Crystal structure of cholera toxin B-pentamer bound to receptor GM1 pentasaccharide.

Cholera toxin (CT) is an AB5 hexameric protein responsible for the symptoms produced by Vibrio cholerae infection. In the first step of cell intoxication, the B-pentamer of the toxin binds specifically to the branched pentasaccharide moiety of ganglioside GM1 on the surface of target human intestinal epithelial cells. We present here the crystal structure of the cholera toxin B-pentamer complexed with the GM1 pentasaccharide. Each receptor binding site on the toxin is found to lie primarily within a single B-subunit, with a single solvent-mediated hydrogen bond from residue Gly 33 of an adjacent subunit. The large majority of interactions between the receptor and the toxin involve the 2 terminal sugars of GM1, galactose and sialic acid, with a smaller contribution from the N-acetyl galactosamine residue. The binding of GM1 to cholera toxin thus resembles a 2-fingered grip: the Gal(beta 1-3)GalNAc moiety representing the "forefinger" and the sialic acid representing the "thumb." The residues forming the binding site are conserved between cholera toxin and the homologous heat-labile enterotoxin from Escherichia coli, with the sole exception of His 13. Some reported differences in the binding affinity of the 2 toxins for gangliosides other than GM1 may be rationalized by sequence differences at this residue. The CTB5:GM1 pentasaccharide complex described here provides a detailed view of a protein:ganglioside specific binding interaction, and as such is of interest not only for understanding cholera pathogenesis and for the design of drugs and development of vaccines but also for modeling other protein:ganglioside interactions such as those involved in GM1-mediated signal transduction.     

Vibrio cholerae cytolysin is composed of an alpha-hemolysin-like core

The enteric pathogen Vibrio cholerae secretes a water-soluble 80-kD cytolysin, Vibrio cholerae cytolysin (VCC) that assembles into pentameric channels following proteolytic activation by exogenous proteases. Until now, VCC has been placed in a unique class of pore-forming toxins, distinct from paradigms such as Staphyloccal alpha-hemolysin. However, as reported here, amino acid sequence analysis and three-dimensional structure modeling indicate that the core component of the VCC toxin is related in sequence and structure to a family of hemolysins from Staphylococcus aureus that include leukocidin F and alpha-hemolysin. Furthermore, our analysis has identified the channel-forming region of VCC and a potential lipid head-group binding site, and suggests a conserved mechanism of assembly and lysis. An additional domain in the VCC toxin is related to plant lectins, conferring additional target cell specificity to the toxin.     

Deduction of functional peptide motifs in scorpion toxins.

Scorpion toxins are important physiological probes for characterizing ion channels. Molecular databases have limited functional annotation of scorpion toxins. Their function can be inferred by searching for conserved motifs in sequence signature databases that are derived statistically but are not necessarily biologically relevant. Mutation studies provide biological information on residues and positions important for structure-function relationship but are not normally used for extraction of binding motifs. 3D structure analyses also aid in the extraction of peptide motifs in which non-contiguous residues are clustered spatially. Here we present new, functionally relevant peptide motifs for ion channels, derived from the analyses of scorpion toxin native and mutant peptides.     

The membrane localization domains of two distinct bacterial toxins form a 4‐helix‐bundle in solution

Membrane localization domain (MLD) was first proposed for a 4‐helix‐bundle motif in the crystal structure of the C1 domain of Pasteurella multocida toxin (PMT). This structure motif is also found in the crystal structures of several clostridial glycosylating toxins (TcdA, TcdB, TcsL, and TcnA). The Ras/Rap1‐specific endopeptidase (RRSP) module of the multifunctional autoprocessing repeats‐in‐toxins (MARTX) toxin produced by Vibrio vulnificus has sequence homology to the C1‐C2 domains of PMT, including a putative MLD. We have determined the solution structure for the MLDs in PMT and in RRSP using solution state NMR. We conclude that the MLDs in these two toxins assume a 4‐helix‐bundle structure in solution.     

Structure-based sequence alignment for the beta-trefoil subdomain of the clostridial neurotoxin family provides residue level information about the putative ganglioside binding site

Clostridial neurotoxins embrace a family of extremely potent toxins comprised of tetanus toxin (TeNT) and seven different serotypes of botulinum toxin (BoNT/A-G). The beta-trefoil subdomain of the C-terminal part of the heavy chain (H(C)), responsible for ganglioside binding, is the most divergent region in clostridial neurotoxins with sequence identity as low as 15%. We re-examined the alignment between family sequences within this subdomain, since in this region all alignments published to date show obvious inconsistencies with the beta-trefoil fold. The final alignment was obtained by considering the general constraints imposed by this fold, and homology modeling studies based on the TeNT structure. Recently solved structures of BoNT/A confirm the validity of this structure-based approach. Taking into account biochemical data and crystal structures of TeNT and BoNT/A, we also re-examined the location of the putative ganglioside binding site and, using the new alignment, characterized this site in other BoNT serotypes.     

Computer modelling of the NAD binding site of ADP-ribosylating toxins: active-site structure and mechanism of NAD binding

Five ADP-ribosylating bacterial toxins, pertussis toxin, cholera toxin, diphtheria toxin, Escherichia LT toxin and Pseudomonas exotoxin A, show significant homology in selected segments of their sequence. Site-directed mutagenesis and chemical modification of residues within these regions cause loss of catalytic activity and of NAD binding. On the basis of these results and of molecular modelling based on the three-dimensional structure of exotoxin A, the geometry of an NAD binding site common to all the toxins is deduced and described in the paper. For diphtheria toxin, sequence similarity with exotoxin A is such that its preliminary structure can be computed by molecular modelling, whereas for the other toxins similarity appears to be restricted to the NAD binding site. Moreover, an analysis of molecular fitting of the NAD molecule into its binding cavity suggests a new model for the conformation of the bound NAD that better accounts for all available experimental information.     

Autoprocessing of the Vibrio cholerae RTX toxin by the cysteine protease domain

Vibrio cholerae RTX is a large multifunctional bacterial toxin that causes actin crosslinking. Due to its size, it was predicted to undergo proteolytic cleavage during translocation into host cells to deliver activity domains to the cytosol. In this study, we identified a domain within the RTX toxin that is conserved in large clostridial glucosylating toxins TcdB, TcdA, TcnA, and TcsL; putative toxins from V. vulnificus, Yersinia sp., Photorhabdus sp., and Xenorhabdus sp.; and a filamentous/hemagglutinin-like protein FhaL from Bordetella sp. In vivo transfection studies and in vitro characterization of purified recombinant protein revealed that this domain from the V. cholerae RTX toxin is an autoprocessing cysteine protease whose activity is stimulated by the intracellular environment. A cysteine point mutation within the RTX holotoxin attenuated actin crosslinking activity suggesting that processing of the toxin is an important step in toxin translocation. Overall, we have uncovered a new mechanism by which large bacterial toxins and proteins deliver catalytic activities to the eukaryotic cell cytosol by autoprocessing after translocation.     

The beta-type toxin Ts II from the scorpion Tityus serrulatus: amino acid sequence determination and assessment of biological and antigenic properties.

The toxin Ts II from the venom of the Brazilian scorpion Tityus serrulatus was purified in two successive chromatographic steps. The amino acid sequence was then determined by automated Edman degradation of the reduced and S-carboxymethylated protein and of proteolytic peptides derived from it. This sequence appears to differ from that of previously characterized toxins found in this venom. However, it is identical to the recently published sequence of protein III-8 from the same venom [Possani et al., J Biol Chem 266:3178-3185, 1991], except that the C-terminus was found to be amidated. Homologies were found between the sequence of Ts II and that of other toxins from Tityus; in particular, the amino acid sequence of Ts II displays 72% sequence identity with Ts VII (also called Titx gamma). Consistent with this structural similarity, some biological properties of Ts II were found to be similar to those of Ts VII: Ts II has an intracerebroventricular LD50 of 6 ng, as compared to 0.6 ng for Ts VII; in a receptor binding assay Ts II, like Ts VII, was found to behave as a beta-type toxin and to inhibit the binding of the reference labelled toxin with a K0.5 of 5 x 10(-9) M, as compared to 7 x 10(-11) M for Ts VII. Nevertheless, Ts II is unable to bind to anti-Ts VII antibodies in radioimmunoassay experiments, indicating the non-conservation between the two toxins of at least some antigenically important residues.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein crystallography and infectious diseases.

The current rapid growth in the number of known 3-dimensional protein structures is producing a database of structures that is increasingly useful as a starting point for the development of new medically relevant molecules such as drugs, therapeutic proteins, and vaccines. This development is beautifully illustrated in the recent book, Protein structure: New approaches to disease and therapy (Perutz, 1992). There is a great and growing promise for the design of molecules for the treatment or prevention of a wide variety of diseases, an endeavor made possible by the insights derived from the structure and function of crucial proteins from pathogenic organisms and from man. We present here 2 illustrations of structure-based drug design. The first is the prospect of developing antitrypanosomal drugs based on crystallographic, ligand-binding, and molecular modeling studies of glycolytic glycosomal enzymes from Trypanosomatidae. These unicellular organisms are responsible for several tropical diseases, including African and American trypanosomiases, as well as various forms of leishmaniasis. Because the target enzymes are also present in the human host, this project is a pioneering study in selective design. The second illustrative case is the prospect of designing anti-cholera drugs based on detailed analysis of the structure of cholera toxin and the closely related Escherichia coli heat-labile enterotoxin. Such potential drugs can be targeted either at inhibiting the toxin's receptor binding site or at blocking the toxin's intracellular catalytic activity. Study of the Vibrio cholerae and E. coli toxins serves at the same time as an example of a general approach to structure-based vaccine design. These toxins exhibit a remarkable ability to stimulate the mucosal immune system, and early results have suggested that this property can be maintained by engineered fusion proteins based on the native toxin structure. The challenge is thus to incorporate selected epitopes from foreign pathogens into the native framework of the toxin such that crucial features of both the epitope and the toxin are maintained. That is, the modified toxin must continue to evoke a strong mucosal immune response, and this response must be directed against an epitope conformation characteristic of the original pathogen.     

The 1.8 Å cholix toxin crystal structure in complex with NAD+ and evidence for a new kinetic model

Certain Vibrio cholerae strains produce cholix, a potent protein toxin that has diphthamide-specific ADP-ribosyltransferase activity against eukaryotic elongation factor 2. Here we present a 1.8 Å crystal structure of cholix in complex with its natural substrate, nicotinamide adenine dinucleotide (NAD⁺). We also substituted hallmark catalytic residues by site-directed mutagenesis and analyzed both NAD⁺ binding and ADP-ribosyltransferase activity using a fluorescence-based assay. These data are the basis for a new kinetic model of cholix toxin activity. Further, the new structural data serve as a reference for continuing inhibitor development for this toxin class.     

Muscarinic toxin-like proteins from cobra venom.

Three new polypeptides were isolated from the venom of the Thailand cobra Naja kaouthia and their amino-acid sequences determined. They consist of 65-amino-acid residues and have four disulfide bridges. A comparison of the amino-acid sequences of the new polypeptides with those of snake toxins shows that two of them (MTLP-1 and MTLP-2) share a high degree of similarity (55-74% sequence identity) with muscarinic toxins from the mamba. The third polypeptide (MTLP-3) is similar to muscarinic toxins with respect to the position of cysteine residues and the size of the disulfide-confined loops, but shows less similarity to these toxins (30-34% sequence identity). It is almost identical with a neurotoxin-like protein from Bungarus multicinctus (TrEMBL accession number Q9W727), the sequence of which has been deduced from cloned cDNA only. The binding affinities of the isolated muscarinic toxin-like proteins towards the different muscarinic acetylcholine receptor (mAChR) subtypes (m1-m5) was determined in competition experiments with N-[3H]methylscopolamine using membrane preparations from CHO-K1 cells, which express these receptors. We found that MTLP-1 competed weakly with radioactive ligand for binding to all mAChR subtypes. The most pronounced effect was observed for the m3 subtype; here an IC50 value of about 3 microM was determined. MTLP-2 had no effect on ligand binding to any of the mAChR subtypes at concentrations up to 1 microM. MTLP-1 showed no inhibitory effect on alpha-cobratoxin binding to the nicotinic acetylcholine receptor from Torpedo californica at concentrations up to 20 microM.     

Induction of cytokines by toxins that have an identical RNA N-glycosidase activity: Shiga toxin, ricin, and modeccin

Shiga toxin (Stx) has an A1-B5 subunit structure, and the A subunit is an RNA N-glycosidase that inhibits cellular protein synthesis. We previously reported that in Caco-2 cells Stx induced cytokines and that the RNA N-glycosidase activity was essential for the cytokine induction. It is known that the binding of the Stx-B subunit to its receptor glycolipid, Gb3, mediates an A subunit-independent signal in some types of cells, but the involvement of this signal in the cytokine induction is unclear. In this study, we investigated whether RNA N-glycosidase itself induces cytokines. IL-8 production was enhanced by Stx, ricin, and modeccin, three toxins that inhibit protein synthesis through an identical RNA N-glycosidase activity, but not by two other types of protein synthesis inhibitors, diphtheria toxin and cycloheximide. The RNA N-glycosidase-type toxins showed a similar induction pattern of cytokine mRNAs. Brefeldin A, a Golgi apparatus inhibitor, completely suppressed the cytokine induction by the toxins. Analysis by using inhibitors of toxin binding and also Stx-B subunit showed that the cytokine-inducing activity was independent of Gb3-mediated signaling. These results indicate that RNA N-glycosidase itself induces the cytokine production and that intracellular transport of toxins through the Golgi apparatus is essential for the activity.     

In vivo covalent cross-linking of cellular actin by the Vibrio cholerae RTX toxin

Enteric pathogens often export toxins that elicit diarrhea as a part of the etiology of disease, including toxins that affect cytoskeletal structure. Recently, we discovered that the intestinal pathogen Vibrio cholerae elicits rounding of epithelial cells that is dependent upon a gene we designated rtxA. Here we investigate the association of rtxA with the cell-rounding effect. We find that V. cholerae exports a large toxin, RTX (repeats-in-toxin) toxin, to culture supernatant fluids and that this toxin is responsible for cell rounding. Furthermore, we find that cell rounding is not due to necrosis, suggesting that RTX toxin is not a typical member of the RTX family of pore-forming toxins. Rather, RTX toxin causes depolymerization of actin stress fibers and covalent cross-linking of cellular actin into dimers, trimers and higher multimers. This RTX toxin-specific cross-linking occurs in cells previously rounded with cytochalasin D, indicating that G-actin is the toxin target. Although several models explain our observations, our simultaneous detection of actin cross-linking and depolymerization points toward a novel mechanism of action for RTX toxin, distinguishing it from all other known toxins.     

Structure based virtual screening of novel inhibitors against multidrug resistant superbugs

Pathogenic microorganisms are persistently expressing resistance towards present generation antibiotics and are on the verge of joining the superbug family. Recent studies revealed that, notorious pathogens such as Salmonella typhi, Shigella dysenteriae and Vibrio cholerae have acquired multiple drug resistance and the treatment became a serious concern. This necessitates an alternative therapeutic solution. Present study investigates the utility of computer aided method to study the mechanism of receptor-ligand interactions and thereby inhibition of virulence factors (shiga toxin of Shigella dysenteriae, cholera toxin of Vibrio cholerae and hemolysin-E of Salmonella typhi) by novel phytoligands. The rational designs of improved therapeutics require the crystal structure for the drug targets. The structures of the virulent toxins were identified as probable drug targets. However, out of the three virulent factors, the structure for hemolysin-E is not yet available in its native form. Thus, we tried to model the structure by homology modeling using Modeller 9v9. After extensive literature survey, we selected 50 phytoligands based on their medicinal significance and drug likenesses. The receptor-ligands interactions between selected leads and toxins were studied by molecular docking using Auto Dock 4.0. We have identified two novel sesquiterpenes, Cadinane [(1S, 4S, 4aS, 6S, 8aS)- 4- Isopropyl- 1, 6- dimethyldecahydronaphthalene] and Cedrol [(8α)-Cedran-8-ol] against Shiga (binding energy -5.56 kcal/mol) and cholera toxins (binding energy -5.33 kcal/mol) respectively which have good inhibitory properties. Similarly, a natural Xanthophyll, Violaxanthin [3S, 3'S, 5R, 5'R, 6S, 6'S)-5, 5', 6, 6'-Tetrahydro-5, 6:5', 6'-diepoxy-β, β-carotene-3, 3'-diol] was identified as novel therapeutic lead for hemolysin-E (binding energy of -5.99 kcal/mol). This data provide an insight for populating the pool of novel inhibitors against various drug targets of superbugs when all current generation drugs seem to have failed.     

Structural representative of the protein family PF14466 has a new fold and establishes links with the C2 and PLAT domains from the widely distant Pfams PF00168 and PF01477

The domain of unknown function (DUF) YP_001302112.1, a protein secreted by the human intestinal microbita, has been determined by NMR and represents the first structure for the Pfam PF14466. Its NMR structure is classified as a new fold, which, nonetheless, shows limited similarities with representatives of the PLAT/LH2 domains from PF01477 and the C2 domains from PF00168, both of which bind Ca²⁺ for their physiological functions. Further experiments revealed affinity of YP_001302112.1 for Ca²⁺, and the NMR structure in the presence of CaCl₂ was better defined than that of the apo‐protein. Overall, these NMR structures establish a new connection between structural representatives from two widely different Pfams that include the calcium‐binding domain of a sialidase from Vibrio cholerae and the α‐toxin from Clostridium perfrigens, whereby these two proteins have only 7% sequence identity. Furthermore, it provides information toward the functional annotation of YP_001302112.1, based on its capacity to bind Ca²⁺, and thus adds to the structural and functional coverage of the protein sequence universe. © 2013 The Protein Society     

Cholera toxin: a paradigm for multi-functional engagement of cellular mechanisms (Review)

Cholera toxin (Ctx) from Vibrio cholerae and its closely related homologue, heat-labile enterotoxin (Etx) from Escherichia coli have become superb tools for illuminating pathways of cellular trafficking and immune cell function. These bacterial protein toxins should be viewed as conglomerates of highly evolved, multi-functional elements equipped to engage the trafficking and signalling machineries of cells. Ctx and Etx are members of a larger family of A-B toxins of bacterial (and plant) origin that are comprised of structurally and functionally distinct enzymatically active A and receptor-binding B sub-units or domains. Intoxication of mammalian cells by Ctx and Etx involves B pentamer-mediated receptor binding and entry into a vesicular pathway, followed by translocation of the enzymatic A1 domain of the A sub-unit into the target cell cytosol, where covalent modification of intracellular targets leads to activation of adenylate cyclase and a sequence of events culminating in life-threatening diarrhoeal disease. Importantly, Ctx and Etx also have the capacity to induce a wide spectrum of remarkable immunological processes. With respect to the latter, it has been found that these toxins activate signalling pathways that modulate the immune system. This review explores the complexities of the cellular interactions that are engaged by these bacterial protein toxins, and highlights some of the new insights to have recently emerged.     

Effects of Midgut-Protein-Preparative and Ligand Binding Procedures on the Toxin Binding Characteristics of BT-R1, a Common High-Affinity Receptor in Manduca sexta for Cry1A Bacillus thuringiensis Toxins

The identity of the physiologically important Cry1A receptor protein(s) in the lepidopteran Manduca sexta has been a matter of dispute due to the multiple proteins which bind the Cry1Ac toxin. Cry1Aa, Cry1Ab, and Cry1Ac exhibit essentially identical toxicities toward M. sexta larvae and show a high degree of sequence and presumed structural identities. These similarities make it likely that there is a common mechanism of toxicity in these lepidopteran-specific toxins in terms of both mode of action and the receptor proteins through which these toxins exert their lepidopteran-specific toxicity. Investigators in our laboratory previously demonstrated that the cloned 210-kDa glycoprotein BT-R₁ binds all three Cry1A toxins (T. P. Keeton and L. A. Bulla, Jr., Appl. Environ. Microbiol. 63:3419–3425, 1997). This protein remains a common binding protein even after being subjected to various midgut membrane preparation and processing protocols. The method used to isolate proteins from the M. sexta larval midgut in no significant way affects the results of ligand binding and vacuum blotting experiments, and we have been unable to detect specific, high-affinity binding of any Cry1A toxin to Cry1Ac binding proteins other than BT-R₁. Alterations in blot substrate and blocking, hybridization, and washing buffers support these conclusions. Collectively, these results indicate that in M. sexta the cadherin-like BT-R₁ protein is a common high-affinity receptor protein for the Cry1A family of toxins.     

Immunity proteins and their specificity for endonuclease colicins: telling right from wrong in protein–protein recognition

Immunity proteins inhibit colicins, protein toxins released by bacteria during times of environmental stress, by binding and inactivating their cytotoxic domains. This protects the producing organism as it attempts to kill off competing bacteria. The cytotoxic domains of related colicins share a high degree of sequence identity, as do their corresponding immunity proteins, yet specificity and affinity are also high, with little non-cognate biological cross-protection evident under physiological conditions. We review recent work on DNase-specific immunity proteins, which shows that, although both cognate and non-cognate proteins can bind a single toxin, their affinities can differ by as much as 12 orders of magnitude. We have termed this mode of binding dual recognition, because the DNase-binding surface of an immunity protein is made up of two components, one conserved and the other variable. The strength of the binding interaction is dominated by the conserved residues, while neighbouring variable residues control specificity. Similar dual recognition systems may exist in other biological contexts, particularly where a protein must discriminate the right binding partner from numerous, structurally homologous alternatives.     

A binding motif for Hsp90 in the A chains of ADP‐ribosylating toxins that move from the endoplasmic reticulum to the cytosol

Cholera toxin (Ctx) is an AB‐type protein toxin that acts as an adenosine diphosphate (ADP)‐ribosyltransferase to disrupt intracellular signalling in the target cell. It moves by vesicle carriers from the cell surface to the endoplasmic reticulum (ER) of an intoxicated cell. The catalytic CtxA1 subunit then dissociates from the rest of the toxin, unfolds, and activates the ER‐associated degradation system for export to the cytosol. Translocation occurs through an unusual ratchet mechanism in which the cytosolic chaperone Hsp90 couples CtxA1 refolding with CtxA1 extraction from the ER. Here, we report that Hsp90 recognises two peptide sequences from CtxA1: an N‐terminal RPPDEI sequence (residues 11–16) and an LDIAPA sequence in the C‐terminal region (residues 153–158) of the 192 amino acid protein. Peptides containing either sequence effectively blocked Hsp90 binding to full‐length CtxA1. Both sequences were necessary for the ER‐to‐cytosol export of CtxA1. Mutagenesis studies further demonstrated that the RPP residues in the RPPDEI motif are required for CtxA1 translocation to the cytosol. The LDIAPA sequence is unique to CtxA1, but we identified an RPPDEI‐like motif at the N‐ or C‐termini of the A chains from four other ER‐translocating toxins that act as ADP‐ribosyltransferases: pertussis toxin, Escherichia coli heat‐labile toxin, Pseudomonas aeruginosa exotoxin A, and Salmonella enterica serovar Typhimurium ADP‐ribosylating toxin. Hsp90 plays a functional role in the intoxication process for most, if not all, of these toxins. Our work has established a defined RPPDEI binding motif for Hsp90 that is required for the ER‐to‐cytosol export of CtxA1 and possibly other toxin A chains as well.