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J. Biol. Chem., Vol. 282, Issue 35, 25540-25547, August 31, 2007

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Multiple Pocket Recognition of SNAP25 by Botulinum Neurotoxin Serotype E^*

From the Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, Wisconsin 53226

Botulinum neurotoxins (BoNTs) are zinc proteases that cleave SNARE proteins to elicit flaccid paralysis by inhibiting the fusion of neurotransmitter-carrying vesicles to the plasma membrane of peripheral neurons. There are seven serotypes of BoNT, termed A–G. The molecular basis for SNAP25 recognition and cleavage by BoNT serotype E is currently unclear. Here we define the multiple pocket recognition of SNAP25 by LC/E. The initial recognition of SNAP25 is mediated by the binding of the B region of SNAP25 to the substrate-binding (B) region of LC/E comprising Leu¹⁶⁶, Arg¹⁶⁷, Asp¹²⁷, Ala¹²⁸, Ser¹²⁹, and Ala¹³⁰. The mutations at these residues affected substrate binding and catalysis. Three additional residues participate in scissile bond cleavage of SNAP25 by LC/E. The P3 site residues, Ile¹⁷⁸, of SNAP25 interacted with the S3 pocket in LC/E through hydrophobic interactions. The S3 pocket included Ile⁴⁷, Ile¹⁶⁴, and Ile¹⁸² and appeared to align the P1' and P2 residues of SNAP25 with the S1' and S2 pockets of LC/E. The S1' pocket of LC/E included three residues, Phe¹⁹¹, Thr¹⁵⁹, and Thr²⁰⁸, which contribute hydrophobic and steric interactions with the SNAP25 P1' residue Ile¹⁸¹. The S2 pocket residue of LC/E, Lys²²⁴, binds the P2 residue of SNAP25, Asp¹⁷⁹, through ionic interactions. Deletion mapping indicates that main chain interaction(s) of residues 182–186 of SNAP25 contribute to substrate recognition by LC/E. Understanding the mechanism for substrate specificity provides insight for the development of inhibitors against the botulinum neurotoxins.

Received for publication, March 6, 2007 , and in revised form, June 7, 2007.

^* This work was supported by the Regional Center of Excellence for Biodefense and Emerging Infectious Diseases Research Program, NIAID, National Institutes of Health, and by National Institutes of Health Grant 1-U54-AI-057153. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed. Tel.: 414-456-8412; Fax: 414-456-6535; E-mail: jtb01{at}mcw.edu.

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