In silico prediction of ARB resistance: A first step in creating personalized ARB therapy

<strong>AT1R Model preparation</strong><br> The crystal structure of human AT1R bound to olmesartan (PDB: 4ZUD) was downloaded from the RCSB Protein Data Bank. 4ZUD contains apocytochrome b562RIL fused to the amino terminus, and many of the flexible regions, as well as helix 8, are not resolved. In order to generate an appropriate starting structure, olmesartan and the apocytochrome b562RIL fusion were removed from 4ZUD, and the missing regions were added to the protein with MOE software (Chemical Computing Group ULC, Montreal, Canada). Specifically, the N-Terminus (residues 1 to 25), intracellular loop 2 (residues 134 to 140), extracellular loop 2 (residues 186 to 188), intracellular loop 3 (residues 223 to 234), and helix 8 (residues 305 to 316) were added to the AT1R in accordance to the human AT1R sequence and PDB:4YAY. The remaining carboxyl-tail of the AT1R (residues 317 to 359) was not modeled. The AT1R model then underwent an energy minimization within MOE using the Amber10:Extended Huckel Theory (EHT) force field. <strong>Molecular dynamic (MD) simulations and analysis</strong><br> The MOE minimized AT1R was loaded into CHARMM-GUI. An 80 Å by 80 Å lipid bi-layer composed of 13% cholesterol and 87% Phosphatidylcholine (POPC) was generated around the receptor. Water was packed 17.5 Å above and below the lipid bi-layer, and 150 mM Na+ and Cl- ions were added to the system via Monte-Carlo ion placing. The all-atom CHARMM C36 force field for proteins and ions, and the CHARMM TIP3P force field for water were selected. A hard non-bonded cutoff of 8.0 angstroms was utilized. All molecular dynamics simulations were performed using the PMEMD module of the AMBER16 package with support for MPI multi-process control and GPU acceleration code. Orthorhombic periodic boundary conditions with a constant pressure of 1 atm was set via the NPT ensemble and temperature was set to 310.15°K (37°C) using Langevin dynamics. The SHAKE algorithm was used to constrain bonds containing hydrogens. The dynamics were propagated using Langevin dynamics with Langevin damping coefficient of 1 ps-1 and a time step of 2 fs. Before the production run, the AT1R model was minimized for 5000 steps using the steepest descent method and then equilibrated for 600 ps. The protein coordinates were saved in 10 ps intervals. The production run lasted 150 ns, at which point all three replicas were stable for at least the last 20 ns.<strong>AT1R 模型制备</strong><br>从RCSB蛋白质数据库（RCSB Protein Data Bank）下载了与奥美沙坦结合的人类AT1R晶体结构（PDB编号：4ZUD）。该PDB编号为4ZUD的结构包含融合至氨基末端的脱细胞色素b562RIL，且诸多柔性区域以及第8螺旋均未获得解析。为构建合适的初始结构，我们从4ZUD中移除了奥美沙坦与脱细胞色素b562RIL融合片段，并通过MOE软件（加拿大蒙特利尔化学计算集团有限公司，Chemical Computing Group ULC）为蛋白补全缺失区域。具体而言，参考人类AT1R序列与PDB编号4YAY，为AT1R补全了N端（残基1至25）、胞内环2（残基134至140）、胞外环2（残基186至188）、胞内环3（残基223至234）及第8螺旋（残基305至316）。AT1R剩余的羧基末端尾部（残基317至359）未进行建模。随后，我们使用Amber10：扩展休克尔理论（Extended Huckel Theory, EHT）力场，在MOE软件中对AT1R模型进行了能量最小化优化。<br><strong>分子动力学（Molecular dynamic, MD）模拟与分析</strong><br>将经MOE完成能量最小化的AT1R模型导入CHARMM-GUI平台。以受体为中心构建了尺寸为80 Å × 80 Å的脂质双层膜，其组成为13%胆固醇与87%磷脂酰胆碱（Phosphatidylcholine, POPC）。在脂质双层膜上下方各填充17.5 Å厚度的水分子体系，并通过蒙特卡洛离子置位法向体系中添加150 mM的Na+与Cl-离子。选用适用于蛋白与离子的全原子CHARMM C36力场，以及适用于水分子的CHARMM TIP3P力场，并采用8.0埃的硬非键相互作用截断半径。所有分子动力学模拟均通过AMBER16软件包的PMEMD模块完成，该模块支持MPI多进程控制与GPU加速代码。通过NPT系综设置正交晶系周期性边界条件与1 atm的恒定压强，采用朗之万动力学将体系温度设定为310.15 K（37 ℃）。使用SHAKE算法约束含氢原子的化学键。模拟动力学采用朗之万动力学传播，朗之万阻尼系数设为1 ps-1，时间步长为2 fs。在生产模拟前，先采用最速下降法对AT1R模型进行5000步能量最小化，随后进行600 ps的平衡模拟。以10 ps为间隔保存蛋白坐标。生产模拟时长为150 ns，所有三个重复实验的体系在最后至少20 ns的时段内均保持稳定。