Substrate recognition of VAMP-2 by botulinum neurotoxin B and tetanus neurotoxin.
Botulinum neurotoxin (BoNT; serotypes A-G) and tetanus neurotoxin elicit flaccid and spastic paralysis, respectively. These neurotoxins are zinc proteases that cleave SNARE proteins to inhibit synaptic vesicle fusion to the plasma membrane. Although BoNT/B and tetanus neurotoxin (TeNT) cleave VAMP-2 at the same scissile bond, their mechanism(s) of VAMP-2 recognition is not clear. Mapping experiments showed that residues 60-87 of VAMP-2 were sufficient for efficient cleavage by BoNT/B and that residues 40-87 of VAMP-2 were sufficient for efficient TeNT cleavage. Alanine-scanning mutagenesis and kinetic analysis identified three regions within VAMP-2 that were recognized by BoNT/B and TeNT: residues adjacent to the site of scissile bond cleavage (cleavage region) and residues located within N-terminal and C-terminal regions relative to the cleavage region. Analysis of residues within the cleavage region showed that mutations at the P7, P4, P2, and P1' residues of VAMP-2 had the greatest inhibition of LC/B cleavage (> or =32-fold), whereas mutations at P7, P4, P1', and P2' residues of VAMP-2 had the greatest inhibition of LC/TeNT cleavage (> or =64-fold). Residues within the cleavage region influenced catalysis, whereas residues N-terminal and C-terminal to the cleavage region influenced binding affinity. Thus, BoNT/B and TeNT possess similar organization but have unique residues to recognize and cleave VAMP-2. These studies provide new insights into how the clostridial neurotoxins recognize their substrates.
Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
Sheng
S
Chen
Cherisse
C
Hall
Joseph T
JT
Barbieri
D000409:Alanine / Q000737:chemistry; D001905:Botulinum Toxins / Q000737:chemistry*; D019274:Botulinum Toxins, Type A; D002462:Cell Membrane / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008666:Metalloendopeptidases / Q000737:chemistry*; D008956:Models, Chemical; D008958:Models, Molecular; D017354:Point Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013744:Tetanus Toxin / Q000737:chemistry*; D014357:Trypsin / Q000737:chemistry; D050988:Vesicle-Associated Membrane Protein 2 / Q000737:chemistry* / Q000378:metabolism
D013744:Tetanus Toxin; D050988:Vesicle-Associated Membrane Protein 2; C096323:rimabotulinumtoxinB; C035979:tetanospasmin; D005982:Glutathione Transferase; D014357:Trypsin; D008666:Metalloendopeptidases; D001905:Botulinum Toxins; D019274:Botulinum Toxins, Type A; D000409:Alanine