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J. Biol. Chem., Vol. 281, Issue 41, 30400-30411, October 13, 2006

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Molecular Mapping of the Chloride-binding Site in von Willebrand Factor (VWF)

ENERGETICS AND CONFORMATIONAL EFFECTS ON THE VWF/ADAMTS-13 INTERACTION^*

Raimondo De Cristofaro¹, Flora Peyvandi, Luciano Baronciani, Roberta Palla, Silvia Lavoretano, Rossana Lombardi, Enrico Di Stasio^¶, Augusto B. Federici, and Pier Mannuccio Mannucci

From the Hemostasis Research Centre, Institute of Internal Medicine and Geriatrics, Catholic University School of Medicine, 00168 Rome, A. Bianchi Bonomi Hemophilia and Thrombosis Center, University of Milan, and the Department of Medicine and Medical Specialties, IRCCS Maggiore Hospital, 20122 Milan, and ^¶Institute of Biochemistry and Clinical Biochemistry, Catholic University School of Medicine, 00168 Rome, Italy

Physiological concentrations of NaCl inhibit the hydrolysis of von Willebrand factor (VWF) by ADAMTS-13. This effect is because of the specific binding of chloride ions to VWF. Urea-induced unfolding was measured in the presence of NaCl, CH₃COONa, and NaClO₄ at pH 8.0, 25 °C, for multimeric VWF, the recombinant A1-A2-A3 VWF domains, and the A1 domain. Chloride stabilizes the folded conformation of the A1-A2-A3 and A1 domains more efficiently than acetate but less strongly than perchlorate. Spectroscopic evidence showed that chloride binds to both the A1 and A1-A2 domain but not to the isolated A2 domain. Binding of Cl^– to both wild type (WT) and the natural mutant p.R1306W A1-A2-A3 domains of VWF has a large heat capacity change equal to –1 and –0.4 kcal mol^–1 K^–1 for WT and p.R1306W A1-A2-A3 domains, respectively. This result implies that a burial of a vast apolar surface area is caused by conformational transitions linked to chloride binding. At any temperature, chloride affinity was higher for WT than for the mutant p.R1306W form. Chloride ions inhibit hydrolysis by ADAMTS-13 of the A1-A2-A3 and A1-A2 domains in the presence of either urea or high shear stress, whereas this effect was either absent or negligible in experiments using A2 and A2-A3 domains. These findings show that the A1 domain contains the binding site of chloride ions that control allosterically the proteolysis by ADAMTS-13 of the Tyr¹⁶⁰⁵–Met¹⁶⁰⁶ bond in the A2 domain and that the R1306W mutation of type 2B VWD quenches the binding of chloride ion to the A1 domain.

Received for publication, April 7, 2006 , and in revised form, August 8, 2006.

^* This work was supported in part by Grant PRIN-2005 from the Italian Ministry of University and Research (to R. D. C.) and a grant from the Fondazione Cariplo (to P. M. M. and F. P.). 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.

¹ To whom correspondence should be addressed: Hemostasis Research Centre, Institute of Internal Medicine and Geriatrics, Catholic University School of Medicine, Largo F. Vito, 1, 00168 Rome, Italy. Tel.: 39-6-30154438; Fax: 39-6-30155915; E-mail: rdecristofaro{at}rm.unicatt.it.

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