title: Two clusters of charged residues located in the electropositive face of the von Willebrand factor A1 domain are essential for heparin binding.
abstract: The VWF A1 domain seems to possess two heparin binding regions (residues 565-587 and 633-648) displaying positively charged amino acids, but the overall polyanion-A1 domain interaction scheme remains essentially elusive. To probe this molecular reaction as well as the role of electrostatic forces in VWF-heparin interaction, we performed mutagenesis and molecular modeling experiments. Fifteen mutated rVWFs were expressed [R571A, K572A, R573A, K585A, R571A/K572A/R573A, R578A/R579A, R578A/R579A/K585A, R571A/K572A/R573A/R578A/R579A/K585A (6A), K642G, K643G, K644G, K645G, K642G/K645G, K643G/K644G, and K642G/K643G/K644G/K645G (4G)]. Experimental results indicate that the multimeric structure of the mutants was similar to that of wild-type (WT) rVWF and that all rVWFs displayed normal binding to four conformation-dependent mAbs directed against the A1 domain. Three variants displayed significant reductions in the level of heparin binding. The 6A variant showed 39.2 +/- 1.3% of the WT rVWF level (p < 0.005), while mutants K643G/K644G and 4G showed 63.6 +/- 3.2 and 53.3 +/- 5% of the WT rVWF level, respectively (p < 0.005). Computational investigations showed that one face of the A1 domain is strongly electropositive, indicating that electrostatic forces should be essential in steering heparin onto its binding site. In agreement with our experimental data, the most striking alterations of the electrostatic potential contours were seen for mutants 4G, K643G/K644G, and 6A. Our data suggest that two clusters, one at positions 571-573, 578, 579, and 585 and the other at positions 642-645, act in concert for the recognition of heparin, forming a single extended binding surface across the electropositive face of the VWF A1 domain. A structural model of the VWF A1 domain-heparin complex is proposed, taking into account both experimental and computer modeling data.
authors.affiliation: INSERM U143, 84 rue du General Leclerc, 94 276 Bicetre Cedex, France.
authors.forename: Ghassem
authors.initials: G
authors.lastname: Rastegar-Lari
authors.forename: Bruno O
authors.initials: BO
authors.lastname: Villoutreix
authors.forename: Anne-Sophie
authors.initials: AS
authors.lastname: Ribba
authors.forename: Paulette
authors.initials: P
authors.lastname: Legendre
authors.forename: Dominique
authors.initials: D
authors.lastname: Meyer
authors.forename: Dominique
authors.initials: D
authors.lastname: Baruch
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001665:Binding Sites; D019556:COS Cells; D002522:Chlorocebus aethiops; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D016296:Mutagenesis; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000378:metabolism*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D055672:Static Electricity; D014162:Transfection; D014841:von Willebrand Factor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D019038:Platelet Glycoprotein GPIb-IX Complex; D011994:Recombinant Proteins; D014841:von Willebrand Factor; D006493:Heparin