Active-site mutagenesis of tetanus neurotoxin implicates TYR-375 and GLU-271 in metalloproteolytic activity.
Tetanus neurotoxin (TeNT) blocks neurotransmitter release by cleaving VAMP/synaptobrevin, a membrane associated protein involved in synaptic vesicle fusion. Such activity is exerted by the N-terminal 50kDa domain of TeNT which is a zinc-dependent endopeptidase (TeNT-L-chain). Based on the three-dimensional structure of botulinum neurotoxin serotype A (BoNT/A) and serotype B (BoNT/B), two proteins closely related to TeNT, and on X-ray scattering studies of TeNT, we have designed mutations at two active site residues to probe their involvement in activity. The active site of metalloproteases is composed of a primary sphere of residues co-ordinating the zinc atom, and a secondary sphere of residues that determines proteolytic specificity and activity. Glu-261 and Glu-267 directly co-ordinates the zinc atom in BoNT/A and BoNT/B respectively and the corresponding residue of TeNT was replaced by Asp or by the non conservative residue Ala. Tyr-365 is 4.3A away from zinc in BoNT/A, and the corresponding residue of TeNT was replaced by Phe or by Ala. The purified mutants had CD, fluorescence and UV spectra closely similar to those of the wild-type molecule. The proteolytic activity of TeNT-Asp-271 (E271D) is similar to that of the native molecule, whereas that of TeNT-Phe-375 (Y375F) is lower than the control. Interestingly, the two Ala mutants are completely devoid of enzymatic activity. These results demonstrate that both Glu-271 and Tyr-375 are essential for the proteolytic activity of TeNT.
Rossetto
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O
Centro CNR Biomembrane and Dipartimento di Scienze Biomediche, Università di Padova, Via G. Colombo 3, 35121, Padova, Italy. rossetto@civ.bio.unipd.it
Caccin
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P
Rigoni
M
M
Tonello
F
F
Bortoletto
N
N
Stevens
R C
RC
Montecucco
C
C
D000595:Amino Acid Sequence; D019274:Botulinum Toxins, Type A / Q000737:chemistry; D008565:Membrane Proteins / Q000378:metabolism; D008666:Metalloendopeptidases / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D050683:R-SNARE Proteins; D013329:Structure-Activity Relationship; D013744:Tetanus Toxin / Q000737:chemistry* / Q000378:metabolism; D015032:Zinc / Q000378:metabolism
D008565:Membrane Proteins; D050683:R-SNARE Proteins; D013744:Tetanus Toxin; C035979:tetanospasmin; D008666:Metalloendopeptidases; D019274:Botulinum Toxins, Type A; D015032:Zinc