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Journal ArticleDOI

Crystal structure of botulinum neurotoxin type A and implications for toxicity.

01 Oct 1998-Nature Structural & Molecular Biology (Nat Struct Biol)-Vol. 5, Iss: 10, pp 898-902
TL;DR: The crystal structure of the entire 1,285 amino acid di-chain neurotoxin was determined and the toxin appears as a hybrid of varied structural motifs and suggests a modular assembly of functional subunits to yield pathogenesis.
Abstract: Botulinum neurotoxin type A (BoNT/A) is the potent disease agent in botulism, a potential biological weapon and an effective therapeutic drug for involuntary muscle disorders. The crystal structure of the entire 1,285 amino acid di-chain neurotoxin was determined at 3.3 A resolution. The structure reveals that the translocation domain contains a central pair of alpha-helices 105 A long and a approximately 50 residue loop or belt that wraps around the catalytic domain. This belt partially occludes a large channel leading to a buried, negative active site--a feature that calls for radically different inhibitor design strategies from those currently used. The fold of the translocation domain suggests a mechanism of pore formation different from other toxins. Lastly, the toxin appears as a hybrid of varied structural motifs and suggests a modular assembly of functional subunits to yield pathogenesis.
Citations
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Journal ArticleDOI
28 Feb 2001-JAMA
TL;DR: People potentially exposed to botulinum toxin should be closely observed, and those with signs of botulism require prompt treatment with antitoxin and supportive care that may include assisted ventilation for weeks or months.
Abstract: ObjectiveThe Working Group on Civilian Biodefense has developed consensus-based recommendations for measures to be taken by medical and public health professionals if botulinum toxin is used as a biological weapon against a civilian population.ParticipantsThe working group included 23 representatives from academic, government, and private institutions with expertise in public health, emergency management, and clinical medicine.EvidenceThe primary authors (S.S.A. and R.S.) searched OLDMEDLINE and MEDLINE (1960–March 1999) and their professional collections for literature concerning use of botulinum toxin as a bioweapon. The literature was reviewed, and opinions were sought from the working group and other experts on diagnosis and management of botulism. Additional MEDLINE searches were conducted through April 2000 during the review and revisions of the consensus statement.Consensus ProcessThe first draft of the working group's consensus statement was a synthesis of information obtained in the formal evidence-gathering process. The working group convened to review the first draft in May 1999. Working group members reviewed subsequent drafts and suggested additional revisions. The final statement incorporates all relevant evidence obtained in the literature search in conjunction with final consensus recommendations supported by all working group members.ConclusionsAn aerosolized or foodborne botulinum toxin weapon would cause acute symmetric, descending flaccid paralysis with prominent bulbar palsies such as diplopia, dysarthria, dysphonia, and dysphagia that would typically present 12 to 72 hours after exposure. Effective response to a deliberate release of botulinum toxin will depend on timely clinical diagnosis, case reporting, and epidemiological investigation. Persons potentially exposed to botulinum toxin should be closely observed, and those with signs of botulism require prompt treatment with antitoxin and supportive care that may include assisted ventilation for weeks or months. Treatment with antitoxin should not be delayed for microbiological testing.

1,659 citations


Cites background from "Crystal structure of botulinum neur..."

  • ...3 A.(38) The toxin’s light chain is a Zncontaining endopeptidase that blocks acetylcholine-containing vesicles from fusingwiththeterminalmembraneof the motorneuron, resulting in flaccidmuscle paralysis (FIGURE 1)....

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Journal ArticleDOI
TL;DR: The mechanism of action of three groups of presynaptic neurotoxins that interfere directly with the process of neurotransmitter release is reviewed, whereas presynapses acting on ion channels are not dealt with here.
Abstract: Nerve terminals are specific sites of action of a very large number of toxins produced by many different organisms. The mechanism of action of three groups of presynaptic neurotoxins that interfere directly with the process of neurotransmitter release is reviewed, whereas presynaptic neurotoxins acting on ion channels are not dealt with here. These neurotoxins can be grouped in three large families: 1) the clostridial neurotoxins that act inside nerves and block neurotransmitter release via their metalloproteolytic activity directed specifically on SNARE proteins; 2) the snake presynaptic neurotoxins with phospholipase A(2) activity, whose site of action is still undefined and which induce the release of acethylcholine followed by impairment of synaptic functions; and 3) the excitatory latrotoxin-like neurotoxins that induce a massive release of neurotransmitter at peripheral and central synapses. Their modes of binding, sites of action, and biochemical activities are discussed in relation to the symptoms of the diseases they cause. The use of these toxins in cell biology and neuroscience is considered as well as the therapeutic utilization of the botulinum neurotoxins in human diseases characterized by hyperfunction of cholinergic terminals.

1,196 citations


Cites background from "Crystal structure of botulinum neur..."

  • ...Here we only mention results that are relevant with respect to the recent identification of a sugar-binding subdomain in BoNT/A and TeNT (326, 610)....

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  • ...Two experimental results do not fit in this model: 1) the L chains of TeNT and BoNT/A, /B, and /E penetrate the lipid bilayer in such a way as to be exposed to the fatty acid chains of phospholipids, i.e., they are not shielded from lipids inside the H chain tunnel (420, 421); and 2) values of the order of a few tens of picoSiemens do not account for the dimensions expected for a protein channel that has to accommodate a polypeptide chain with lateral groups of different volume, charge, and hydrophilicity....

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  • ...The HC domains of TeNT and BoNT/A, /B, and /E appear to be sufficient for the internalization process in murine spinal cord neurons (328)....

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  • ...The sugarbinding and protein binding subdomains present in the HC domain of TeNT and BoNT/A (326, 611) and the protection experiments mentioned above support the suggestion that CNT may bind strongly and specifically to the presynaptic membrane because they display multiple interactions with sugar and protein binding sites (413)....

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  • ...Another characteritic of the active site of BoNT/A is that it is 20–24 Å deep in the protein and that it is accessible via an anionic channel, not accessible in the intact molecule because it is shielded by HN and its wrapping belt (326)....

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Journal ArticleDOI
David S. Auld1
TL;DR: The influence of zinc on quaternary protein structure has led to the identification of a fourth type of zinc binding site, protein interface, which is formed from ligands supplied from amino acid residues residing in the binding surface of two proteins.
Abstract: Zinc is known to be indispensable to growth and development and transmission of the genetic message. It does this through a remarkable mosaic of zinc binding motifs that orchestrate all aspects of metabolism. There are now nearly 200 three dimensional structures for zinc proteins, representing all six classes of enzymes and covering a wide range of phyla and species. These structures provide standards of reference for the identity and nature of zinc ligands in other proteins for which only the primary structure is known. Three primary types of zinc sites are apparent from examination of these structures: structural, catalytic and cocatalytic. The most common amino acids that supply ligands to these sites are His, Glu, Asp and Cys. In catalytic sites zinc generally forms complexes with water and any three nitrogen, oxygen and sulfur donors with His being the predominant amino acid chosen. Water is always a ligand to such sites. Structural zinc sites have four protein ligands and no bound water molecule. Cys is the preferred ligand in such sites. Cocatalytic sites contain two or three metals in close proximity with two of the metals bridged by a side chain moiety of a single amino acid residue, such as Asp, Glu or His and sometimes a water molecule. Asp and His are the preferred amino acids for these sites. No Cys ligands are found in such sites. The scaffolding of the zinc sites is also important to the function and reactivity of the bound metal. The influence of zinc on quaternary protein structure has led to the identification of a fourth type of zinc binding site, protein interface. In this case zinc sites are formed from ligands supplied from amino acid residues residing in the binding surface of two proteins. The resulting zinc site usually has the coordination properties of a catalytic or structural zinc binding site.

699 citations

Journal ArticleDOI
TL;DR: This review seeks to identify and characterize all major steps in toxin action, from initial absorption to eventual paralysis of cholinergic transmission, as well as an agent that can be used to treat disease.
Abstract: Botulinum toxin is a uniquely potent substance synthesized by the organisms Clostridium botulinum, Clostridium baratii, and Clostridium butyricum. This toxin, which acts preferentially on peripheral cholinergic nerve endings to block acetylcholine release, is both an agent that causes disease (i.e., botulism) as well as an agent that can be used to treat disease (e.g., dystonia). The ability of botulinum toxin to produce its effects is largely dependent on its ability to penetrate cellular and intracellular membranes. Thus, toxin that is ingested or inhaled can bind to epithelial cells and be transported to the general circulation. Toxin that reaches peripheral nerve endings binds to the cell surface then penetrates the plasma membrane by receptor-mediated endocytosis and the endosome membrane by pH-induced translocation. Internalized toxin acts in the cytosol as a metalloendoprotease to cleave polypeptides that are essential for exocytosis. This review seeks to identify and characterize all major steps in toxin action, from initial absorption to eventual paralysis of cholinergic transmission.

520 citations

Journal ArticleDOI
TL;DR: How the proteolytic attack at specific sites of the targets for BoNTs and TeNT induces perturbations of the fusogenic SNARE complex dynamics and how these alterations can account for the inhibition of spontaneous and evoked quantal neurotransmitter release by the neurotoxins are explained.

492 citations


Cites background from "Crystal structure of botulinum neur..."

  • ...Examination of the three-dimensional structure of BoNT/A shows that when the interchain disulphide bridge that links heavy and light chains is intact, the N-terminal portion of the heavy chain masks the catalytic cleft in the light chain, thereby preventing substrate access [15]....

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  • ...Finally, the tridimensional structure of botulinum neurotoxin type A has now been resolved [15]....

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  • ...Only the tridimensional structure of BoNT/A has been resolved [15]....

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References
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Journal ArticleDOI
TL;DR: The MOLSCRIPT program as discussed by the authors produces plots of protein structures using several different kinds of representations, including simple wire models, ball-and-stick models, CPK models and text labels.
Abstract: The MOLSCRIPT program produces plots of protein structures using several different kinds of representations. Schematic drawings, simple wire models, ball-and-stick models, CPK models and text labels can be mixed freely. The schematic drawings are shaded to improve the illusion of three dimensionality. A number of parameters affecting various aspects of the objects drawn can be changed by the user. The output from the program is in PostScript format.

13,971 citations

Journal ArticleDOI
TL;DR: In this paper, the authors describe strategies and tools that help to alleviate this problem and simplify the model-building process, quantify the goodness of fit of the model on a per-residue basis and locate possible errors in peptide and side-chain conformations.
Abstract: Map interpretation remains a critical step in solving the structure of a macromolecule. Errors introduced at this early stage may persist throughout crystallographic refinement and result in an incorrect structure. The normally quoted crystallographic residual is often a poor description for the quality of the model. Strategies and tools are described that help to alleviate this problem. These simplify the model-building process, quantify the goodness of fit of the model on a per-residue basis and locate possible errors in peptide and side-chain conformations.

12,936 citations

Journal ArticleDOI
01 Dec 1991-Proteins
TL;DR: It is demonstrated in this work that the surface tension, water‐organic solvent, transfer‐free energies and the thermodynamics of melting of linear alkanes provide fundamental insights into the nonpolar driving forces for protein folding and protein binding reactions.
Abstract: We demonstrate in this work that the surface tension, water-organic solvent, transfer-free energies and the thermodynamics of melting of linear alkanes provide fundamental insights into the nonpolar driving forces for protein folding and protein binding reactions. We first develop a model for the curvature dependence of the hydrophobic effect and find that the macroscopic concept of interfacial free energy is applicable at the molecular level. Application of a well-known relationship involving surface tension and adhesion energies reveals that dispersion forces play little or no net role in hydrophobic interactions; rather, the standard model of disruption of water structure (entropically driven at 25 degrees C) is correct. The hydrophobic interaction is found, in agreement with the classical picture, to provide a major driving force for protein folding. Analysis of the melting behavior of hydrocarbons reveals that close packing of the protein interior makes only a small free energy contribution to folding because the enthalpic gain resulting from increased dispersion interactions (relative to the liquid) is countered by the freezing of side chain motion. The identical effect should occur in association reactions, which may provide an enormous simplification in the evaluation of binding energies. Protein binding reactions, even between nearly planar or concave/convex interfaces, are found to have effective hydrophobicities considerably smaller than the prediction based on macroscopic surface tension. This is due to the formation of a concave collar region that usually accompanies complex formation. This effect may preclude the formation of complexes between convex surfaces.

5,295 citations

Journal ArticleDOI
TL;DR: In this article, a method is given to estimate the parameter σA in these phase probability expressions from the observed and calculated structure factor amplitudes, from which one can estimate the mean coordinate error for the model, and when there are coordinate errors, a new expression for the non-centric Fourier coefficients is required to suppress this model bias.
Abstract: Unrefined or partially refined models of macromolecules are generally incomplete and typically have large coordinate errors. It is shown that phase probability equations appropriate for a perfect partial structure lead to inaccurate estimates of phase probabilities in such cases. Therefore, it is necessary to use equations that have been derived allowing for errors in the partial structure. A method is given to estimate the parameter σA in these phase probability expressions from the observed and calculated structure factor amplitudes. From the variation of σA with resolution, one can estimate the mean coordinate error for the model. Electron density maps calculated using partial structure phases are biased towards the partial structure. When there are coordinate errors, a new expression for the non-centric Fourier coefficients [(2m|FN| - D|FcP|) exp(iαcP)] is required to suppress this model bias. Judged by correlation coefficients comparing electron density maps with the correct and the partial structure maps, the Fourier coefficients derived here are superior to others currently in use.

1,931 citations

Journal ArticleDOI
22 May 1997-Nature
TL;DR: X-ray crystallography determines the structure of the protease-resistant part of a gp41 ectodomain solubilized with a trimeric GCN4 coiled coil in place of the amino-terminal fusion peptide, and suggests a common mechanism for initiating fusion.
Abstract: Fusion of viral and cellular membranes by the envelope glycoprotein gp120/gp41 effects entry of HIV-1 into the cell. The precursor, gp160, is cleaved post-translationally into gp120 and gp41 which remain non-covalently associated. Binding to both CD4 and a co-receptor leads to the conformational changes in gp120/gp41 needed for membrane fusion. We used X-ray crystallography to determine the structure of the protease-resistant part of a gp41 ectodomain solubilized with a trimeric GCN4 coiled coil in place of the amino-terminal fusion peptide. The core of the molecule is found to be an extended, triple-stranded alpha-helical coiled coil with the amino terminus at its tip. A carboxy-terminal alpha-helix packs in the reverse direction against the outside of the coiled coil, placing the amino and carboxy termini near each other at one end of the long rod. These features, and the existence of a similar reversal of chain direction in the fusion pH-induced conformation of influenza virus HA2 and in the transmembrane subunit of Moloney murine leukaemia virus (Fig. 1a-d), suggest a common mechanism for initiating fusion.

1,666 citations


"Crystal structure of botulinum neur..." refers background in this paper

  • ...In fact, the long pair of a-helices with their triple helix bundles at either end bear more resemblance to the coiled coil viral proteins: HIV-1 gp41/GCN4, influenza hemagglutinin, and the MoMuLV TM fragmen...

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