Role of glycosphingolipid conformational change in membrane pore forming activity of cobra cardiotoxin.
The major cardiotoxin from Taiwan cobra (CTX A3) is a pore forming beta-sheet polypeptide that requires sulfatide (sulfogalactosylceramide, SGC) on the plasma membrane of cardiomyocytes for CTX-induced membrane leakage and cell internalization. Herein, we demonstrate by fluorescence spectroscopic studies that sulfatides induce CTX A3 oligomerization in sulfatide containing phosphatidylcholine (PC) vesicles to form transient pores with pore size and lifetime in the range of about 30 A and 10(-2) s, respectively. These values are consistent with the CTX A3-induced conductance and mean lifetime determined previously by using patch-clamp electrophysiological experiments on the plasma membrane of H9C2 cells. We also derived the peripheral binding structural model of CTX A3-sulfatide complex in sulfatide containing PC micelles by NMR and molecular docking method and compared with other CTX A3-sulfatide complex structure determined previously by X-ray in membrane-like environment. The NMR results indicate that sulfatide head group conformation changes from a bent shovel (-sc/ap) to an extended (sc/ap) conformation upon initial binding of CTX A3. An additional global reorientation of sulfatide molecule is also needed for CTX A3 dimer formation as inferred by the difference between the X-ray and NMR complex structure. Since the overall folding of CTX A3 molecules remained the same, sulfatide in phospholipid bilayer is proposed to play an active role by involving its local and global conformational changes to promote both the oligomerization and reorientation of CTX A3 molecule for its transient pore formation and cell internalization.
Tjong
Siu-Cin
SC
Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu, Taiwan.
Wu
Po-Long
PL
Wang
Chang-Mao
CM
Huang
Wei-Ning
WN
Ho
Nan-Lu
NL
Wu
Wen-guey
WG
D000818:Animals; D054715:Cardiotoxins / Q000737:chemistry*; D002460:Cell Line; D017815:Elapidae; D006028:Glycosphingolipids / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D008968:Molecular Conformation; D019906:Nuclear Magnetic Resonance, Biomolecular; D013050:Spectrometry, Fluorescence
D054715:Cardiotoxins; D006028:Glycosphingolipids