Department of Physiology and Biophysics, Weill Cornell Medical School, New York, NY 10065, USA.
Molecules. 2021 Nov 18;26(22):6956. doi: 10.3390/molecules26226956.
Intracellular transport of chloride by members of the CLC transporter family involves a coupled exchange between a Cl anion and a proton (H), which makes the transport function dependent on ambient pH. Transport activity peaks at pH 4.5 and stalls at neutral pH. However, a structure of the WT protein at acidic pH is not available, making it difficult to assess the global conformational rearrangements that support a pH-dependent gating mechanism. To enable modeling of the CLC-ec1 dimer at acidic pH, we have applied molecular dynamics simulations (MD) featuring a new force field modification scheme-termed an Equilibrium constant pH approach (ECpH). The ECpH method utilizes linear interpolation between the force field parameters of protonated and deprotonated states of titratable residues to achieve a representation of pH-dependence in a narrow range of physiological pH values. Simulations of the CLC-ec1 dimer at neutral and acidic pH comparing ECpH-MD to canonical MD, in which the pH-dependent protonation is represented by a binary scheme, substantiates the better agreement of the conformational changes and the final model with experimental data from NMR, cross-link and AFM studies, and reveals structural elements that support the gate-opening at pH 4.5, including the key glutamates Glu and Glu.
CLC 转运蛋白家族成员的氯离子细胞内转运涉及氯离子（Cl-）与质子（H+）的偶联交换，这使得转运功能依赖于环境 pH 值。转运活性在 pH4.5 时达到峰值，并在中性 pH 值时停滞。然而，酸性 pH 值下 WT 蛋白的结构不可用，这使得评估支持 pH 依赖性门控机制的全局构象重排变得困难。为了能够在酸性 pH 值下对 CLC-ec1 二聚体进行建模，我们应用了分子动力学模拟（MD），该模拟采用了一种新的力场修正方案，称为平衡常数 pH 方法（ECpH）。ECpH 方法利用可滴定残基质子化和去质子化状态的力场参数之间的线性插值来实现对生理 pH 值范围内 pH 依赖性的表示。在中性和酸性 pH 值下对 CLC-ec1 二聚体进行的 ECpH-MD 与经典 MD 的模拟比较，证实了 ECpH-MD 与实验数据（来自 NMR、交联和 AFM 研究）的更好一致性，揭示了支持在 pH4.5 时打开门的结构元素，包括关键的谷氨酸 Glu 和 Glu。
