title: Structural basis of citrate-dependent and heparan sulfate-mediated cell surface retention of cobra cardiotoxin A3.
abstract: 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.
authors.affiliation: Institute of Bioinformatics and Structural Biology, National TsingHua University, Hsinchu 30043, Taiwan.
authors.forename: Shao-Chen
authors.initials: SC
authors.lastname: Lee
authors.forename: Hong-Hsiang
authors.initials: HH
authors.lastname: Guan
authors.forename: Chia-Hui
authors.initials: CH
authors.lastname: Wang
authors.forename: Wei-Ning
authors.initials: WN
authors.lastname: Huang
authors.forename: Siu-Cin
authors.initials: SC
authors.lastname: Tjong
authors.forename: Chun-Jung
authors.initials: CJ
authors.lastname: Chen
authors.forename: Wen-guey
authors.initials: WG
authors.lastname: Wu
mesh_terms: 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
chemical_list: D002951:Citrates; D004179:Cobra Cardiotoxin Proteins; D004546:Elapid Venoms; D006497:Heparan Sulfate; D009844:Oligosaccharides