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Mode of VAMP substrate recognition and inhibition of Clostridium botulinum neurotoxin F

Nature Structural & Molecular Biology volume 16, pages 789–794 (2009)Cite this article

Abstract

Clostridium botulinum neurotoxins (BoNTs) cleave neuronal proteins responsible for neurotransmitter release, causing the neuroparalytic disease botulism. BoNT serotypes B, D, F and G cleave and inactivate vesicle-associated membrane protein (VAMP), each at a unique peptide bond. The specificity of BoNTs depends on the mode of substrate recognition. We have investigated the mechanism of substrate recognition of BoNT F by determining the crystal structures of its complex with two substrate-based inhibitors, VAMP 22-58/Gln58D-cysteine and 27-58/Gln58D-cysteine. The inhibitors bind to BoNT F in the canonical direction (as seen for BoNTs A and E substrates) but are positioned specifically via three major exosites away from the active site. The cysteine sulfur of the inhibitors interacts with the zinc and exists as sulfinic acid in the inhibitor VAMP 27-58/Gln58D-cysteine. Arg133 and Arg171, which form part of two separate exosites, are crucial for substrate binding and catalysis.

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Figure 1: BoNT F in complex with the VAMP substrate inhibitors.
Figure 2: Interactions of VAMP inhibitors with BoNT F.
Figure 3: Schematic representation of VAMP substrate inhibitor binding interactions with BoNT F.
Figure 4: Major VAMP recognition and binding exosites in BoNT F.

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Acknowledgements

Research was supported by award from the US Defense Threat Reduction Agency (DTRA) BO742081 under Department of Energy prime contract N0. DEAC02-98CH10886 with Brookhaven National Laboratory (BNL) (to S.S.). It was also funded in part by the DTRA under research plan 3.10023_06_RD_B (J.J.S. and R.G.S.). Opinions, interpretations, conclusions and recommendations are those of the authors and are not necessarily endorsed by the US Army. We gratefully acknowledge the data collection support from X12c and X29 (National Synchrotron Light Source). We thank M.G. Blewitt for DNA sequencing of mutants, the Proteomic Center of Stony Brook University for their help with MALDI-TOF measurements and V. Graziano (BNL) for help in tryptophan florescence experiments and discussion.

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Authors and Affiliations

  1. Biology Department, Brookhaven National Laboratory, Upton, New York, USA

    Rakhi Agarwal & Subramanyam Swaminathan

  2. Integrated Toxicology Division, US Army Research Institute of Infectious Diseases, Fort Detrick, Maryland, USA

    James J Schmidt & Robert G Stafford

Authors
  1. Rakhi Agarwal
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  2. James J Schmidt
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  3. Robert G Stafford
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  4. Subramanyam Swaminathan
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Contributions

R.A. performed the experiments; S.S. conceived and designed the experiments; R.A., S.S. and J.J.S. wrote the manuscript; R.G.S. and J.J.S. designed the inhibitors.

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Correspondence to Subramanyam Swaminathan.

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Agarwal, R., Schmidt, J., Stafford, R. et al. Mode of VAMP substrate recognition and inhibition of Clostridium botulinum neurotoxin F. Nat Struct Mol Biol 16, 789–794 (2009). https://doi.org/10.1038/nsmb.1626

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