A correlation between differential structural features and the degree of endopeptidase activity of type A botulinum neurotoxin in aqueous solution.
Botulinum neurotoxin type A is one of the most toxic substances known to man (LD(50) for mouse 0.1 ng/kg). It is also an effective therapeutic drug against involuntary muscle disorders and for pain management. BoNT/A is a Zn(2+) endopeptidase which selectively cleaves SNAP-25 (synaptosomal-associated protein of 25 kDa), a critical component of the exocytotic machinery. Based on nucleotide sequence, BoNT/A is a 145 kDa protein, which appears as a 145 kDa protein band on sodium dodecyl sulfate--polyacrylamide gel electrophoresis. We have examined the structure of BoNT/A in aqueous solution, and found the structure in aqueous solution differs dramatically from that resolved by X-ray crystallography, both at secondary and at quaternary levels. In terms of secondary structure, BoNT/A in aqueous solution has about 47% beta-sheet structure as revealed by infrared spectroscopy, while X-ray crystallography revealed only 17% beta-sheet structure. In terms of quaternary structure, the estimated molecular mass of the native BoNT/A in aqueous solution ranged between 230 and 314 kDa, based on results from different chemical and biophysical techniques (native gel electrophoresis, chemical cross-linking, size exclusion chromatography, and fluorescence anisotropy). These results indicate that BoNT/A exists as a dimer in aqueous solution, which contrasts with the reported monomeric structure of BoNT/A based on X-ray crystallography. The dimeric form of BoNT/A can self-dissociate into the monomeric form at a concentration lower than 50 nM. This concentration-dependent structural change has a significant impact on the endopeptidase activity of BoNT/A: the catalytic efficiency of the monomeric BoNT/A is about 4-fold higher than that of its dimeric form. This difference implies a sterically restricted catalytic site of BoNT/A in the dimeric form of BoNT/A.
Cai
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Department of Chemistry and Biochemistry, University of Massachusetts Dartmouth, North Dartmouth, Massachusetts 02747, USA.
Singh
B R
BR
D019274:Botulinum Toxins, Type A / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D002850:Chromatography, Gel; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D005454:Fluorescence Polarization; D008666:Metalloendopeptidases / Q000737:chemistry*; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012996:Solutions; D013050:Spectrometry, Fluorescence; D017550:Spectroscopy, Fourier Transform Infrared; D014364:Tryptophan; D014867:Water
D012996:Solutions; D014867:Water; D014364:Tryptophan; D008666:Metalloendopeptidases; C022510:thimet oligopeptidase; D019274:Botulinum Toxins, Type A