Structural basis of citrate-dependent and heparan sulfate-mediated cell surface retention of cobra cardiotoxin A3.
Anionic citrate is a major component of venom, but the role of venom citrate in toxicity other than its inhibitory effect on the cation-dependent action of venom toxins is poorly understood. By immobilizing Chinese hamster ovary cells in microcapillary tubes and heparin on sensor chips, we demonstrated that heparan sulfate-mediated cell retention of the major cardiotoxin (CTX) from the Taiwan cobra, CTX A3, near membrane surfaces is citrate-dependent. X-ray determination of a CTX A3-heparin hexasaccharide complex structure at 2.4 A resolution revealed a molecular mechanism for toxin retention in which heparin-induced conformational changes of CTX A3 lead to citrate-mediated dimerization. A citrate ion bound to Lys-23 and Lys-31 near the tip of loop II stabilizes hydrophobic contact of the CTX A3 homodimer at the functionally important loop I and II regions. Additionally, the heparin hexasaccharide interacts with five CTX A3 molecules in the crystal structure, providing another mechanism whereby the toxin establishes a complex network of interactions that result in a strong interaction with cell surfaces presenting heparan sulfate. Our results suggest a novel role for venom citrate in biological activity and reveal a structural model that explains cell retention of cobra CTX A3 through heparan sulfate-CTX interactions.
Lee
Shao-Chen
SC
Institute of Bioinformatics and Structural Biology, National TsingHua University, Hsinchu 30043, Taiwan.
Guan
Hong-Hsiang
HH
Wang
Chia-Hui
CH
Huang
Wei-Ning
WN
Tjong
Siu-Cin
SC
Chen
Chun-Jung
CJ
Wu
Wen-guey
WG
D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism*; D002951:Citrates / Q000494:pharmacology*; D004179:Cobra Cardiotoxin Proteins / Q000737:chemistry* / Q000378:metabolism*; D004546:Elapid Venoms / Q000737:chemistry; D017815:Elapidae; D006497:Heparan Sulfate / Q000494:pharmacology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013624:Taiwan
D002951:Citrates; D004179:Cobra Cardiotoxin Proteins; D004546:Elapid Venoms; D006497:Heparan Sulfate; D009844:Oligosaccharides