
subjecting sheets to uniaxial loading followed by balanced biaxial deformation, resulting in a total
deformation close to plane-strain. A combined strain of approximately double that of proportional
loading was achieved. The evolution of macrostrain, microstrain and texture during non-proportional
loading were evaluated by in-situ high energy synchrotron diffraction. The results have demonstrated
that the inhomogeneous strain accumulation from non-proportional deformation is strongly dependent
on texture and the applied strain-ratio of the first deformation pass. Experimental diffraction evidence is
supported by results produced by a novel method of CPFE-derived diffraction simulation. Using
constitutive laws selected on the basis of good agreement with measured lattice strain development, the
CPFE model demonstrated the capability to replicate ductility gains measured experimentally.
© 2016 Acta Materialia Inc. Published by Elsevier Ltd. This is an open access article under the CC BY
license
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Nov 2016
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I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[1221, 7864]
Abstract: The parasite Plasmodium vivax is the most widely distributed cause of recurring malaria. N-Myristoyltransferase (NMT), an enzyme that catalyses the covalent attachment of myristate to the N-terminal glycine of substrate proteins, has been described as a potential target for the treatment of this disease. Herein, we report the synthesis and the structure-guided optimization of a series of quinolines with balanced activity against both Plasmodium vivax and Plasmodium falciparum N-myristoyltransferase (NMT).
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Nov 2016
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I12-JEEP: Joint Engineering, Environmental and Processing
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Diamond Proposal Number(s):
[9333]
Open Access
Abstract: A methodology to simulate X-ray diffraction lattice strains using crystal plasticity, replicating in-situ synchrotron experimental measurements during the deformation of a low-carbon steel, has been developed. Uniquely, the model calculated lattice strains for full Debye-Scherrer diffraction rings, providing the in-plane lattice strain distributions determined from crystal plasticity. Thus, a direct method of comparison between experimental and crystal plasticity results becomes possible. The model considered two forms of hardening whilst subjecting the material to two dissimilar proportional strain-paths; uniaxial and balanced biaxial deformation. Both deformation paths showed influence on resulting lattice strain distributions which were also found to depend upon texture. Biaxial straining led to a stronger dependence on the material’s hardening behaviour and this was attributed to the higher rate of work hardening seen under biaxial compared to uniaxial straining. However, biaxial deformation showed quite isotropic lattice strains distribution, irrespective of initial texture or hardening. Quantitatively, good agreement between the computed and experimentally determined lattice strain distributions was obtained for each strain path. This success demonstrates the possibility of calibrating crystal plasticity model parameters using such methodologies, or simply to provide insight into the governing mechanisms in polycrystal deformation.
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Apr 2016
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I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[7864, 1221]
Abstract: N-Myristoyltransferase (NMT) represents an attractive drug target in parasitic infections such as malaria due to its genetic essentiality and amenability to inhibition by drug-like small molecules. Scaffold simplification from previously reported inhibitors containing bicyclic cores identified phenyl derivative 3, providing a versatile platform to study the effects of substitution on the scaffold, which yielded pyridyl 19. This molecule exhibited improved enzyme and cellular potency, and reduced lipophilicity compared to inhibitor 3. Further structure-based inhibitor design led to the discovery of 30, the most potent inhibitor in this series, which showed single-digit nM enzyme affinity and sub-μM anti-plasmodial activity
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Aug 2015
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I02-Macromolecular Crystallography
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Open Access
Abstract: Virulence and host range in Rhodococcus equi depends on the variable pathogenicity island of their virulence plasmids. Notable gene products are a family of small secreted virulence-associated proteins (Vaps) that are critical to intramacrophagic proliferation. Equine-adapted strains, which cause severe pyogranulomatous pneumonia in foals, produce a cell-associated VapA that is necessary for virulence, alongside five other secreted homologues. In the absence of biochemical insight, attention has turned to the structures of these proteins to develop a functional hypothesis. Recent studies have described crystal structures for VapD and a truncate of the VapA orthologue of porcine-adapted strains, VapB. Here, we crystallised the full-length VapG and determined its structure by molecular replacement. Electron density corresponding to the N-terminal domain was not visible suggesting that it is disordered. The protein core adopted a compact elliptical, anti-parallel β-barrel fold with β1–β2–β3–β8–β5–β6–β7–β4 topology decorated by a single peripheral α-helix unique to this family. The high glycine content of the protein allows close packing of secondary structural elements. Topologically, the surface has no indentations that indicate a nexus for molecular interactions. The distribution of polar and apolar groups on the surface of VapG is markedly uneven. One-third of the surface is dominated by exposed apolar side-chains, with no ionisable and only four polar side-chains exposed, giving rise to an expansive flat hydrophobic surface. Other surface regions are more polar, especially on or near the α-helix and a belt around the centre of the β-barrel. Possible functional significance of these recent structures is discussed.
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Aug 2015
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