Department of Orthopaedic Surgery, University of California, Los Angeles, CA, USA.
Sanofi-Aventis Pharma Deutschland GmbH, Frankfurt am Main, Germany.
J Thromb Haemost. 2019 Apr;17(4):574-584. doi: 10.1111/jth.14401. Epub 2019 Mar 6.
Essentials Consensus sequence and biochemical data suggest a Na -site in the factor (F) IXa protease domain. X-ray structure of the FIXa EGF2/protease domain at 1.37 Å reveals a Na -site not observed earlier. Molecular dynamics simulations data support that Na ± Ca promote FIXa protease domain stability. Sulfate ions found in the protease domain mimic heparin sulfate binding mode in FIXa. SUMMARY: Background Activated coagulation factor IX (FIXa) consists of a γ-carboxyglutamic acid domain, two epidermal growth factor-like (EGF) domains, and a C-terminal protease domain. Consensus sequence and biochemical data support the existence of a Na -site in the FIXa protease domain. However, soaking experiments or crystals grown in high concentration of ammonium sulfate did not reveal a Na -site in wild-type or mutant FIXa EGF2/protease domain structure. Objective Determine the structure of the FIXa EGF2/protease domain in the presence of Na ; perform molecular dynamics (MD) simulations to explore the role of Na in stabilizing FIXa structure. Methods Crystallography, MD simulations, and modeling heparin binding to FIXa. Results Crystal structure at 1.37-Å resolution revealed that Na is coordinated to carbonyl groups of residues 184A, 185, 221A, and 224 in the FIXa protease domain. The Na -site in FIXa is similar to that of FXa and is linked to the Asp189 S1-site. In MD simulations, Na reduced fluctuations in residues 217-225 (Na -loop) and 70-80 (Ca -loop), whereas Ca reduced fluctuations only in residues of the Ca -loop. Ca and Na together reduced fluctuations in residues of the Ca -loop and Na -loop (residues 70-80, 183-194, and 217-225). Moreover, we observed four sulfate ions that make salt bridges with FIXa protease domain Arg/Lys residues, which have been implicated in heparin binding. Based upon locations of the sulfate ions, we modeled heparin binding to FIXa, which is similar to the heparin binding in thrombin. Conclusions The FIXa Na -site in association with Ca contributes to stabilization of the FIXa protease domain. The heparin binding mode in FIXa is similar to that in thrombin.
凝血因子 IXa（FIXa）由一个 γ-羧基谷氨酸结构域、两个表皮生长因子样（EGF）结构域和一个 C 端蛋白酶结构域组成。共识序列和生化数据支持 FIXa 蛋白酶结构域中存在一个钠离子结合位点。然而，在野生型或突变型 FIXa EGF2/蛋白酶结构域的浸泡实验或晶体生长实验中，并未在高浓度硫酸铵中发现钠离子结合位点。本研究旨在确定 FIXa EGF2/蛋白酶结构域在钠离子存在下的结构，并通过分子动力学（MD）模拟探索钠离子在稳定 FIXa 结构中的作用。采用晶体学、MD 模拟和肝素结合到 FIXa 的建模方法。结果显示，在 1.37-Å 的分辨率下，晶体结构揭示了钠离子与 FIXa 蛋白酶结构域中的残基 184A、185、221A 和 224 的羰基形成配位。FIXa 中的钠离子结合位点类似于 FXa，并与 Asp189 S1 位点相连。在 MD 模拟中，钠离子减少了残基 217-225（钠离子结合环）和 70-80（钙离子结合环）的波动，而钙离子仅减少了钙离子结合环中残基的波动。钙离子和钠离子共同减少了钙离子结合环和钠离子结合环（残基 70-80、183-194 和 217-225）中残基的波动。此外，我们观察到四个硫酸根离子与 FIXa 蛋白酶结构域的精氨酸/赖氨酸残基形成盐桥，这些残基与肝素结合有关。根据硫酸根离子的位置，我们模拟了肝素与 FIXa 的结合，其与凝血酶中的肝素结合相似。结论 FIXa 与钙离子结合的钠离子结合位点有助于稳定 FIXa 蛋白酶结构域。FIXa 中的肝素结合模式与凝血酶中的肝素结合模式相似。
