Handley Lindsey D, Treuheit Nicholas A, Venkatesh Varun J, Komives Elizabeth A
Department of Chemistry and Biochemistry, University of California at San Diego , 9500 Gilman Drive, La Jolla, California 92093-0378, United States.
Department of Medicinal Chemistry, University of Washington , Health Sciences Building H072A, 1959 Northeast Pacific Street, Seattle, Washington 98195, United States.
Biochemistry. 2015 Nov 3;54(43):6650-8. doi: 10.1021/acs.biochem.5b00825. Epub 2015 Oct 26.
Human α-thrombin is a serine protease with dual functions. Thrombin acts as a procoagulant, cleaving fibrinogen to make the fibrin clot, but when bound to thrombomodulin (TM), it acts as an anticoagulant, cleaving protein C. A minimal TM fragment consisting of the fourth, fifth, and most of the sixth EGF-like domain (TM456m) that has been prepared has much improved solubility, thrombin binding capacity, and anticoagulant activity versus those of previous TM456 constructs. In this work, we compare backbone amide exchange of human α-thrombin in three states: apo, D-Phe-Pro-Arg-chloromethylketone (PPACK)-bound, and TM456m-bound. Beyond causing a decreased level of amide exchange at their binding sites, TM and PPACK both cause a decreased level of amide exchange in other regions including the γ-loop and the adjacent N-terminus of the heavy chain. The decreased level of amide exchange in the N-terminus of the heavy chain is consistent with the historic model of activation of serine proteases, which involves insertion of this region into the β-barrel promoting the correct conformation of the catalytic residues. Contrary to crystal structures of thrombin, hydrogen-deuterium exchange mass spectrometry results suggest that the conformation of apo-thrombin does not yet have the N-terminus of the heavy chain properly inserted for optimal catalytic activity, and that binding of TM allosterically promotes the catalytically active conformation.
人α-凝血酶是一种具有双重功能的丝氨酸蛋白酶。凝血酶作为促凝剂，裂解纤维蛋白原形成纤维蛋白凝块，但当与血栓调节蛋白（TM）结合时，它作为抗凝剂，裂解蛋白C。已制备的由第四、第五和大部分第六个表皮生长因子样结构域组成的最小TM片段（TM456m），其溶解性、凝血酶结合能力和抗凝活性比以前的TM456构建体有了很大提高。在这项工作中，我们比较了人α-凝血酶在三种状态下的主链酰胺交换情况：无配体状态、与D-苯丙氨酸-脯氨酸-精氨酸-氯甲基酮（PPACK）结合状态以及与TM456m结合状态。除了导致其结合位点处酰胺交换水平降低外，TM和PPACK还导致其他区域包括γ-环和重链相邻N端的酰胺交换水平降低。重链N端酰胺交换水平的降低与丝氨酸蛋白酶激活的传统模型一致，该模型涉及该区域插入β-桶以促进催化残基的正确构象。与凝血酶的晶体结构相反，氢-氘交换质谱结果表明，无配体凝血酶的构象尚未使重链的N端正确插入以实现最佳催化活性，并且TM的结合通过变构促进催化活性构象。
