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Substrate recognition strategy for botulinum neurotoxin serotype A

Abstract

Clostridal neurotoxins (CNTs) are the causative agents of the neuroparalytic diseases botulism and tetanus1,2. CNTs impair neuronal exocytosis through specific proteolysis of essential proteins called SNAREs3. 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 proteolysis4,5,6,7. Site-specific SNARE hydrolysis is catalysed by the CNT light chains, a unique group of zinc-dependent endopeptidases3. 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)8,9,10. Here we report 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. Substrate orientation is similar to that of the general zinc-dependent metalloprotease thermolysin11. We observe significant structural changes near the toxin's catalytic pocket upon substrate binding, probably serving to render the protease competent for catalysis. The novel structures of the substrate-recognition exosites could be used for designing inhibitors specific to BoNT/A.

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Figure 1: The nearly identical active sites of different BoNT light chains.
Figure 2: The interface between sn2 and BoNT/A.
Figure 3: Detailed views of α- and β-exosites.
Figure 4: Kinetic characterization of sn2 mutants.
Figure 5: Wall-eyed stereo view of β-exosite conformational changes.
Figure 6: Exosite-based model of BoNT/A substrate recognition.

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Acknowledgements

We thank T. Binz and J. Ernst for providing initial BoNT/A and sn2 constructs and P. Adams, T. Fenn, S. Kaiser, Z. Panepucci, P. Strop and W. Weis for technical assistance and critical reading. Portions of this research were carried out at the Stanford Synchrotron Radiation Laboratory, a national user facility operated by Stanford University on behalf of the US Department of Energy (Office of Basic Energy Sciences). The SSRL Structural Molecular Biology Program is supported by the Department of Energy (Office of Biological and Environmental Research) and by the National Institutes of Health (National Center for Research Resources, Biomedical Technology Program) and the National Institute of General Medical Sciences. Portions of this research were conducted at the Advanced Light Source which is supported by the Office of Energy Research (Office of Basic Energy Sciences, Materials Sciences Division) of the US Department of Energy at Lawrence Berkeley National Laboratory. This work was supported in part by an NIH grant to A.T.B.

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Correspondence to Axel T. Brunger.

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This file contains the Supplementary Methods section of the paper with references, along with Supplementary Tables 1 and 2, and Supplementary Figures 1, 2 and 3. (PDF 428 kb)

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Breidenbach, M., Brunger, A. Substrate recognition strategy for botulinum neurotoxin serotype A. Nature 432, 925–929 (2004). https://doi.org/10.1038/nature03123

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