Publications by authors named "Ambrose R Cole"

13 Publications

  • Page 1 of 1

Terminal Regions Confer Plasticity to the Tetrameric Assembly of Human HspB2 and HspB3.

J Mol Biol 2018 09 30;430(18 Pt B):3297-3310. Epub 2018 Jun 30.

Department of Biological Sciences, Crystallography, Institute of Structural & Molecular Biology, Birkbeck College, Malet Street, London, WC1E 7HX, UK.

Heterogeneity in small heat shock proteins (sHsps) spans multiple spatiotemporal regimes-from fast fluctuations of part of the protein, to conformational variability of tertiary structure, plasticity of the interfaces, and polydispersity of the inter-converting, and co-assembling oligomers. This heterogeneity and dynamic nature of sHsps has significantly hindered their structural characterization. Atomic coordinates are particularly lacking for vertebrate sHsps, where most available structures are of extensively truncated homomers. sHsps play important roles in maintaining protein levels in the cell and therefore in organismal health and disease. HspB2 and HspB3 are vertebrate sHsps that are found co-assembled in neuromuscular cells, and variants thereof are associated with disease. Here, we present the structure of human HspB2/B3, which crystallized as a hetero-tetramer in a 3:1 ratio. In the HspB2/B3 tetramer, the four α-crystallin domains (ACDs) assemble into a flattened tetrahedron which is pierced by two non-intersecting approximate dyads. Assembly is mediated by flexible "nuts and bolts" involving IXI/V motifs from terminal regions filling ACD pockets. Parts of the N-terminal region bind in an unfolded conformation into the anti-parallel shared ACD dimer grooves. Tracts of the terminal regions are not resolved, most likely due to their disorder in the crystal lattice. This first structure of a full-length human sHsp heteromer reveals the heterogeneous interactions of the terminal regions and suggests a plasticity that is important for the cytoprotective functions of sHsps.
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http://dx.doi.org/10.1016/j.jmb.2018.06.047DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6119766PMC
September 2018

Structure of the stationary phase survival protein YuiC from B.subtilis.

BMC Struct Biol 2015 Jul 11;15:12. Epub 2015 Jul 11.

Institute for Structural and Molecular Biology, Crystallography, Department of Biological Sciences, Birkbeck University of London, Malet Street, London, WC1E 7HX, UK.

Background: Stationary phase survival proteins (Sps) were found in Firmicutes as having analogous domain compositions, and in some cases genome context, as the resuscitation promoting factors of Actinobacteria, but with a different putative peptidoglycan cleaving domain.

Results: The first structure of a Firmicute Sps protein YuiC from B. subtilis, is found to be a stripped down version of the cell-wall peptidoglycan hydrolase MltA. The YuiC structures are of a domain swapped dimer, although some monomer is also found in solution. The protein crystallised in the presence of pentasaccharide shows a 1,6-anhydrodisaccharide sugar product, indicating that YuiC cleaves the sugar backbone to form an anhydro product at least on lengthy incubation during crystallisation.

Conclusions: The structural simplification of MltA in Sps proteins is analogous to that of the resuscitation promoting factor domains of Actinobacteria, which are stripped down versions of lysozyme and soluble lytic transglycosylase proteins.
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http://dx.doi.org/10.1186/s12900-015-0039-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4499186PMC
July 2015

Crystal structure of the human, FIC-domain containing protein HYPE and implications for its functions.

Structure 2014 Dec;22(12):1831-1843

Division of Biosciences, Institute of Structural and Molecular Biology, University College London, Gower Street, London WC1E 6BT, UK. Electronic address:

Protein AMPylation, the transfer of AMP from ATP to protein targets, has been recognized as a new mechanism of host-cell disruption by some bacterial effectors that typically contain a FIC-domain. Eukaryotic genomes also encode one FIC-domain protein,HYPE, which has remained poorly characterized.Here we describe the structure of human HYPE, solved by X-ray crystallography, representing the first structure of a eukaryotic FIC-domain protein. We demonstrate that HYPE forms stable dimers with structurally and functionally integrated FIC-domains and with TPR-motifs exposed for protein-protein interactions. As HYPE also uniquely possesses a transmembrane helix, dimerization is likely to affect its positioning and function in the membrane vicinity. The low rate of auto AMPylation of the wild-type HYPE could be due to autoinhibition, consistent with the mechanism proposed for a number of putative FIC AMPylators. Our findings also provide a basis to further consider possible alternative cofactors of HYPE and distinct modes of target-recognition.
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http://dx.doi.org/10.1016/j.str.2014.10.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4342408PMC
December 2014

Clostridium perfringens epsilon toxin mutant Y30A-Y196A as a recombinant vaccine candidate against enterotoxemia.

Vaccine 2014 May 5;32(23):2682-7. Epub 2014 Apr 5.

College of Life and Environmental Sciences, University of Exeter, Stocker Road, Exeter EX4 4QD, United Kingdom.

Epsilon toxin (Etx) is a β-pore-forming toxin produced by Clostridium perfringens toxinotypes B and D and plays a key role in the pathogenesis of enterotoxemia, a severe, often fatal disease of ruminants that causes significant economic losses to the farming industry worldwide. This study aimed to determine the potential of a site-directed mutant of Etx (Y30A-Y196A) to be exploited as a recombinant vaccine against enterotoxemia. Replacement of Y30 and Y196 with alanine generated a stable variant of Etx with significantly reduced cell binding and cytotoxic activities in MDCK.2 cells relative to wild type toxin (>430-fold increase in CT50) and Y30A-Y196A was inactive in mice after intraperitoneal administration of trypsin activated toxin at 1000× the expected LD50 dose of trypsin activated wild type toxin. Moreover, polyclonal antibody raised in rabbits against Y30A-Y196A provided protection against wild type toxin in an in vitro neutralisation assay. These data suggest that Y30A-Y196A mutant could form the basis of an improved recombinant vaccine against enterotoxemia.
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http://dx.doi.org/10.1016/j.vaccine.2014.03.079DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4022833PMC
May 2014

Crystal structures of the human Dysferlin inner DysF domain.

BMC Struct Biol 2014 Jan 17;14. Epub 2014 Jan 17.

Crystallography, Biological Sciences, Institute for Structural and Molecular Biology, Birkbeck University of London, Malet Street, London WC1E 7HX, UK.

Background: Mutations in dysferlin, the first protein linked with the cell membrane repair mechanism, causes a group of muscular dystrophies called dysferlinopathies. Dysferlin is a type two-anchored membrane protein, with a single C terminal trans-membrane helix, and most of the protein lying in cytoplasm. Dysferlin contains several C2 domains and two DysF domains which are nested one inside the other. Many pathogenic point mutations fall in the DysF domain region.

Results: We describe the crystal structure of the human dysferlin inner DysF domain with a resolution of 1.9 Ångstroms. Most of the pathogenic mutations are part of aromatic/arginine stacks that hold the domain in a folded conformation. The high resolution of the structure show that these interactions are a mixture of parallel ring/guanadinium stacking, perpendicular H bond stacking and aliphatic chain packing.

Conclusions: The high resolution structure of the Dysferlin DysF domain gives a template on which to interpret in detail the pathogenic mutations that lead to disease.
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http://dx.doi.org/10.1186/1472-6807-14-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3898210PMC
January 2014

Architecturally diverse proteins converge on an analogous mechanism to inactivate Uracil-DNA glycosylase.

Nucleic Acids Res 2013 Oct 26;41(18):8760-75. Epub 2013 Jul 26.

Department of Biological Sciences, Institute of Structural and Molecular Biology, Birkbeck College, University of London, Malet Street, London WC1E 7HX, UK and Research Department of Structural and Molecular Biology, Institute of Structural and Molecular Biology, University College London, Gower Street, London WC1E 6BT, UK.

Uracil-DNA glycosylase (UDG) compromises the replication strategies of diverse viruses from unrelated lineages. Virally encoded proteins therefore exist to limit, inhibit or target UDG activity for proteolysis. Viral proteins targeting UDG, such as the bacteriophage proteins ugi, and p56, and the HIV-1 protein Vpr, share no sequence similarity, and are not structurally homologous. Such diversity has hindered identification of known or expected UDG-inhibitory activities in other genomes. The structural basis for UDG inhibition by ugi is well characterized; yet, paradoxically, the structure of the unbound p56 protein is enigmatically unrevealing of its mechanism. To resolve this conundrum, we determined the structure of a p56 dimer bound to UDG. A helix from one of the subunits of p56 occupies the UDG DNA-binding cleft, whereas the dimer interface forms a hydrophobic box to trap a mechanistically important UDG residue. Surprisingly, these p56 inhibitory elements are unexpectedly analogous to features used by ugi despite profound architectural disparity. Contacts from B-DNA to UDG are mimicked by residues of the p56 helix, echoing the role of ugi's inhibitory beta strand. Using mutagenesis, we propose that DNA mimicry by p56 is a targeting and specificity mechanism supporting tight inhibition via hydrophobic sequestration.
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http://dx.doi.org/10.1093/nar/gkt633DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3794593PMC
October 2013

Clostridium perfringens epsilon toxin H149A mutant as a platform for receptor binding studies.

Protein Sci 2013 May 8;22(5):650-9. Epub 2013 Apr 8.

Biosciences, College of Life and Environmental Sciences, University of Exeter, Exeter EX4 4QD, United Kingdom.

Clostridium perfringens epsilon toxin (Etx) is a pore-forming toxin responsible for a severe and rapidly fatal enterotoxemia of ruminants. The toxin is classified as a category B bioterrorism agent by the U.S. Government Centres for Disease Control and Prevention (CDC), making work with recombinant toxin difficult. To reduce the hazard posed by work with recombinant Etx, we have used a variant of Etx that contains a H149A mutation (Etx-H149A), previously reported to have reduced, but not abolished, toxicity. The three-dimensional structure of H149A prototoxin shows that the H149A mutation in domain III does not affect organisation of the putative receptor binding loops in domain I of the toxin. Surface exposed tyrosine residues in domain I of Etx-H149A (Y16, Y20, Y29, Y30, Y36 and Y196) were mutated to alanine and mutants Y30A and Y196A showed significantly reduced binding to MDCK.2 cells relative to Etx-H149A that correlated with their reduced cytotoxic activity. Thus, our study confirms the role of surface exposed tyrosine residues in domain I of Etx in binding to MDCK cells and the suitability of Etx-H149A for further receptor binding studies. In contrast, binding of all of the tyrosine mutants to ACHN cells was similar to that of Etx-H149A, suggesting that Etx can recognise different cell surface receptors. In support of this, the crystal structure of Etx-H149A identified a glycan (β-octyl-glucoside) binding site in domain III of Etx-H149A, which may be a second receptor binding site. These findings have important implications for developing strategies designed to neutralise toxin activity.
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http://dx.doi.org/10.1002/pro.2250DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3649266PMC
May 2013

Molecular architecture and functional analysis of NetB, a pore-forming toxin from Clostridium perfringens.

J Biol Chem 2013 Feb 13;288(5):3512-22. Epub 2012 Dec 13.

Department of Biological Sciences, School of Crystallography, Institute of Structural and Molecular Biology, Birkbeck College, Malet Street, London, WC1E 7HX, United Kingdom.

NetB is a pore-forming toxin produced by Clostridium perfringens and has been reported to play a major role in the pathogenesis of avian necrotic enteritis, a disease that has emerged due to the removal of antibiotics in animal feedstuffs. Here we present the crystal structure of the pore form of NetB solved to 3.9 Å. The heptameric assembly shares structural homology to the staphylococcal α-hemolysin. However, the rim domain, a region that is thought to interact with the target cell membrane, shows sequence and structural divergence leading to the alteration of a phosphocholine binding pocket found in the staphylococcal toxins. Consistent with the structure we show that NetB does not bind phosphocholine efficiently but instead interacts directly with cholesterol leading to enhanced oligomerization and pore formation. Finally we have identified conserved and non-conserved amino acid positions within the rim loops that significantly affect binding and toxicity of NetB. These findings present new insights into the mode of action of these pore-forming toxins, enabling the design of more effective control measures against necrotic enteritis and providing potential new tools to the field of bionanotechnology.
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http://dx.doi.org/10.1074/jbc.M112.430223DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3561570PMC
February 2013

Structure of a bacterial voltage-gated sodium channel pore reveals mechanisms of opening and closing.

Nat Commun 2012 ;3:1102

Department of Crystallography, Institute of Structural and Molecular Biology, Birkbeck College, University of London, Malet Street, London WC1E 7HX, UK.

Voltage-gated sodium channels are vital membrane proteins essential for electrical signalling; in humans, they are key targets for the development of pharmaceutical drugs. Here we report the crystal structure of an open-channel conformation of NavMs, the bacterial channel pore from the marine bacterium Magnetococcus sp. (strain MC-1). It differs from the recently published crystal structure of a closed form of a related bacterial sodium channel (NavAb) by having its internal cavity accessible to the cytoplasmic surface as a result of a bend/rotation about a central residue in the carboxy-terminal transmembrane segment. This produces an open activation gate of sufficient diameter to allow hydrated sodium ions to pass through. Comparison of the open and closed structures provides new insight into the features of the functional states present in the activation cycles of sodium channels and the mechanism of channel opening and closing.
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http://dx.doi.org/10.1038/ncomms2077DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3493636PMC
February 2013

Structural analysis of human FANCL, the E3 ligase in the Fanconi anemia pathway.

J Biol Chem 2011 Sep 20;286(37):32628-37. Epub 2011 Jul 20.

Protein Structure and Function Laboratory, Lincoln's Inn Fields Laboratories of the London Research Institute, Cancer Research UK, 44 Lincoln's Inn Fields, London WC2A 3LY, United Kingdom.

The Fanconi anemia (FA) pathway is essential for the repair of DNA interstrand cross-links. At the heart of this pathway is the monoubiquitination of the FANCI-FANCD2 (ID) complex by the multiprotein "core complex" containing the E3 ubiquitin ligase FANCL. Vertebrate organisms have the eight-protein core complex, whereas invertebrates apparently do not. We report here the structure of the central domain of human FANCL in comparison with the recently solved Drosophila melanogaster FANCL. Our data represent the first structural detail into the catalytic core of the human system and reveal that the central fold of FANCL is conserved between species. However, there are macromolecular differences between the FANCL proteins that may account for the apparent distinctions in core complex requirements between the vertebrate and invertebrate FA pathways. In addition, we characterize the binding of human FANCL with its partners, Ube2t, FANCD2, and FANCI. Mutational analysis reveals which residues are required for substrate binding, and we also show the domain required for E2 binding.
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http://dx.doi.org/10.1074/jbc.M111.244632DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3173227PMC
September 2011

The structure of the catalytic subunit FANCL of the Fanconi anemia core complex.

Nat Struct Mol Biol 2010 Mar 14;17(3):294-8. Epub 2010 Feb 14.

Protein Structure and Function Laboratory, Lincoln's Inn Fields Laboratories of the London Research Institute, Cancer Research UK, London Research Institute, London, UK.

The Fanconi anemia (FA) pathway is activated in response to DNA damage, leading to monoubiquitination of the substrates FANCI and FANCD2 by the FA core complex. Here we report the crystal structure of FANCL, the catalytic subunit of the FA core complex, at 3.2 A. The structure reveals an architecture fundamentally different from previous sequence-based predictions. The molecule is composed of an N-terminal E2-like fold, which we term the ELF domain, a novel double-RWD (DRWD) domain, and a C-terminal really interesting new gene (RING) domain predicted to facilitate E2 binding. Binding assays show that the DRWD domain, but not the ELF domain, is responsible for substrate binding.
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http://dx.doi.org/10.1038/nsmb.1759DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2929457PMC
March 2010

Clostridium perfringens epsilon-toxin shows structural similarity to the pore-forming toxin aerolysin.

Nat Struct Mol Biol 2004 Aug 18;11(8):797-8. Epub 2004 Jul 18.

Department of Crystallography, Birkbeck College, Malet Street, London WC1E 7HX, UK.

Epsilon-toxin from Clostridium perfringens is a lethal toxin. Recent studies suggest that the toxin acts via an unusually potent pore-forming mechanism. Here we report the crystal structure of epsilon-toxin, which reveals structural similarity to aerolysin from Aeromonas hydrophila. Pore-forming toxins can change conformation between soluble and transmembrane states. By comparing the two toxins, we have identified regions important for this transformation.
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http://dx.doi.org/10.1038/nsmb804DOI Listing
August 2004

Clostridium absonum alpha-toxin: new insights into clostridial phospholipase C substrate binding and specificity.

J Mol Biol 2003 Oct;333(4):759-69

School of Crystallography, Birkbeck College, Malet Street, London WC1E 7HX, UK.

Clostridium absonum phospholipase C (Caa) is a 42.7 kDa protein, which shows 60% amino acid sequence identity with the Clostridium perfringens phospholipase C, or alpha-toxin (Cpa), and has been isolated from patients suffering from gas gangrene. We report the cloning and sequencing, purification, characterisation and crystal structure of the Caa enzyme. Caa had twice the phospholipid-hydrolysing (lecithinase) activity, 1.5 times the haemolytic activity and over seven times the activity towards phosphatidylcholine-based liposomes when compared with Cpa. However, the Caa enzyme had a lower activity than Cpa to the free (i.e. not in lipid bilayer) substrate para-nitrophenylphosphorylcholine, towards sphingomyelin-based liposomes and showed half the cytotoxicity. The lethal dose (LD(50)) of Caa in mice was approximately twice that of Cpa. The crystal structure of Caa shows that the 72-93 residue loop is in a conformation different from those of previously determined open-form alpha-toxin structures. This conformational change suggests a role for W84 in membrane binding and a possible route of entry into the active site along a hydrophobic channel created by the re-arrangement of this loop. Overall, the properties of Caa are compatible with a role as a virulence-determinant in gas gangrene caused by C.absonum.
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http://dx.doi.org/10.1016/j.jmb.2003.07.016DOI Listing
October 2003
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