Roles of disulfide bridges in scorpion toxin BmK M1 analyzed by mutagenesis.
The unique fold of scorpion toxins is a natural scaffold for protein engineering, in which multiple disulfide bonds are crucial structural elements. To understand the respective roles of these disulfide bridges, a mutagenesis analysis for the four disulfide bonds, 12-63, 16-36, 22-46 and 26-48, of a representative toxin BmK M1 from the scorpion Buthus martensii Karsch was carried out. All cysteines were replaced by serine with double mutations. The recombinant mutants were expressed in the Saccharomyces cerevisiae S-78 system. Toxic activities of the expressed mutants were tested on ICR mice in vivo and on neuronal Na+ channels (rNav1.2) by electrophysiological analysis. Recombinant variants M1 (C22S,C46S) and M1 (C26S,C48S) were not expressed at all; M1 (C16S,C36S) could be expressed at trace levels but was extremely unstable. Variant M1 (C12S,C63S) could be expressed in an amount comparable with that of unmodified rBmK M1, but had no detectable bioactivities. The results indicated that among the four disulfide bonds for long-chain scorpion toxins, loss of either bridge C22-C46 or C26-C48 is fatal for the general folding of the molecule. Bridge C16-C36 mainly contributes to the global stability of the folded scaffold, and bridge C12-C63 plays an essential role in the functional performance of scorpion toxins.
Sun
Y-M
YM
Center for Molecular Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China.
Liu
W
W
Zhu
R-H
RH
Goudet
C
C
Tytgat
J
J
Wang
D-C
DC
D000595:Amino Acid Sequence; D000818:Animals; D002942:Circular Dichroism; D003553:Cystine / Q000737:chemistry* / Q000235:genetics; D019476:Insect Proteins; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009474:Neurons / Q000187:drug effects; D009498:Neurotoxins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D011487:Protein Conformation; D012604:Scorpion Venoms / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D012605:Scorpions / Q000737:chemistry*; D016415:Sequence Alignment; D015222:Sodium Channels / Q000187:drug effects; D014981:Xenopus
C403888:BmK M1 neurotoxin; D019476:Insect Proteins; D009498:Neurotoxins; D012604:Scorpion Venoms; D015222:Sodium Channels; D003553:Cystine