title: Saturation Mutagenesis of the Antithrombin Reactive Center Loop P14 Residue Supports a Three-step Mechanism of Heparin Allosteric Activation Involving Intermediate and Fully Activated States.
abstract: Past studies have suggested that a key feature of the mechanism of heparin allosteric activation of the anticoagulant serpin, antithrombin, is the release of the reactive center loop P14 residue from a native state stabilizing interaction with the hydrophobic core. However, more recent studies have indicated that this structural change plays a secondary role in the activation mechanism. To clarify this role, we expressed and characterized 15 antithrombin P14 variants. The variants exhibited basal reactivities with factors Xa and IXa, heparin affinities and thermal stabilities that were dramatically altered from wild type, consistent with the P14 mutations perturbing native state stability and shifting an allosteric equilibrium between native and activated states. Rapid kinetic studies confirmed that limiting rate constants for heparin allosteric activation of the mutants were altered in conjunction with the observed shifts of the allosteric equilibrium. However, correlations of the P14 mutations' effects on parameters reflecting the allosteric activation state of the serpin were inconsistent with a two-state model of allosteric activation and suggested multiple activated states. Together, these findings support a minimal three-state model of allosteric activation in which the P14 mutations perturb equilibria involving distinct native, intermediate, and fully activated states wherein the P14 residue retains an interaction with the hydrophobic core in the intermediate state but is released from the core in the fully activated state, and the bulk of allosteric activation has occurred in the intermediate.
authors.affiliation: Center for Molecular Biology of Oral Diseases and Departments of Periodontics.
authors.forename: Ryan
authors.initials: R
authors.lastname: Roth
authors.affiliation: Center for Molecular Biology of Oral Diseases and Departments of Periodontics.
authors.forename: Richard
authors.initials: R
authors.lastname: Swanson
authors.affiliation: Center for Molecular Biology of Oral Diseases and Departments of Periodontics.
authors.forename: Gonzalo
authors.initials: G
authors.lastname: Izaguirre
authors.affiliation: Departments of Medicine and Bioengineering, University of Utah, Salt Lake City, Utah 84132.
authors.forename: Susan C
authors.initials: SC
authors.lastname: Bock
authors.affiliation: Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, Illinois 60612.
authors.forename: Peter G W
authors.initials: PGW
authors.lastname: Gettins
authors.affiliation: Center for Molecular Biology of Oral Diseases and Departments of Periodontics. Electronic address: stolson@uic.edu.
authors.forename: Steven T
authors.initials: ST
authors.lastname: Olson
mesh_terms: D000494:Allosteric Regulation; D000991:Antithrombins / Q000737:chemistry / Q000378:metabolism*; D006493:Heparin / Q000378:metabolism*; D007700:Kinetics; D009154:Mutation; D010447:Peptide Hydrolases / Q000378:metabolism; D011485:Protein Binding
chemical_list: D000991:Antithrombins; D006493:Heparin; D010447:Peptide Hydrolases
keywords: allosteric regulation; anticoagulant; antithrombin (AT); coagulation factor; heparin; protease; protease inhibitor; serine protease; serpin