SSRL Science Highlight - December 2004

Mark A. Breidenbach1 and Axel T. Brunger2

1Department of Molecular and Cellular Physiology and 2Howard Hughes Medical Institute and Departments of Molecular and Cellular Physiology, Neurology and Neurological Sciences and Stanford Synchrotron Radiation Laboratory, Stanford University, Stanford, California 94305, USA

Clostridal neurotoxins (CNTs) are the causative agents of the neuroparalytic diseases botulism and tetanus1,2 CNTs impair neuronal exocytosis (a process by which neurotransmitter is released into a synapse) through specific proteolysis of essential proteins called SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors)3. SNARE assembly into a low-energy ternary complex is believed to catalyse membrane fusion, precipitating neurotransmitter release; this process is attenuated in response to SNARE proteolysis4-7. Site-specific SNARE hydrolysis is catalysed by the CNT light chains, a unique group of zinc-dependent endopeptidases (enzymes that catalyse hydrolysis of peptide bonds within other proteins)3. The means by which a CNT properly identifies and cleaves its target SNARE has been a subject of much speculation; it is thought to use one or more regions of enzyme-substrate interaction remote from the active site (exosites)8-10. Using x-ray diffraction data collected in part at SSRL beamline 9-2, we have determined the first structure of a CNT endopeptidase in complex with its target SNARE at a resolution of 2.1 Å: botulinum neurotoxin serotype A (BoNT/A) protease bound to human SNAP-25. The structure, together with enzyme kinetic data, reveals an array of exosites that determine substrate specificity (figure 1).

Figure 1. Three different views of the SNAP-25-BoNT/A complex are shown; these are related by the specified rotations around a vertical axis in the plane of the figure that goes through the centre of the complex.

The a-exosite (indicated by a green arrow in Fig. 1) is formed by BoNT/A light-chain helices (tan) a1-a4 that bind to the helical N-terminus of the substrate (red). Green areas indicate the approximate locations of contacting side chains involved in the a-exosite. On the opposite face of BoNT/A, the b-exosite is indicated by a blue arrow (Fig. 1). The C-terminus of SNAP-25 forms an anti-parallel b sheet along with a portion of the '250 loop' (light-blue), which is separated from the active site (indicated by Zn2+, purple sphere) by the '370 loop' (light-blue). Dark-blue areas indicate the approximate locations of contacting side chains involved in the b-exosite. Yellow areas indicate the approximate locations of other exosites (anchor points) involved in side-chain contacts between the SNAP-25 substrate and the BoNT/A light chain.

Based on our structure and available kinetic data for several mutant SNAP-25 substrates, we conclude that most of this unusually large enzyme-substrate interface serves to provide a substrate-specific boost to catalytic efficiency by reducing Km (the Michaelis constant). We also observe significant structural changes near the toxin's catalytic pocket upon substrate binding, probably serving to render the protease competent for catalysis. A general model of the strategy used by BoNT/A to recognize and cleave SNAP-25 is presented in figure 2. SNAP-25 is shown attached to a presynaptic membrane via palmitylation sites (shown in black) on its linker domain (purple). The N-terminal (sn1, cyan) and C-terminal (sn2, green) domains are unstructured or flexible in uncomplexed SNAP-25 (ref. 11). Binding of BoNT/A (blue) is probably initiated by helix formation at the a-exosite, and anchor points along the extended portion of SNAP-25 (green notches) are additional determinants of substrate specificity. These sites reduce Km and enhance binding at the b-exosite, inducing conformational changes at the active site, which render the endopeptidase competent to cleave its substrate.

Figure 2. Exosite-based model of BoNT/A substrate recognition.

Ultimately, the novel structures of the substrate-recognition exosites could be used for designing inhibitors specific to BoNT/A.

Primary Citation:

Breidenbach, M. A. & Brunger, A. T. Substrate recognition strategy for botulinum neurotoxin serotype A. Nature 432, 925-929 (2004)


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Last Updated: 13 December 2004
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