Title
Importance of the Conserved Aromatic Residues in the Scorpion α-Like Toxin BmK M1 THE HYDROPHOBIC SURFACE REGION REVISITED
Author
Yan-Mei Sun, Frank Bosmans, Rong-Huan Zhu, Cyril Goudet, Yu-Mei Xiong, Jan Tytgat, Da-Cheng Wang
Year
2003
Journal
The Journal of Biological Chemistry
Abstract
About one-third of the amino acid residues conserved in all scorpion long chain Na channel toxins are aromatic residues, some of which constitute the so-called “conserved hydrophobic surface.” At present, in-depth structure-function studies of these aromatic residues using site-directed mutagenesis are still rare. In this study, an effective yeast expression system was used to study the role of seven conserved aromatic residues (Tyr5 , Tyr14, Tyr21, Tyr35, Trp38, Tyr42, and Trp47) from the scorpion toxin BmK M1. Using site-directed mutagenesis, all of these aromatic residues were individually substituted with Gly in association with a more conservative substitution of Phe for Tyr5 , Tyr14, Tyr35, or Trp47. The mutants, which were expressed in Saccharomyces cerevisiae S-78 cells, were then subjected to a bioassay in mice, electrophysiological characterization on cloned Na channels (Nav1.5), and CD analysis. Our results show an eye-catching correlation between the LD50 values in mice and the EC50 values on Nav1.5 channels in oocytes, indicating large mutant-dependent differences that emphasize important specific roles for the conserved aromatic residues in BmK M1. The aromatic side chains of the Tyr5 , Tyr35, and Trp47 cluster protruding from the three-stranded -sheet seem to be essential for the structure and function of the toxin. Trp38 and Tyr42 (located in the 2-sheet and in the loop between the 2- and 3-sheets, respectively) are most likely involved in the pharmacological function of the toxin. Scor
Full Article
Instrument
J-720
Keywords
Circular dichroism, Secondary structure, Biochemistry