Site-directed mutagenesis identifies active-site residues of the light chain of botulinum neurotoxin type A.
Botulinum neurotoxins (BoNTs) are metalloproteases which block neuroexocytosis via specific cleavage and inactivation of SNARE proteins. Such proteolysis accounts for the extreme toxicity of these neurotoxins and of their prolonged effect. The recently determined structures of BoNT/A and/B allows one to design active-site mutants to probe the role of specific residues in the proteolysis of SNARE proteins. Here we present the results of mutations of the second glutamyl residue involved in zinc coordination and of a tyrosine and a phenylalanine residues that occupy critical positions within the active site of BoNT/A. The spectroscopic properties of the purified mutants are closely similar to those of the wild-type molecule indicating the acquisition of a correct tertiary structure. Mutation of the Glu-262* nearly abolishes SNAP-25 hydrolysis as expected for a residue involved in zinc coordination. The Phe-266 and Tyr-366 mutants have reduced proteolytic activity indicating a direct participation in the proteolytic reaction, and their possible role in catalysis is discussed.
Rigoni
M
M
Dipartimento di Scienze Biomediche Sperimentali, Università di Padova, Viale G. Colombo, 3, 35121 Padua, Italy.
Caccin
P
P
Johnson
E A
EA
Montecucco
C
C
Rossetto
O
O
D000595:Amino Acid Sequence; D001665:Binding Sites; D001905:Botulinum Toxins / Q000737:chemistry; D019274:Botulinum Toxins, Type A / Q000737:chemistry* / Q000235:genetics* / Q000494:pharmacology; D002942:Circular Dichroism; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009465:Neuromuscular Agents / Q000737:chemistry* / Q000494:pharmacology; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000737:chemistry / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D013744:Tetanus Toxin / Q000737:chemistry; D015032:Zinc / Q000032:analysis
D009465:Neuromuscular Agents; D021122:Protein Subunits; D011994:Recombinant Proteins; D013744:Tetanus Toxin; C096323:rimabotulinumtoxinB; D001905:Botulinum Toxins; D019274:Botulinum Toxins, Type A; D015032:Zinc