Publications by authors named "Claire E Naylor"

24 Publications

  • Page 1 of 1

The pore structure of Clostridium perfringens epsilon toxin.

Nat Commun 2019 06 14;10(1):2641. Epub 2019 Jun 14.

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

Epsilon toxin (Etx), a potent pore forming toxin (PFT) produced by Clostridium perfringens, is responsible for the pathogenesis of enterotoxaemia of ruminants and has been suggested to play a role in multiple sclerosis in humans. Etx is a member of the aerolysin family of β-PFTs (aβ-PFTs). While the Etx soluble monomer structure was solved in 2004, Etx pore structure has remained elusive due to the difficulty of isolating the pore complex. Here we show the cryo-electron microscopy structure of Etx pore assembled on the membrane of susceptible cells. The pore structure explains important mutant phenotypes and suggests that the double β-barrel, a common feature of the aβ-PFTs, may be an important structural element in driving efficient pore formation. These insights provide the framework for the development of novel therapeutics to prevent human and animal infections, and are relevant for nano-biotechnology applications.
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http://dx.doi.org/10.1038/s41467-019-10645-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6572795PMC
June 2019

Ultra-high resolution X-ray structures of two forms of human recombinant insulin at 100 K.

Chem Cent J 2017 Aug 1;11(1):73. Epub 2017 Aug 1.

Department of Crystallography, Biochemical Sciences, Birkbeck College, Malet St, London, WC1E7HX, UK.

The crystal structure of a commercially available form of human recombinant (HR) insulin, Insugen (I), used in the treatment of diabetes has been determined to 0.92 Å resolution using low temperature, 100 K, synchrotron X-ray data collected at 16,000 keV (λ = 0.77 Å). Refinement carried out with anisotropic displacement parameters, removal of main-chain stereochemical restraints, inclusion of H atoms in calculated positions, and 220 water molecules, converged to a final value of R = 0.1112 and R = 0.1466. The structure includes what is thought to be an ordered propanol molecule (POL) only in chain D(4) and a solvated acetate molecule (ACT) coordinated to the Zn atom only in chain B(2). Possible origins and consequences of the propanol and acetate molecules are discussed. Three types of amino acid representation in the electron density are examined in detail: (i) sharp with very clearly resolved features; (ii) well resolved but clearly divided into two conformations which are well behaved in the refinement, both having high quality geometry; (iii) poor density and difficult or impossible to model. An example of type (ii) is observed for the intra-chain disulphide bridge in chain C(3) between Sγ6-Sγ11 which has two clear conformations with relative refined occupancies of 0.8 and 0.2, respectively. In contrast the corresponding S-S bridge in chain A(1) shows one clearly defined conformation. A molecular dynamics study has provided a rational explanation of this difference between chains A and C. More generally, differences in the electron density features between corresponding residues in chains A and C and chains B and D is a common observation in the Insugen (I) structure and these effects are discussed in detail. The crystal structure, also at 0.92 Å and 100 K, of a second commercially available form of human recombinant insulin, Intergen (II), deposited in the Protein Data Bank as 3W7Y which remains otherwise unpublished is compared here with the Insugen (I) structure. In the Intergen (II) structure there is no solvated propanol or acetate molecule. The electron density of Intergen (II), however, does also exhibit the three types of amino acid representations as in Insugen (I). These effects do not necessarily correspond between chains A and C or chains B and D in Intergen (II), or between corresponding residues in Insugen (I). The results of this comparison are reported. Graphical abstract Conformations of PheB25 and PheD25 in three insulin structures: implications for biological activity? Insulin residues PheB25 and PheD25 are considered to be important for insulin receptor binding and changes in biological activity occur when these residues are modified. In porcine insulin and Intergen (II) PheB25 adopts conformation B and PheD25 conformation D. However, unexpectedly PheB25 in Insugen (I) human recombinant insulin adopts two distinct conformations corresponding to B and D, Figure 1 and PheD25 adopts a single conformation corresponding to B not D, Figure 2. Conformations of this residue in the ultra-high resolution structure of Insugen (I) are therefore unique within this set. Figures were produced with Biovia, Discovery Studio 2016.
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http://dx.doi.org/10.1186/s13065-017-0296-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5539060PMC
August 2017

The complete structure of an activated open sodium channel.

Nat Commun 2017 02 16;8:14205. Epub 2017 Feb 16.

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

Voltage-gated sodium channels (Navs) play essential roles in excitable tissues, with their activation and opening resulting in the initial phase of the action potential. The cycling of Navs through open, closed and inactivated states, and their closely choreographed relationships with the activities of other ion channels lead to exquisite control of intracellular ion concentrations in both prokaryotes and eukaryotes. Here we present the 2.45 Å resolution crystal structure of the complete NavMs prokaryotic sodium channel in a fully open conformation. A canonical activated conformation of the voltage sensor S4 helix, an open selectivity filter leading to an open activation gate at the intracellular membrane surface and the intracellular C-terminal domain are visible in the structure. It includes a heretofore unseen interaction motif between W77 of S3, the S4-S5 interdomain linker, and the C-terminus, which is associated with regulation of opening and closing of the intracellular gate.
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http://dx.doi.org/10.1038/ncomms14205DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5316852PMC
February 2017

Cryo-EM structure of lysenin pore elucidates membrane insertion by an aerolysin family protein.

Nat Commun 2016 Apr 6;7:11293. Epub 2016 Apr 6.

MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge CB2 0QH, UK.

Lysenin from the coelomic fluid of the earthworm Eisenia fetida belongs to the aerolysin family of small β-pore-forming toxins (β-PFTs), some members of which are pathogenic to humans and animals. Despite efforts, a high-resolution structure of a channel for this family of proteins has been elusive and therefore the mechanism of activation and membrane insertion remains unclear. Here we determine the pore structure of lysenin by single particle cryo-EM, to 3.1 Å resolution. The nonameric assembly reveals a long β-barrel channel spanning the length of the complex that, unexpectedly, includes the two pre-insertion strands flanking the hypothetical membrane-insertion loop. Examination of other members of the aerolysin family reveals high structural preservation in this region, indicating that the membrane-insertion pathway in this family is conserved. For some toxins, proteolytic activation and pro-peptide removal will facilitate unfolding of the pre-insertion strands, allowing them to form the β-barrel of the channel.
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http://dx.doi.org/10.1038/ncomms11293DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4823867PMC
April 2016

Red Blood Cell Susceptibility to Pneumolysin: CORRELATION WITH MEMBRANE BIOCHEMICAL AND PHYSICAL PROPERTIES.

J Biol Chem 2016 May 16;291(19):10210-27. Epub 2016 Mar 16.

the College of Engineering, Mathematics and Physical Sciences, School of Physics, University of Exeter, Exeter EX4 4QL, United Kingdom.

This study investigated the effect of the biochemical and biophysical properties of the plasma membrane as well as membrane morphology on the susceptibility of human red blood cells to the cholesterol-dependent cytolysin pneumolysin, a key virulence factor of Streptococcus pneumoniae, using single cell studies. We show a correlation between the physical properties of the membrane (bending rigidity and surface and dipole electrostatic potentials) and the susceptibility of red blood cells to pneumolysin-induced hemolysis. We demonstrate that biochemical modifications of the membrane induced by oxidative stress, lipid scrambling, and artificial cell aging modulate the cell response to the toxin. We provide evidence that the diversity of response to pneumolysin in diabetic red blood cells correlates with levels of glycated hemoglobin and that the mechanical properties of the red blood cell plasma membrane are altered in diabetes. Finally, we show that diabetic red blood cells are more resistant to pneumolysin and the related toxin perfringolysin O relative to healthy red blood cells. Taken together, these studies indicate that the diversity of cell response to pneumolysin within a population of human red blood cells is influenced by the biophysical and biochemical status of the plasma membrane and the chemical and/or oxidative stress pre-history of the cell.
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http://dx.doi.org/10.1074/jbc.M115.691899DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4858971PMC
May 2016

Molecular basis of ion permeability in a voltage-gated sodium channel.

EMBO J 2016 Apr 12;35(8):820-30. Epub 2016 Feb 12.

Institute of Structural and Molecular Biology, Birkbeck College University of London, London, UK

Voltage-gated sodium channels are essential for electrical signalling across cell membranes. They exhibit strong selectivities for sodium ions over other cations, enabling the finely tuned cascade of events associated with action potentials. This paper describes the ion permeability characteristics and the crystal structure of a prokaryotic sodium channel, showing for the first time the detailed locations of sodium ions in the selectivity filter of a sodium channel. Electrostatic calculations based on the structure are consistent with the relative cation permeability ratios (Na(+) ≈ Li(+) ≫ K(+), Ca(2+), Mg(2+)) measured for these channels. In an E178D selectivity filter mutant constructed to have altered ion selectivities, the sodium ion binding site nearest the extracellular side is missing. Unlike potassium ions in potassium channels, the sodium ions in these channels appear to be hydrated and are associated with side chains of the selectivity filter residues, rather than polypeptide backbones.
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http://dx.doi.org/10.15252/embj.201593285DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4972137PMC
April 2016

Structural model of the open-closed-inactivated cycle of prokaryotic voltage-gated sodium channels.

J Gen Physiol 2015 Jan 15;145(1):5-16. Epub 2014 Dec 15.

Institute of Structural and Molecular Biology, Birkbeck College, University of London, London WC1E 7HX, England, UK

In excitable cells, the initiation of the action potential results from the opening of voltage-gated sodium channels. These channels undergo a series of conformational changes between open, closed, and inactivated states. Many models have been proposed for the structural transitions that result in these different functional states. Here, we compare the crystal structures of prokaryotic sodium channels captured in the different conformational forms and use them as the basis for examining molecular models for the activation, slow inactivation, and recovery processes. We compare structural similarities and differences in the pore domains, specifically in the transmembrane helices, the constrictions within the pore cavity, the activation gate at the cytoplasmic end of the last transmembrane helix, the C-terminal domain, and the selectivity filter. We discuss the observed differences in the context of previous models for opening, closing, and inactivation, and present a new structure-based model for the functional transitions. Our proposed prokaryotic channel activation mechanism is then compared with the activation transition in eukaryotic sodium channels.
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http://dx.doi.org/10.1085/jgp.201411242DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4278185PMC
January 2015

Structure of a C. perfringens enterotoxin mutant in complex with a modified Claudin-2 extracellular loop 2.

J Mol Biol 2014 Sep 11;426(18):3134-3147. Epub 2014 Jul 11.

Department of Biological Sciences, Birkbeck College, London WC1E 7HX, UK. Electronic address:

CPE (Clostridium perfringens enterotoxin) is the major virulence determinant for C. perfringens type-A food poisoning, the second most common bacterial food-borne illness in the UK and USA. After binding to its receptors, which include particular human claudins, the toxin forms pores in the cell membrane. The mature pore apparently contains a hexamer of CPE, claudin and, possibly, occludin. The combination of high binding specificity with cytotoxicity has resulted in CPE being investigated, with some success, as a targeted cytotoxic agent for oncotherapy. In this paper, we present the X-ray crystallographic structure of CPE in complex with a peptide derived from extracellular loop 2 of a modified, CPE-binding Claudin-2, together with high-resolution native and pore-formation mutant structures. Our structure provides the first atomic-resolution data on any part of a claudin molecule and reveals that claudin's CPE-binding fingerprint (NPLVP) is in a tight turn conformation and binds, as expected, in CPE's C-terminal claudin-binding groove. The leucine and valine residues insert into the binding groove while the first residue, asparagine, tethers the peptide via an interaction with CPE's aspartate 225 and the two prolines are required to maintain the tight turn conformation. Understanding the structural basis of the contribution these residues make to binding will aid in engineering CPE to target tumor cells.
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http://dx.doi.org/10.1016/j.jmb.2014.07.001DOI Listing
September 2014

Prokaryotic NavMs channel as a structural and functional model for eukaryotic sodium channel antagonism.

Proc Natl Acad Sci U S A 2014 Jun 21;111(23):8428-33. Epub 2014 May 21.

Institute of Structural and Molecular Biology, School of Biological Sciences, Birkbeck College, University of London, London WC1E 7HX, United Kingdom;

Voltage-gated sodium channels are important targets for the development of pharmaceutical drugs, because mutations in different human sodium channel isoforms have causal relationships with a range of neurological and cardiovascular diseases. In this study, functional electrophysiological studies show that the prokaryotic sodium channel from Magnetococcus marinus (NavMs) binds and is inhibited by eukaryotic sodium channel blockers in a manner similar to the human Nav1.1 channel, despite millions of years of divergent evolution between the two types of channels. Crystal complexes of the NavMs pore with several brominated blocker compounds depict a common antagonist binding site in the cavity, adjacent to lipid-facing fenestrations proposed to be the portals for drug entry. In silico docking studies indicate the full extent of the blocker binding site, and electrophysiology studies of NavMs channels with mutations at adjacent residues validate the location. These results suggest that the NavMs channel can be a valuable tool for screening and rational design of human drugs.
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http://dx.doi.org/10.1073/pnas.1406855111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4060673PMC
June 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

Identification of a key residue for oligomerisation and pore-formation of Clostridium perfringens NetB.

Toxins (Basel) 2014 Mar 12;6(3):1049-61. Epub 2014 Mar 12.

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

Necrotic enteritis toxin B (NetB) is a β-pore-forming toxin produced by Clostridium perfringens and has been identified as a key virulence factor in the pathogenesis of avian necrotic enteritis, a disease causing significant economic damage to the poultry industry worldwide. In this study, site-directed mutagenesis was used to identify amino acids that play a role in NetB oligomerisation and pore-formation. NetB K41H showed significantly reduced toxicity towards LMH cells and human red blood cells relative to wild type toxin. NetB K41H was unable to oligomerise and form pores in liposomes. These findings suggest that NetB K41H could be developed as a genetic toxoid vaccine to protect against necrotic enteritis.
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http://dx.doi.org/10.3390/toxins6031049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3968376PMC
March 2014

Role of the C-terminal domain in the structure and function of tetrameric sodium channels.

Nat Commun 2013 ;4:2465

1] School of Biological Sciences, Institute of Structural and Molecular Biology, Birkbeck College, University of London, London WC1E 7HX, UK [2].

Voltage-gated sodium channels have essential roles in electrical signalling. Prokaryotic sodium channels are tetramers consisting of transmembrane (TM) voltage-sensing and pore domains, and a cytoplasmic carboxy-terminal domain. Previous crystal structures of bacterial sodium channels revealed the nature of their TM domains but not their C-terminal domains (CTDs). Here, using electron paramagnetic resonance (EPR) spectroscopy combined with molecular dynamics, we show that the CTD of the NavMs channel from Magnetococcus marinus includes a flexible region linking the TM domains to a four-helix coiled-coil bundle. A 2.9 Å resolution crystal structure of the NavMs pore indicates the position of the CTD, which is consistent with the EPR-derived structure. Functional analyses demonstrate that the coiled-coil domain couples inactivation with channel opening, and is enabled by negatively charged residues in the linker region. A mechanism for gating is proposed based on the structure, whereby splaying of the bottom of the pore is possible without requiring unravelling of the coiled-coil.
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http://dx.doi.org/10.1038/ncomms3465DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3791462PMC
April 2014

Structural Insights into Clostridium perfringens Delta Toxin Pore Formation.

PLoS One 2013 21;8(6):e66673. Epub 2013 Jun 21.

Department of Biological Sciences, Birkbeck College, London, United Kingdom.

Clostridium perfringens Delta toxin is one of the three hemolysin-like proteins produced by C. perfringens type C and possibly type B strains. One of the others, NetB, has been shown to be the major cause of Avian Nectrotic Enteritis, which following the reduction in use of antibiotics as growth promoters, has become an emerging disease of industrial poultry. Delta toxin itself is cytotoxic to the wide range of human and animal macrophages and platelets that present GM2 ganglioside on their membranes. It has sequence similarity with Staphylococcus aureus β-pore forming toxins and is expected to heptamerize and form pores in the lipid bilayer of host cell membranes. Nevertheless, its exact mode of action remains undetermined. Here we report the 2.4 Å crystal structure of monomeric Delta toxin. The superposition of this structure with the structure of the phospholipid-bound F component of S. aureus leucocidin (LukF) revealed that the glycerol molecules bound to Delta toxin and the phospholipids in LukF are accommodated in the same hydrophobic clefts, corresponding to where the toxin is expected to latch onto the membrane, though the binding sites show significant differences. From structure-based sequence alignment with the known structure of staphylococcal α-hemolysin, a model of the Delta toxin pore form has been built. Using electron microscopy, we have validated our model and characterized the Delta toxin pore on liposomes. These results highlight both similarities and differences in the mechanism of Delta toxin (and by extension NetB) cytotoxicity from that of the staphylococcal pore-forming toxins.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0066673PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3689675PMC
October 2017

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

Structure of the food-poisoning Clostridium perfringens enterotoxin reveals similarity to the aerolysin-like pore-forming toxins.

J Mol Biol 2011 Oct 3;413(1):138-49. Epub 2011 Aug 3.

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

Clostridium perfringens enterotoxin (CPE) is a major cause of food poisoning and antibiotic-associated diarrhea. Upon its release from C. perfringens spores, CPE binds to its receptor, claudin, at the tight junctions between the epithelial cells of the gut wall and subsequently forms pores in the cell membranes. A number of different complexes between CPE and claudin have been observed, and the process of pore formation has not been fully elucidated. We have determined the three-dimensional structure of the soluble form of CPE in two crystal forms by X-ray crystallography, to a resolution of 2.7 and 4.0 Å, respectively, and found that the N-terminal domain shows structural homology with the aerolysin-like β-pore-forming family of proteins. We show that CPE forms a trimer in both crystal forms and that this trimer is likely to be biologically relevant but is not the active pore form. We use these data to discuss models of pore formation.
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http://dx.doi.org/10.1016/j.jmb.2011.07.066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3235586PMC
October 2011

Molecular basis of toxicity of Clostridium perfringens epsilon toxin.

FEBS J 2011 Dec 19;278(23):4589-601. Epub 2011 May 19.

Biosciences, College of Life and Environmental Sciences, University of Exeter, UK.

Clostridium perfringens ε-toxin is produced by toxinotypes B and D strains. The toxin is the aetiological agent of dysentery in newborn lambs but is also associated with enteritis and enterotoxaemia in goats, calves and foals. It is considered to be a potential biowarfare or bioterrorism agent by the US Government Centers for Disease Control and Prevention. The relatively inactive 32.9 kDa prototoxin is converted to active mature toxin by proteolytic cleavage, either by digestive proteases of the host, such as trypsin and chymotrypsin, or by C. perfringens λ-protease. In vivo, the toxin appears to target the brain and kidneys, but relatively few cell lines are susceptible to the toxin, and most work has been carried out using Madin-Darby canine kidney (MDCK) cells. The binding of ε-toxin to MDCK cells and rat synaptosomal membranes is associated with the formation of a stable, high molecular weight complex. The crystal structure of ε-toxin reveals similarity to aerolysin from Aeromonas hydrophila, parasporin-2 from Bacillus thuringiensis and a lectin from Laetiporus sulphureus. Like these toxins, ε-toxin appears to form heptameric pores in target cell membranes. The exquisite specificity of the toxin for specific cell types suggests that it binds to a receptor found only on these cells.
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http://dx.doi.org/10.1111/j.1742-4658.2011.08140.xDOI Listing
December 2011

Identification of zyklopen, a new member of the vertebrate multicopper ferroxidase family, and characterization in rodents and human cells.

J Nutr 2010 Oct 4;140(10):1728-35. Epub 2010 Aug 4.

Department of Nutritional Science and Toxicology, University of California, Berkeley, CA 94720, USA.

We previously detected a membrane-bound, copper-containing oxidase that may be involved in iron efflux in BeWo cells, a human placental cell line. We have now identified a gene encoding a predicted multicopper ferroxidase (MCF) with a putative C-terminal membrane-spanning sequence and high sequence identity to hephaestin (Heph) and ceruloplasmin (Cp), the other known vertebrate MCF. Molecular modeling revealed conservation of all type I, II, and III copper-binding sites as well as a putative iron-binding site. Protein expression was observed in multiple diverse mouse tissues, including placenta and mammary gland, and the expression pattern was distinct from that of Cp and Heph. The protein possessed ferroxidase activity, and protein levels decreased in cellular copper deficiency. Knockdown with small interfering RNA in BeWo cells indicates that this gene represents the previously detected oxidase. We propose calling this new member of the MCF family "zyklopen."
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http://dx.doi.org/10.3945/jn.109.117531DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2937573PMC
October 2010

Structural relationships and cellular tropism of staphylococcal superantigen-like proteins.

Infect Immun 2004 Jul;72(7):4261-70

Department of Immunology and Molecular Pathology, University College London, 46 Cleveland Street, London W1T 4JF, United Kingdom.

The staphylococcal superantigen-like proteins (SSLs) are a family of polymorphic paralogs encoded in the Staphylococcus aureus genome whose function is unknown. The crystal structure of SSL7 was determined and compared to that of SSL5 and that of a classical superantigen, streptococcal pyrogenic exotoxin. Although the overall architecture of the superantigen family is retained in both SSL7 and SSL5, there are significant differences in the structures which suggest that the characteristic major histocompatibility complex binding site of superantigens has been lost. To complement these data, the abilities of SSL7 and a closely related paralog, SSL9, to interact with cells of the immune system were investigated. In populations of human white blood cells, both SSLs interacted selectively with monocytes via specific saturable but separate binding sites, which led to rapid uptake of the SSLs. In addition, SSLs were rapidly taken up by dendritic cells, but not by macrophages, into the same endosomal compartment as dextran. The ability of these secreted proteins to target antigen-presenting cells may enhance a misplaced antibody response against the proteins, which may facilitate bacterial colonization rather than contribute to host protection. Like classical superantigens, therefore, SSLs may distract the host's immune system, but they may do so via entirely different molecular mechanisms.
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http://dx.doi.org/10.1128/IAI.72.7.4261-4270.2004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC427445PMC
July 2004

Hephaestin is a ferroxidase that maintains partial activity in sex-linked anemia mice.

Blood 2004 May 29;103(10):3933-9. Epub 2004 Jan 29.

Department of Nutrition and Toxicology, University of California, Berkeley, Berkeley, CA 94720, USA.

Hephaestin (Hp) plays an important role in intestinal iron absorption and is predicted to be a ferroxidase based on significant sequence identity to the serum multicopper ferroxidase ceruloplasmin. Here, we demonstrate that Hp has both amine oxidase and ferroxidase activity in cultured cells and primary intestinal enterocytes with the use of both gel and solution assays. The specificity of the activity is shown by immunoblotting, immunoprecipitation, and immunodepletion experiments. Surprisingly, the truncated hephaestin expressed in sex-linked anemia (sla) mice still has measurable, but decreased, oxidase activity. Molecular modeling of the truncated hephaestin suggests retention of a minimum catalytic core required for enzymatic activity. We suggest that hephaestin, by way of its ferroxidase activity, facilitates iron export from intestinal enterocytes, most likely in cooperation with the basolateral iron transporter, Ireg1.
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http://dx.doi.org/10.1182/blood-2003-09-3139DOI Listing
May 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

Crystal structure of the C. perfringens alpha-toxin with the active site closed by a flexible loop region.

J Mol Biol 2002 May;319(2):275-81

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

Clostridium perfringens biotype A strains are the causative agents of gas-gangrene in man and are also implicated as etiological agents in sudden death syndrome in young domestic livestock. The main virulence factor produced by these strains is a zinc-dependent, phosphatidylcholine-preferring phospholipase C (alpha-toxin). The crystal structure of alpha-toxin, at pH 7.5, with the active site open and therefore accessible to substrate has previously been reported, as has calcium-binding to the C-terminal domain of the enzyme at pH 4.7. Here we focus on conformation changes in the N-terminal domain of alpha-toxin in crystals grown at acidic pH. These changes result in both the obscuring of the toxin active site and the loss of one of three zinc ions from it. Additionally, this "closed" form contains a small alpha helix, not present in the open structure, which hydrogen bonds to both the N and C-terminal domains. In conjunction with the previously reported findings that alpha-toxin can exist in active and inactive forms and that Thr74Ile and Phe69Cys substitutions markedly reduced the haemolytic activity of the enzyme, our work suggests that these loop conformations play a critical role in the activity of the toxin.
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http://dx.doi.org/10.1016/S0022-2836(02)00290-5DOI Listing
May 2002

Analysis of the human hephaestin gene and protein: comparative modelling of the N-terminus ecto-domain based upon ceruloplasmin.

Protein Eng 2002 Mar;15(3):205-14

Department of Biochemistry and Molecular Biology, Royal Free and University College Medical School, London NW3 2PF, UK.

Hephaestin was implicated in mammalian iron homeostasis following its identification as the defective gene in murine sex-linked anaemia. It is a member of the family of copper oxidases that includes mammalian ceruloplasmin, factors V and VIII, yeast fet3 and fet5 and bacterial ascorbate oxidase. Hephaestin is different from ceruloplasmin, a soluble ferroxidase, in having a membrane-spanning region towards the C-terminus. Here we report the gene structure, spanning approximately 100 kb, of the human homologue of mouse hephaestin. The sequence was assembled from the cDNA clones and the chromosome X genomic sequence data available at the Sanger Centre. It has an open reading frame that encodes a protein of 1158 residues, 85% identical with the murine homologue. A model of the N-terminal ecto-domain has been built based on the known three-dimensional structure of human ceruloplasmin. The overall tertiary structure for the hephaestin and the putative residues involved in binding copper and iron appear to be highly conserved between these proteins, which suggests they share the same fold and a conserved function.
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http://dx.doi.org/10.1093/protein/15.3.205DOI Listing
March 2002
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