Unique substrate recognition by botulinum neurotoxins serotypes A and 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. BoNT serotype A and serotype E cleave SNAP25 at residues 197-198 and 180-181, respectively. Unlike other zinc proteases, the BoNTs recognize extended regions of SNAP25 for cleavage. The basis for this extended substrate recognition and specificity is unclear. Saturation mutagenesis and deletion mapping identified residues 156-202 of SNAP25 as the optimal cleavage domain for BoNT/A, whereas the optimal cleavage domain for BoNT/E was shorter, comprising residues 167-186 of SNAP25. Two sub-sites were resolved within each optimal cleavage domain, which included a recognition or active site (AS) domain that contained the site of cleavage and a binding (B) domain, which contributed to substrate affinity. Within the AS domains, the P1', P3, and P5 sites of SNAP25 contributed to scissile bond cleavage by LC/A, whereas the P1' and P2 sites of SNAP25 contributed to scissile bond cleavage by LC/E. These studies provide insight into the development of strategies for small molecule inhibitors of the BoNTs.
Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
Sheng
S
Chen
Joseph T
JT
Barbieri
D000595:Amino Acid Sequence; D001665:Binding Sites; D001905:Botulinum Toxins / Q000737:chemistry* / Q000378:metabolism; D019274:Botulinum Toxins, Type A / Q000737:chemistry* / Q000378:metabolism; D002384:Catalysis; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D003014:Clostridium botulinum / Q000378:metabolism*; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000378:metabolism; D017353:Gene Deletion; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D050825:Synaptosomal-Associated Protein 25 / Q000737:chemistry*; D015032:Zinc / Q000737:chemistry
D010455:Peptides; D050825:Synaptosomal-Associated Protein 25; D001905:Botulinum Toxins; D019274:Botulinum Toxins, Type A; D015032:Zinc; C096326:botulinum toxin type E