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 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.
Chen
Sheng
S
Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
Barbieri
Joseph T
JT
D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D001665:Binding Sites / Q000502:physiology; D001905:Botulinum Toxins / Q000737:chemistry* / Q000378:metabolism; D002384:Catalysis; D006801:Humans; D045726:Metalloproteases / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D009498:Neurotoxins / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D020836:Protein Structure, Quaternary; D013379:Substrate Specificity / Q000502:physiology; D050825:Synaptosomal-Associated Protein 25 / Q000737:chemistry* / Q000378:metabolism; D015032:Zinc / Q000737:chemistry* / Q000378:metabolism
D000596:Amino Acids; D009498:Neurotoxins; C494208:SNAP25 protein, human; D050825:Synaptosomal-Associated Protein 25; D045726:Metalloproteases; D001905:Botulinum Toxins; D015032:Zinc; C096326:botulinum toxin type E