Multiple pocket recognition of SNAP25 by botulinum neurotoxin serotype E

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 Leu(166), Arg(167), Asp(127), Ala(128), Ser(129), and Ala(130). 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, Ile(178), of SNAP25 interacted with the S3 pocket in LC/E through hydrophobic interactions. The S3 pocket included Ile(47), Ile(164), and Ile(182) 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, Phe(191), Thr(159), and Thr(208), which contribute hydrophobic and steric interactions with the SNAP25 P1 ' residue Ile(181). The S2 pocket residue of LC/E, Lys(224), binds the P2 residue of SNAP25, Asp(179), 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.