Receptor and substrate interactions of clostridial neurotoxins
Introduction
The family of clostridial neurotoxins (CNTs) consists of tetanus neurotoxin (TeNT) and the seven botulinum neurotoxin serotypes (BoNT/A-G), and represents the most toxic agents known. The median lethal dose is below 1 ng per kg of body weight (Gill, 1982). The disease tetanus is caused by germination of Gram-positive, anaerobic spore-forming Clostridium tetani in infected tissue lesions, thereby producing and releasing TeNT into the blood stream. In contrast, botulism is evoked by ingestion of acid resistant BoNT progenitor toxins, generated by various strains of C. botulinum, C. butyricum and C. barati, and subsequent transcytosis of this complex or the released BoNT through the intestinal epithelial barrier (Bigalke and Shoer, 2000). The CNTs reach the motoneurons via circulation and specifically bind to unmyelinated areas of nerve terminals (Dolly et al., 1984). Here, BoNTs inhibit acetylcholine release followed by flaccid paralysis while TeNT is transported retrogradely to inhibitory neurons and blocks release of glycine or γ-aminobutyric acid which results in spastic paralysis.
The crystal structures of the BoNT/A, B and E holotoxins (Lacy et al., 1998, Swaminathan and Eswaramoorthy, 2000, Kumaran et al., 2009) revealed that most likely all CNTs are composed of four functionally independent domains that perform individual tasks in the multi-step intoxication process (Fig. 1). All CNTs are produced as ∼150 kDa single chain (sc) proteins. They are post-translationally proteolysed into a ∼100 kDa heavy chain (HC) and a ∼50 kDa light chain (LC). Both chains remain associated by a single disulphide bridge, non-covalent interactions and an HC derived peptide loop wrapping around the LC. The HCs are responsible for neurospecific binding, uptake and translocation of the LCs into the cytosol. Following cell attachment, internalisation via receptor-mediated endocytosis brings the BoNTs into the synaptic vesicles. Here, the acidic environment eliminates repulsive electrostatic interactions between the largely α-helical amino-terminal half of the HC, the HN-domain and the membrane, allowing its penetration into the membrane, without triggering detectable structural changes (Galloux et al., 2008). At the same time the LC is partially unfolded (Koriazova and Montal, 2003). Translocation of LC by HC can be observed in real time as an increase of channel conductance. The HC channel is occluded by the LC during transit, then unoccluded after completion of translocation and release of LC (Fischer and Montal, 2007b). Upon reduction of the disulphide bond, the LC functions as a zinc dependent endopeptidase in the cytosol (Fischer and Montal, 2007a, Schiavo et al., 1990).
Section snippets
Gangliosides as receptors for CNTs
The specific binding to peripheral nerve endings at the neuromuscular junction solely involves the 50 kDa C-terminal half of the HC, the HC-fragment (Evinger and Erichsen, 1986, Fishman and Carrigan, 1987, Lalli et al., 1999, Simpson, 1984a, Simpson, 1984b, Simpson, 1985) and complex polysialo-gangliosides, glycosphingolipids that are found particularly in membranes of neuronal cells (Simpson and Rapport, 1971, van Heyningen and Miller, 1961). The interaction of gangliosides with CNTs was
A protein is the second receptor for CNTs
The discrepancy in affinity between binding of CNTs to isolated gangliosides and neuronal tissue prompted predictions of a second receptor component. The protease-sensitive binding of BoNT/A and TeNT to rat brain synaptosomes (Dolly et al., 1982, Kitamura, 1976, Lazarovici and Yavin, 1986, Pierce et al., 1986) resulted in a dual receptor model. First, polysialo-gangliosides were considered to accumulate CNTs on the plasma membrane surface. Then, CNTs would simply stay on the surface until
Characterisation of binding sites in CNTs
The crystal structure of the TeNT HC-fragment revealed that it is composed of two domains, an N-terminal lectin-like jelly-roll domain (HCN, residues 865–1110) and a C-terminal β-trefoil domain (HCC, residues 1110–1315) (Knapp et al., 1998, Umland et al., 1997). Deletion mutagenesis studies showed that the TeNT HCC-domain binds to gangliosides and neuronal cells even more efficiently than the complete HC-fragment (Halpern and Loftus, 1993), whereas the HCN-domain does not bind at all (
CNT LC proteases
BoNT and TeNT LCs are amongst the most selective proteases known (Oost et al., 2003). Primary sequence and structural analysis of LCs suggest that their enzymatic mechanism is related to that of other Zn2+-metalloproteases (Agarwal et al., 2004, Breidenbach and Brunger, 2005, Lacy et al., 1998, Rao et al., 2005, Swaminathan and Eswaramoorthy, 2000), but the structural basis of SNARE target selectivity is unusual. Remarkably, the LCs do not appear to recognise a consensus site, or even have
LC–substrate interactions
The structure of a BoNT/A·LC–SNAP-25 complex (PDB ID 1XTG) (Breidenbach and Brunger, 2004) for the first time provided molecular insights into the basis of LC substrate selectivity (Fig. 4). To date, this is the only structure of a complex between a CNT-LC and its substrate. A previous report of the structure of a complex between BoNT/B-LC and synaptobrevin 2 (Hanson and Stevens, 2000) is not supported by the experimental data (Breidenbach and Brunger, 2004, Rupp and Segelke, 2001) and the
LC–inhibitor interactions and implications for drug development
For many years, complexes between LC proteases and inhibitors resisted attempts at co-crystallisation. Over the past two years, dramatic progress has been made resulting in co-crystal structures of several inhibitor-BoNT/A LC complexes (Fu et al., 2006, Kumaran et al., 2008a, Kumaran et al., 2008b, Silvaggi et al., 2007, Silvaggi et al., 2008, Zuniga et al., 2008). Co-crystal structures with small hydroxamate compounds and tetrapeptides, all including an Arg moiety, have now uniquely identified
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