title: Conformational activation of antithrombin by heparin involves an altered exosite interaction with protease.
abstract: Heparin allosterically activates antithrombin as an inhibitor of factors Xa and IXa by enhancing the initial Michaelis complex interaction of inhibitor with protease through exosites. Here, we investigate the mechanism of this enhancement by analyzing the effects of alanine mutations of six putative antithrombin exosite residues and three complementary protease exosite residues on antithrombin reactivity with these proteases in unactivated and heparin-activated states. Mutations of antithrombin Tyr(253) and His(319) exosite residues produced massive 10-200-fold losses in reactivity with factors Xa and IXa in both unactivated and heparin-activated states, indicating that these residues made critical attractive interactions with protease independent of heparin activation. By contrast, mutations of Asn(233), Arg(235), Glu(237), and Glu(255) exosite residues showed that these residues made both repulsive and attractive interactions with protease that depended on the activation state and whether the critical Tyr(253)/His(319) residues were mutated. Mutation of factor Xa Arg(143), Lys(148), and Arg(150) residues that interact with the exosite in the x-ray structure of the Michaelis complex confirmed the importance of all residues for heparin-activated antithrombin reactivity and Arg(150) for native serpin reactivity. These results demonstrate that the exosite is a key determinant of antithrombin reactivity with factors Xa and IXa in the native as well as the heparin-activated state and support a new model of allosteric activation we recently proposed in which a balance between attractive and repulsive exosite interactions in the native state is shifted to favor the attractive interactions in the activated state through core conformational changes induced by heparin binding.
authors.affiliation: From the Department of Periodontics, Center for Molecular Biology of Oral Diseases and goniza@uic.edu.
authors.forename: Gonzalo
authors.initials: G
authors.lastname: Izaguirre
authors.affiliation: the Centro Regional de Hemodonación, University of Murcia, Murcia 30003, Spain, and.
authors.forename: Sonia
authors.initials: S
authors.lastname: Aguila
authors.affiliation: From the Department of Periodontics, Center for Molecular Biology of Oral Diseases and.
authors.forename: Lixin
authors.initials: L
authors.lastname: Qi
authors.affiliation: From the Department of Periodontics, Center for Molecular Biology of Oral Diseases and.
authors.forename: Richard
authors.initials: R
authors.lastname: Swanson
authors.affiliation: From the Department of Periodontics, Center for Molecular Biology of Oral Diseases and.
authors.forename: Ryan
authors.initials: R
authors.lastname: Roth
authors.affiliation: the Department of Biochemistry and Molecular Biology, St. Louis University, St. Louis, Missouri 63104.
authors.forename: Alireza R
authors.initials: AR
authors.lastname: Rezaie
authors.affiliation: the Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, Illinois 60612.
authors.forename: Peter G W
authors.initials: PG
authors.lastname: Gettins
authors.affiliation: From the Department of Periodontics, Center for Molecular Biology of Oral Diseases and.
authors.forename: Steven T
authors.initials: ST
authors.lastname: Olson
mesh_terms: D000494:Allosteric Regulation; D000596:Amino Acids / Q000737:chemistry* / Q000378:metabolism; D000991:Antithrombins / Q000737:chemistry* / Q000378:metabolism; D016367:Baculoviridae / Q000235:genetics; D001665:Binding Sites; D015949:Factor IXa / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015951:Factor Xa / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D065427:Factor Xa Inhibitors / Q000737:chemistry* / Q000378:metabolism; D015870:Gene Expression; D006493:Heparin / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship
chemical_list: D000596:Amino Acids; D000991:Antithrombins; D065427:Factor Xa Inhibitors; D011994:Recombinant Proteins; D006493:Heparin; D015949:Factor IXa; D015951:Factor Xa
keywords: Allosteric Regulation; Anticoagulant; Antithrombin (AT); Blood Coagulation; Exosite; Heparin; Protease; Serpin