Unknown

Dataset Information

0

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.

SUBMITTER: Roth R 

PROVIDER: S-EPMC4653662 | biostudies-literature | 2015 Nov

REPOSITORIES: biostudies-literature

altmetric image

Publications

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.

Roth Ryan R   Swanson Richard R   Izaguirre Gonzalo G   Bock Susan C SC   Gettins Peter G W PGW   Olson Steven T ST  

The Journal of biological chemistry 20150910 47


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 basa  ...[more]

Similar Datasets

| S-EPMC1456925 | biostudies-literature
| S-EPMC10921935 | biostudies-literature
| S-EPMC6363533 | biostudies-literature
| S-EPMC4758949 | biostudies-literature
| S-EPMC10791936 | biostudies-literature
| S-EPMC4826033 | biostudies-literature
| S-EPMC2965262 | biostudies-literature
2019-02-15 | GSE126550 | GEO
| S-EPMC2743457 | biostudies-literature
| S-EPMC5666567 | biostudies-literature