Structure of Clostridial Neurotoxins

Clostridial neurotoxins share a common structural organization. They are produced as inactive polypeptide chains of 150 kDa. Upon bacterial lysis, the CNTs are released and cleaved by endogenous or exogenous proteases at an exposed loop. An active di-chain neurotoxin is thus generated (Fig. 1) (DasGupta, 1994). The heavy chain (H, 100 kDa) and the light chain (L, 50 kDa) are bridged by a single interchain disulfide bridge. This interchain bond is essential for neurotoxicity of CNTs when the toxins are applied to the extracellular space (Schiavo et al., 1990; De Paiva et al., 1993). Biochemical studies, as well as low resolution electron microscopic analysis, led to the hypothesis that TeTx and BoNTs are folded into three distinct 50 kDa domains, each with a different set of physiological functions (Fig. 1). mechanism of action of The mechanism of neurotoxin cell intoxication consists of four dis-neurotoxins tinct steps: (a) binding to the neuronal membrane, (b) internalization into an acidic compartment, (c) membrane translocation and (d) enzymatic target modification (Montecucco etal., 1994). The L chain is responsible for the intracellular catalytic activity of CNTs. The amino-terminal 50 kDa domain of the H chain (HN) is implicated in membrane translocation, while the carboxy-terminal part (Hc) is mainly responsible for the neurospecific binding, structure of clostridial The amino acid sequence of all eight CNTs has been derived from neurotoxins their corresponding genes (Minton, 1995). The L chains and H chains are composed on average of 439 and 843 residues, respectively. Both chains contain homologous domains separated by regions of very little similarity (Fig. 2). The most conserved portions of the L chains are the amino-terminal and central regions (residues 216-244,

ENZYMATIC ACTIVITY

L 50 kDa

Fig. 1. The mechanism of activation of tetanus and botulinum neurotoxins. The toxins are produced as an inactive single polypeptide chain of 150 kDa, composed of three 50 kDa domains, connected by protease-sensitive loops. The toxins are activated by selective proteolytic cleavage which generates two disulfide-linked chains: L (50kDa) and H (100kDa). The three domains play different functional roles in cell penetration: Hc is responsible for cell binding and HN for cell penetration. Reduction takes place inside the nerve cells and liberates the metallo-protease activity of the L chain in the cytosol

ACTIVE

Fig. 1. The mechanism of activation of tetanus and botulinum neurotoxins. The toxins are produced as an inactive single polypeptide chain of 150 kDa, composed of three 50 kDa domains, connected by protease-sensitive loops. The toxins are activated by selective proteolytic cleavage which generates two disulfide-linked chains: L (50kDa) and H (100kDa). The three domains play different functional roles in cell penetration: Hc is responsible for cell binding and HN for cell penetration. Reduction takes place inside the nerve cells and liberates the metallo-protease activity of the L chain in the cytosol

BONT/A LAHELIHAGHRLYG

BONT/B LMHELIHVLHGLYG

BONT/C LMHELNHAMHNLYG

BoNTVD LMHELTHSLHQLYG

BoNT/E LMHELIHSLHGLYG

BoNT/F LAHELIHALHGLYG

BoNT/G LMHELIHVLHGLYG

TeNT LMHELIHVLHGLYG

Zincins HExxH

Metzincins hxHExhHxhGhxH

Fig. 2. Structure and active site of clostridial neurotoxins. The upper panel shows the structure of CNTs, and the segments that show significant homology between the different serotypes are in black (Minton, 1995). The highest homology is shown by a short segment corresponding to the amino acid residues 216-244 in TeTx. This segment contains the zinc-binding motif of metallo-proteinases (zincins) and it is dissimilar to the consensus sequence of the metzincin metallo-proteinase family (Jiang and Bond, 1992)

numbering of TeTx). The latter region contains the His-Glu-Xaa-Xaa-His binding motif of zinc-endopeptidases (Fig. 2) (Jongeneel et al., 1989; Jiang and Bond, 1992). This observation led to the demonstration that CNTs are zinc-containing proteins (Schiavo et al., 1992 b, c, 1993 c, 1994, 1995 a; Wright et al., 1992; Yamasaki et al., 1994 b). One atom of zinc is bound to the L chain of TeTx, BoNT/A, B, and F. These neurotoxins show a single zinc binding site with a dissociation constant (Kd) of 50-100 nM. In addition, multiple divalent cation binding sites with a lower affinity are also present (Schiavo et al., 1992 c; Wright et al., 1992). BoNT/C binds two atoms of zinc (Schiavo et al., 1995 a), similar to the neutrophil collagenase, whose three-dimensional structure has been recently determined (Lovejoy et al., 1994). One atom of zinc is present at the active site of this metallo-proteinase and is exchangeable, while the second one is bound very strongly and is thought to play a structural role. Zinc can be removed from the CNTs with heavy metal chelators, thus generating an inactive apo-neurotoxin. The active holo-toxin can be reformed upon incubation of the apo-neurotoxin in zinc-containing buffers (Schiavo et al., 1992 b, c, 1993 c, 1994, 1995 a; Simpson et al., 1993; Hôhne-Zell et al., 1994).

The zinc atom of zinc-endopeptidases is coordinated by either two or three histidine residues (Jiang and Bond, 1992). In thermolysin-like enzymes, the zinc atom is coordinated by two histidines within the consensus sequence of zinc-endopeptidases, by a water molecule bound to the glutamic acid of the same motif and by another (distal) glutamate residue. Astacin, a crayfish metallo-protease, penta-coordinates zinc via three histidine residues, one tyrosine residue and one water molecule. Adamalysin, the alkaline protease of Pseudomonas aeruginosa, and collagenase adopt a tetrahedral zinc coordination via three histidines and a glutamate-bound water molecule. The active site of zinc-endopeptidases resides in a cleft with the zinc atom in its center and the residues of the zinc-binding motif forming an a-helix. The orientation and volume of the amino-acid side-chains at the active site determine the peptide bond specificity of the toxin cleavage. The water molecule bound to the glutamate residue of the motif is involved in peptide bond hydrolysis by a mechanism that has been studied in detail only for thermolysin (Matthews, 1988).

In order to determine the number of histidine residues involved in zinc coordination, the L chains of TeTx and BoNT/A, B and E were modified with diethyl pyrocarbonate (DEPC), a reagent that specifically modifies histidine residues. In each case, two additional histidines were modified in the apo-toxin that were not affected in the holo-neurotoxin (Schiavo et al., 1992 b, c). These results indicate that the zinc atom of CNTs is coordinated via two histidines and a Glu-bound water molecule, as in thermolysin. Mutations at the two histidines of the motif inactivate TeTx and suppress its ability to bind radiolabeled 65Zn2+ (Yamasaki eta/., 1994 b). In addition, mutations of the conserved Glu-271 and Glu-272 of TeTx, predicted to be in an a-helical segment (Lebeda and Olson, 1994), result in decreased zinc binding and loss of activity. Based on these experimental results, it has been suggested that CNTs are thermolysin-like proteases and that one of these two Glu residues represents the fourth zinc ligand (Yamasaki etal., 1994 b). In contrast, a comparison of the extended X-ray absorption fine structure (EXAFS) spectra of TeTx, astacin, alkaline protease and thermolysin shows a close similarity of TeTx with astacin and alkaline protease (Morante et al., 1996). This result indicates that at least one additional aromatic residue is present around the zinc atom of TeTx, in addition to the two histidines required for cation coordination. Sequence comparison indicates that a Tyr residue (Tyr-243 in TeTx), conserved among all CNTs, is located in the same position as the third histidine in astacin and astacin-like proteinases. Moreover Tyr-243 replacement in TeTx leads to a great loss of toxicity (Yamasaki etal., 1994 b). Taken together, these results suggest a novel manner of zinc coordination among metallo-proteinases. In addition, studies on the denaturant sensitivity of the holo- and apo-L chain indicate that the zinc atom does not contribute significantly to the structural stability of the CNTs (De Filippis et al., 1995).

The H chains are less conserved than the L chains and the carboxyl-terminal part of the H chain (Hc) is the most variable region of the toxin (Fig. 2) (Minton, 1995). This is consistent with the notions that the Hc domain is involved in binding to the nerve terminals and that different neurotoxins bind to different cognate receptors. On this basis it may be suggested that the receptor binding regions of TeTx and BoNTs are mainly located within the 180 carboxy-terminal residues of the H chain.

Nucleotide and amino acid sequence comparisons of the CNTs clearly indicate that they derive from a common ancestral gene. In this respect, it is significant that the CNT genes are located on mobile genetic elements (Minton, 1995). Bacteriophages, plasmids, and conjugation transposons may have spread these genes among bacteria of the Clostridium genus. Mutations in CNT genes are apparent from the discovery of variants of the seven BoNTs among the same serotypes, which have being detected with the methods of modern molecular genetics (Minton, 1995). Moreover, strains that harbour more that one BoNT gene have been identified (Hatheway, 1995).

The scant knowledge about the ecology of toxigenic strains of Clostridia allows us only to speculate on the role of CÑTs in Clostridia natural history life cycles. A successful bacterium is capable of multiplying and of Clostridia spreading rapidly. In general, well-established infectious agents cause the smallest alteration in host physiology compatible with their need to enter and multiply in the host and spread to other individuals. This state is defined as "balanced pathogenicity" and is of paramount importance for the ecology of the infectious agent (Mims, 1987). Living vertebrates offer only very small anaerobic habitats within their bodies where Clostridia can survive. The release of a neurotoxin that kills the animal host converts it into an anaerobic fermen-tor able to support the massive growth of Clostridia of endogenous as well as exogenous origin. In this simplified view, the production of neurotoxins is crucial to create a new habitat. Since the cadaver cannot support bacterial spread to other hosts, Clostridia sporulate and the spores are dispersed. On this basis toxigenic Clostridia do not appear to be "balanced pathogens". However, it should be considered that killing the host is necessary to the life cycle of a strictly anaerobic organism in an oxygen-rich habitat, and that the production of spores is essential to their survival and spreading in the environment.

The finding that CNTs are zinc-endopeptidases specific for different proteins of the neuroexocytotic apparatus suggests a possible evolutionary origin of these neurotoxins. Clostridia produce a variety of proteinases that act outside cells. At a certain stage of evolution a metallo-proteinase gene fused with another gene, giving rise to a protein able to act specifically at the level of the nervous system. Further genetic rearrangements may have led to a molecule able to cleave selected proteins of the exocytotic apparatus. Different sites of attack on the same supramolecular structure ensures that an animal species cannot become resistant to all CNTs by single point mutations of the target.

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