Pozzi Nicola, Chen Zhiwei, Pelc Leslie A, Shropshire Daniel B, Di Cera Enrico
Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, MO 63104.
Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, MO 63104
Proc Natl Acad Sci U S A. 2014 May 27;111(21):7630-5. doi: 10.1073/pnas.1403779111. Epub 2014 May 12.
The zymogen prothrombin is proteolytically converted by factor Xa to the active protease thrombin in a reaction that is accelerated >3,000-fold by cofactor Va. This physiologically important effect is paradigmatic of analogous cofactor-dependent reactions in the coagulation and complement cascades, but its structural determinants remain poorly understood. Prothrombin has three linkers connecting the N-terminal Gla domain to kringle-1 (Lnk1), the two kringles (Lnk2), and kringle-2 to the C-terminal protease domain (Lnk3). Recent developments indicate that the linkers, and particularly Lnk2, confer on the zymogen significant flexibility in solution and enable prothrombin to sample alternative conformations. The role of this flexibility in the context of prothrombin activation was tested with several deletions. Removal of Lnk2 in almost its entirety (ProTΔ146-167) drastically reduces the enhancement of thrombin generation by cofactor Va from >3,000-fold to 60-fold because of a significant increase in the rate of activation in the absence of cofactor. Deletion of Lnk2 mimics the action of cofactor Va and offers insights into how prothrombin is activated at the molecular level. The crystal structure of ProTΔ146-167 reveals a contorted architecture where the domains are not vertically stacked, kringle-1 comes within 9 Å of the protease domain, and the Gla-domain primed for membrane binding comes in contact with kringle-2. These findings broaden our molecular understanding of a key reaction of the blood coagulation cascade where cofactor Va enhances activation of prothrombin by factor Xa by compressing Lnk2 and morphing prothrombin into a conformation similar to the structure of ProTΔ146-167.
凝血酶原酶原在因子Xa的作用下通过蛋白水解作用转化为活性蛋白酶凝血酶，该反应在辅因子Va的作用下加速>3000倍。这种具有重要生理意义的效应是凝血和补体级联中类似的辅因子依赖性反应的典型代表，但其结构决定因素仍知之甚少。凝血酶原有三个连接子，将N端的Gla结构域连接到kringle-1（Lnk1）、两个kringle结构域（Lnk2）以及kringle-2连接到C端蛋白酶结构域（Lnk3）。最近的研究进展表明这些连接子，尤其是Lnk2，赋予了酶原在溶液中显著的灵活性，并使凝血酶原能够呈现出不同的构象状态。通过几个缺失突变体测试了这种灵活性在凝血酶原激活过程中的作用。几乎完全去除Lnk2（ProTΔ146-167）会使辅因子Va对凝血酶生成的增强作用从>3000倍急剧降低至60倍，这是因为在没有辅因子的情况下激活速率显著增加。Lnk2的缺失模拟了辅因子Va的作用，并为凝血酶原在分子水平上的激活方式提供了见解。ProTΔ146-167的晶体结构揭示了一种扭曲的结构，其中各结构域并非垂直堆叠，kringle-1与蛋白酶结构域的距离在9 Å以内，而准备与膜结合的Gla结构域与kringle-2接触。这些发现拓宽了我们对血液凝固级联关键反应的分子理解，即辅因子Va通过压缩Lnk2并将凝血酶原转变为类似于ProTΔ146-167结构的构象，从而增强因子Xa对凝血酶原的激活作用。
