Structural and biochemical studies of botulinum neurotoxin serotype C1 light chain protease: implications for dual substrate specificity.
Clostridial neurotoxins are the causative agents of the neuroparalytic disease botulism and tetanus. They block neurotransmitter release through specific proteolysis of one of the three soluble N-ethylmaleimide-sensitive-factor attachment protein receptors (SNAREs) SNAP-25, syntaxin, and synaptobrevin, which constitute part of the synaptic vesicle fusion machinery. The catalytic component of the clostridial neurotoxins is their light chain (LC), a Zn2+ endopeptidase. There are seven structurally and functionally related botulinum neurotoxins (BoNTs), termed serotype A to G, and tetanus neurotoxin (TeNT). Each of them exhibits unique specificity for their target SNAREs and peptide bond(s) they cleave. The mechanisms of action for substrate recognition and target cleavage are largely unknown. Here, we report structural and biochemical studies of BoNT/C1-LC, which is unique among BoNTs in that it exhibits dual specificity toward both syntaxin and SNAP-25. A distinct pocket (S1') near the active site likely achieves the correct register for the cleavage site by only allowing Ala as the P1' residue for both SNAP-25 and syntaxin. Mutations of this SNAP-25 residue dramatically reduce enzymatic activity. The remote alpha-exosite that was previously identified in the complex of BoNT/A-LC and SNAP-25 is structurally conserved in BoNT/C1. However, mutagenesis experiments show that the alpha-exosite of BoNT/C1 plays a less stringent role in substrate discrimination in comparison to that of BoNT/A, which could account for its dual substrate specificity.
Jin
Rongsheng
R
Howard Hughes Medical Institute and Department of Molecular and Cellular Physiology, Stanford Synchrotron Radiation Laboratory, Stanford University, Stanford, California 94305, USA.
Sikorra
Stefan
S
Stegmann
Christian M
CM
Pich
Andreas
A
Binz
Thomas
T
Brunger
Axel T
AT
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001905:Botulinum Toxins / Q000737:chemistry*; D018360:Crystallography, X-Ray; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000737:chemistry*; D017433:Protein Structure, Secondary; D050765:Qa-SNARE Proteins / Q000737:chemistry; D051381:Rats; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D050825:Synaptosomal-Associated Protein 25 / Q000737:chemistry
D050765:Qa-SNARE Proteins; D050825:Synaptosomal-Associated Protein 25; D010447:Peptide Hydrolases; D001905:Botulinum Toxins; C096324:botulinum toxin type C