Department of Physics, Wake Forest University, Winston-Salem, North Carolina 27106 United States.
Freenome, South San Francisco, California 94080 United States.
J Chem Inf Model. 2021 Feb 22;61(2):950-965. doi: 10.1021/acs.jcim.0c01303. Epub 2021 Jan 15.
Thrombin plays an important role in the process of hemostasis and blood coagulation. Studies in thrombin can help us find ways to treat cancer because thrombin is able to reduce the characteristic hypercoagulability of cancer. Thrombin is composed of two chains, the light chain and the heavy chain. The function of the heavy chain has been largely explored, while the function of the light chain was obscured until several disease-associated mutations in the light chain come to light. In this study, we want to explore the dynamic and conformation effects of mutations on the light chain further to determine possible associations between mutation, conformational changes, and disease. The study, which is a follow-up for our studies on apo thrombin and the mutant, ΔK9, mainly focuses on the mutants E8K and R4A. E8K is a disease-associated mutation, and R4A is used to study the role of Arg4, which is suggested experimentally to play a critical role for thrombin's catalytic activities. We performed five all-atom one microsecond-scale molecular dynamics (MD) simulations for both E8K and R4A, and quantified the changes in the conformational ensemble of the mutants. From the root-mean-square fluctuations (RMSF) for the α-carbons, we find that the atomic fluctuations change in the mutants in the 60s loop and γ loop. The correlation coefficients for the α-carbons indicate that the correlation relation for atom-pairs in the protein is also impacted. The clustering analysis and the principal component analysis (PCA) consistently tell us that the catalytic pocket and the regulatory loops are destabilized by the mutations. We also find that there are two binding modes for Na by clustering the vector difference between the Na ions and the 220s loop. After further analysis, we find that there is a relation between the Na binding and the rigidification of the γ loop, which may shed light on the mysterious role of the γ loop in thrombin.
凝血酶在止血和血液凝固过程中起着重要作用。对凝血酶的研究可以帮助我们找到治疗癌症的方法，因为凝血酶能够降低癌症的特征性高凝状态。凝血酶由两条链组成，轻链和重链。重链的功能已经得到了很大的探索，而轻链的功能则在轻链中出现几种与疾病相关的突变后变得模糊不清。在这项研究中，我们希望进一步探索突变对轻链的动态和构象效应，以确定突变、构象变化和疾病之间可能存在的联系。这项研究是我们对脱辅基凝血酶和突变体ΔK9 的研究的后续，主要集中在突变体 E8K 和 R4A 上。E8K 是一种与疾病相关的突变，而 R4A 则用于研究 Arg4 的作用，实验表明 Arg4 对凝血酶的催化活性起着关键作用。我们对 E8K 和 R4A 进行了五次全原子、一微秒尺度的分子动力学（MD）模拟，并量化了突变体构象集合的变化。从α-碳原子的均方根波动（RMSF）来看，我们发现突变体中 60s 环和γ环的原子波动发生了变化。α-碳原子的相关系数表明，蛋白质中原子对的相关关系也受到了影响。聚类分析和主成分分析（PCA）一致表明，催化口袋和调节环被突变所破坏。我们还发现，通过对 Na 离子和 220s 环之间的向量差进行聚类，可以得到两种 Na 结合方式。进一步分析后，我们发现 Na 结合与γ环的僵化之间存在关系，这可能揭示了γ环在凝血酶中的神秘作用。
