ORCID Profile
0000-0002-6726-2078
Current Organisation
University of Nottingham
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Publisher: Cold Spring Harbor Laboratory
Date: 03-04-2023
DOI: 10.1101/2023.03.31.534560
Abstract: The periplasmic chaperone SilF has been identified as part of an Ag(I) detoxification system in Gram negative bacteria. Sil proteins also bind Cu(I), but with reported weaker affinity, therefore leading to the designation of a specific detoxification system for Ag(I). Using isothermal titration calorimetry we show that binding of both ions is not only tighter than previously thought, but of very similar affinities. We investigated the structural origins of ion binding using molecular dynamics and QM/MM simulations underpinned by structural and biophysical experiments. The results of this analysis showed that the binding site adapts to accommodate either ion, with key interactions with the solvent in the case of Cu(I). The implications of this are that Gram negative bacteria do not appear to have evolved a specific Ag(I) efflux system but take advantage of the existing Cu(I) detoxification system. Therefore, there are consequences for how we define a particular metal resistance mechanism and understand its evolution in the environment.
Publisher: Elsevier BV
Date: 05-2007
DOI: 10.1016/J.JMB.2007.03.025
Abstract: Cell ision is a fundamental process for both eukaryotic and prokaryotic cells. In bacteria, cell ision is driven by a dynamic, ring-shaped, cytoskeletal element (the Z-ring) made up of polymers of the tubulin-like protein FtsZ. It is thought that lateral associations between FtsZ polymers are important for function of the Z-ring in vivo, and that these interactions are regulated by accessory cell ision proteins such as ZipA, EzrA and ZapA. We demonstrate that the putative Escherichia coli ZapA orthologue, YgfE, exists in a dimer/tetramer equilibrium in solution, binds to FtsZ polymers, strongly promotes FtsZ polymer bundling and is a potent inhibitor of the FtsZ GTPase activity. We use linear dichroism, a technique that allows structure analysis of molecules within linear polymers, to reveal a specific conformational change in GTP bound to FtsZ polymers, upon bundling by YgfE. We show that the consequences of FtsZ polymer bundling by YgfE and alent cations are very similar in terms of GTPase activity, bundle morphology and GTP orientation and therefore propose that this conformational change in bound GTP reveals a general mechanism of FtsZ bundling.
Publisher: American Chemical Society (ACS)
Date: 13-08-2013
DOI: 10.1021/CB400508K
Publisher: Wiley
Date: 06-01-2005
Publisher: Elsevier BV
Date: 10-2023
Publisher: Public Library of Science (PLoS)
Date: 21-05-2015
Publisher: Oxford University Press (OUP)
Date: 02-02-2023
DOI: 10.1093/NAR/GKAD030
Abstract: Altered eIF4A1 activity promotes translation of highly structured, eIF4A1-dependent oncogene mRNAs at root of oncogenic translational programmes. It remains unclear how these mRNAs recruit and activate eIF4A1 unwinding specifically to facilitate their preferential translation. Here, we show that single-stranded RNA sequence motifs specifically activate eIF4A1 unwinding allowing local RNA structural rearrangement and translation of eIF4A1-dependent mRNAs in cells. Our data demonstrate that eIF4A1-dependent mRNAs contain AG-rich motifs within their 5’UTR which specifically activate eIF4A1 unwinding of local RNA structure to facilitate translation. This mode of eIF4A1 regulation is used by mRNAs encoding components of mTORC-signalling and cell cycle progression, and renders these mRNAs particularly sensitive to eIF4A1-inhibition. Mechanistically, we show that binding of eIF4A1 to AG-rich sequences leads to multimerization of eIF4A1 with eIF4A1 subunits performing distinct enzymatic activities. Our structural data suggest that RNA-binding of multimeric eIF4A1 induces conformational changes in the RNA resulting in an optimal positioning of eIF4A1 proximal to the RNA duplex enabling efficient unwinding. Our data proposes a model in which AG-motifs in the 5’UTR of eIF4A1-dependent mRNAs specifically activate eIF4A1, enabling assembly of the helicase-competent multimeric eIF4A1 complex, and positioning these complexes proximal to stable localised RNA structure allowing ribosomal subunit scanning.
Publisher: Springer Science and Business Media LLC
Date: 28-08-2007
Abstract: The cell wall of Mycobacterium tuberculosis contains a wide range of phosphatidyl inositol-based glycolipids that play critical structural roles and, in part, govern pathogen-host interactions. Synthesis of phosphatidyl inositol is dependent on free myo-inositol, generated through dephosphorylation of myo-inositol-1-phosphate by inositol monophosphatase (IMPase). Human IMPase, the putative target of lithium therapy, has been studied extensively, but the function of four IMPase-like genes in M. tuberculosis is unclear. We determined the crystal structure, to 2.6 Å resolution, of the IMPase M. tuberculosis SuhB in the apo form, and analysed self-assembly by analytical ultracentrifugation. Contrary to the paradigm of constitutive dimerization of IMPases, SuhB is predominantly monomeric in the absence of the physiological activator Mg 2+ , in spite of a conserved fold and apparent dimerization in the crystal. However, Mg 2+ concentrations that result in enzymatic activation of SuhB decisively promote dimerization, with the inhibitor Li + lifying the effect of Mg 2+ , but failing to induce dimerization on its own. The correlation of Mg 2+ -driven enzymatic activity with dimerization suggests that catalytic activity is linked to the dimer form. Current models of lithium inhibition of IMPases posit that Li + competes for one of three catalytic Mg 2+ sites in the active site, stabilized by a mobile loop at the dimer interface. Our data suggest that Mg 2+ /Li + -induced ordering of this loop may promote dimerization by expanding the dimer interface of SuhB. The dynamic nature of the monomer-dimer equilibrium may also explain the extended concentration range over which Mg 2+ maintains SuhB activity.
Publisher: Elsevier BV
Date: 03-2007
DOI: 10.1016/J.JMB.2006.12.057
Abstract: A recently identified class of proteins conferring insecticidal activity to several bacteria within the Enterobacteriaceae family have potential for control of commercially important insect pests. Here, we report the first purification, biophysical characterisation and 3-D structural analysis of one of the toxin components, XptA1, from Xenorhabdus nematophila PMFI296 to a resolution of 23 A. Membrane binding studies indicate that the three-component toxin system has a different mode of action from that of proteins from Bacillus thuringiensis (Bt). Biophysical characterisation of XptA1 suggests a mechanism of action of XptA1 whereby it first binds to the cell membrane forming a structure with a central cavity and forms a complex with its partners XptB1 and XptC1 producing the full insecticidal toxin. The structure of XptA1 is shown by a combination of electron microscopy, ultracentrifugation and circular dichroism spectroscopy to be a 1.15 MDa tetramer with a cage-like structure. Each of the four symmetry-related subunits has three well-defined domains and a longitudinal twist with one end narrower than the other. One third of the residues of XptA1 are alpha-helical and it is suggested the subunits associate partly via an alpha-helical coiled-coil interaction. XptA1 itself shows the same secondary structure at neutral pH and in an alkaline environment up to pH10.5. This pH tolerance indicates that the folded XptA1 can pass through the midgut of Lepidopteran insects susceptible to the insecticidal toxin complex. This implies therefore that its folded structure is important for its biological activity.
Publisher: Springer Science and Business Media LLC
Date: 26-01-2013
DOI: 10.1007/S12154-012-0090-1
Abstract: In this paper we report the molecular profiling, lipidome and proteome, of the plant organelle known as an oil body (OB). The OB is remarkable in that it is able to perform its biological role (storage of triglycerides) whilst resisting the physical stresses caused by changes during desiccation (dehydration) and germination (rehydration). The molecular profile that confers such extraordinary physical stability on OBs was determined using a combination of 31 P/ 1 H nuclear magnetic resonance (NMR), high-resolution mass spectrometry and nominal mass-tandem mass spectrometry for the lipidome, and gel-electrophoresis-chromatography-tandem mass spectrometry for the proteome. The integrity of the procedure for isolating OBs was supported by physical evidence from small-angle neutron-scattering experiments. Suppression of lipase activity was crucial in determining the lipidome. There is conclusive evidence that the latter is dominated by phosphatidylcholine (∼60 %) and phosphatidylinositol (∼20 %), with a variety of other head groups (∼20 %). The fatty acid profile of the surface monolayer comprised palmitic, linoleic and oleic acids (2:1:0.25, 1 H NMR) with only traces of other fatty acids (C24:0, C22:0, C18:0, C18:3, C16:2 by MS). The proteome is rich in oleosins (78 %) with the remainder being made up of caleosins and steroleosins. These data are sufficiently detailed to inform an update of the understood model of this organelle and can be used to inform the use of such components in a range of molecular biological, biotechnological and food industry applications. The techniques used in this study for profiling the lipidome throw a new light on the lipid profile of plant cellular compartments.
Publisher: International Union of Crystallography (IUCr)
Date: 24-02-2005
Location: United Kingdom of Great Britain and Northern Ireland
Location: United Kingdom of Great Britain and Northern Ireland
No related grants have been discovered for David Scott.