3HKJ

Crystal structure of human thrombin mutant W215A/E217A in complex with the extracellular fragment of human PAR1

Summary for 3HKJ

• Entry DOI: 10.2210/pdb3hkj/pdb
• Related: 1TQ0 3HK3 3HK6 3HKI
• Descriptor: Thrombin light chain, Thrombin heavy chain, Proteinase-activated receptor 1, ... (5 entities in total)
• Functional Keywords: serine protease, acute phase, blood coagulation, cleavage on pair of basic residues, disease mutation, disulfide bond, gamma-carboxyglutamic acid, glycoprotein, hydrolase, kringle, protease, secreted, zymogen, cell membrane, g-protein coupled receptor, membrane, phosphoprotein, receptor, transducer, transmembrane
• Biological source: Homo sapiens (human); More
• Total number of polymer chains: 6
• Total formula weight: 72298.40

Authors

Gandhi, P.S.,Page, M.J.,Chen, Z.,Bush-Pelc, L.,Di Cera, E. (deposition date: 2009-05-23, release date: 2009-07-07, Last modification date: 2024-11-27)

Primary citation

Gandhi, P.S.,Page, M.J.,Chen, Z.,Bush-Pelc, L.,Di Cera, E.
Mechanism of the Anticoagulant Activity of Thrombin Mutant W215A/E217A.
J.Biol.Chem., 284:24098-24105, 2009

PubMed Abstract: The thrombin mutant W215A/E217A (WE) is a potent anticoagulant both in vitro and in vivo. Previous x-ray structural studies have shown that WE assumes a partially collapsed conformation that is similar to the inactive E* form, which explains its drastically reduced activity toward substrate. Whether this collapsed conformation is genuine, rather than the result of crystal packing or the mutation introduced in the critical 215-217 beta-strand, and whether binding of thrombomodulin to exosite I can allosterically shift the E* form to the active E form to restore activity toward protein C are issues of considerable mechanistic importance to improve the design of an anticoagulant thrombin mutant for therapeutic applications. Here we present four crystal structures of WE in the human and murine forms that confirm the collapsed conformation reported previously under different experimental conditions and crystal packing. We also present structures of human and murine WE bound to exosite I with a fragment of the platelet receptor PAR1, which is unable to shift WE to the E form. These structural findings, along with kinetic and calorimetry data, indicate that WE is strongly stabilized in the E* form and explain why binding of ligands to exosite I has only a modest effect on the E*-E equilibrium for this mutant. The E* --> E transition requires the combined binding of thrombomodulin and protein C and restores activity of the mutant WE in the anticoagulant pathway.

PubMed: 19586901
DOI: 10.1074/jbc.M109.025403

Experimental method

X-RAY DIFFRACTION (2.6 Å)