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Crystal structure of human thrombin mutant W215A/E217A in complex with the extracellular fragment of human PAR1
Structural highlights
DiseaseTHRB_HUMAN Defects in F2 are the cause of factor II deficiency (FA2D) [MIM:613679. It is a very rare blood coagulation disorder characterized by mucocutaneous bleeding symptoms. The severity of the bleeding manifestations correlates with blood factor II levels.[1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] Genetic variations in F2 may be a cause of susceptibility to ischemic stroke (ISCHSTR) [MIM:601367; also known as cerebrovascular accident or cerebral infarction. A stroke is an acute neurologic event leading to death of neural tissue of the brain and resulting in loss of motor, sensory and/or cognitive function. Ischemic strokes, resulting from vascular occlusion, is considered to be a highly complex disease consisting of a group of heterogeneous disorders with multiple genetic and environmental risk factors.[13] Defects in F2 are the cause of thrombophilia due to thrombin defect (THPH1) [MIM:188050. It is a multifactorial disorder of hemostasis characterized by abnormal platelet aggregation in response to various agents and recurrent thrombi formation. Note=A common genetic variation in the 3-prime untranslated region of the prothrombin gene is associated with elevated plasma prothrombin levels and an increased risk of venous thrombosis. Defects in F2 are associated with susceptibility to pregnancy loss, recurrent, type 2 (RPRGL2) [MIM:614390. A common complication of pregnancy, resulting in spontaneous abortion before the fetus has reached viability. The term includes all miscarriages from the time of conception until 24 weeks of gestation. Recurrent pregnancy loss is defined as 3 or more consecutive spontaneous abortions.[14] FunctionTHRB_HUMAN Thrombin, which cleaves bonds after Arg and Lys, converts fibrinogen to fibrin and activates factors V, VII, VIII, XIII, and, in complex with thrombomodulin, protein C. Functions in blood homeostasis, inflammation and wound healing.[15] Evolutionary Conservation Check, as determined by ConSurfDB. You may read the explanation of the method and the full data available from ConSurf. Publication Abstract from PubMedThe 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. Mechanism of the anticoagulant activity of thrombin mutant W215A/E217A.,Gandhi PS, Page MJ, Chen Z, Bush-Pelc L, Di Cera E J Biol Chem. 2009 Sep 4;284(36):24098-105. Epub 2009 Jul 8. PMID:19586901[16] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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