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J. Biol. Chem., Vol. 280, Issue 10, 9567-9577, March 11, 2005

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Structural Basis of Citrate-dependent and Heparan Sulfate-mediated Cell Surface Retention of Cobra Cardiotoxin A3^*

Shao-Chen Lee, Hong-Hsiang Guan¶, Chia-Hui Wang, Wei-Ning Huang, Siu-Cin Tjong, Chun-Jung Chen¶||**, and Wen-guey Wu

From the Institute of Bioinformatics and Structural Biology, National TsingHua University, Hsinchu 30043, ^¶Biology Group, Research Division, National Synchroton Radiation Research Center, Hsinchu 30077, and ^||Department of Physics and Structural Biology Program, National TsingHua University, Hsinchu 30043, Taiwan

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 Å 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.

Received for publication, November 2, 2004 , and in revised form, December 2, 2004.

The atomic coordinates and structure factors (code 1XT3) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by National Science Council Grants NSC-92-2311-B-007-014, 91-EC-17-A-17-SE-0009 (to W.-G. W.), NSC-92-2321-B-213-001, and National Synchroton Radiation Research Center Grant 924RSB02 (to C.-J. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Both authors contributed equally to this work.

^** To whom correspondence may be addressed. Tel.: 886-3-578-0281 (ext. 7330); Fax: 886-3-578-3813; E-mail: cjchen{at}nsrrc.org.tw. To whom correspondence may be addressed. Tel.: 886-3-573-1040; Fax: 886-3-571-5934; E-mail: wgwu{at}life.nthu.edu.tw.

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