Molecular mapping of the chloride-binding site in von Willebrand factor (VWF): Energetics and conformational effects on the VWF/ADAMTS-13 interaction

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

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish
Pages (from-to)30400-30411
Number of pages12
JournalJournal of Biological Chemistry
Volume281
Issue number41
DOIs
Publication statusPublished - Oct 13 2006

    Fingerprint

ASJC Scopus subject areas

  • Biochemistry

Cite this