Department of Chemistry, Stanford University, Stanford, California, United States of America.
Department of Molecular & Cellular Physiology, Stanford University, Stanford, California, United States of America.
PLoS Comput Biol. 2020 Mar 30;16(3):e1007530. doi: 10.1371/journal.pcbi.1007530. eCollection 2020 Mar.
This work reports a dynamical Markov state model of CLC-2 "fast" (pore) gating, based on 600 microseconds of molecular dynamics (MD) simulation. In the starting conformation of our CLC-2 model, both outer and inner channel gates are closed. The first conformational change in our dataset involves rotation of the inner-gate backbone along residues S168-G169-I170. This change is strikingly similar to that observed in the cryo-EM structure of the bovine CLC-K channel, though the volume of the intracellular (inner) region of the ion conduction pathway is further expanded in our model. From this state (inner gate open and outer gate closed), two additional states are observed, each involving a unique rotameric flip of the outer-gate residue GLUex. Both additional states involve conformational changes that orient GLUex away from the extracellular (outer) region of the ion conduction pathway. In the first additional state, the rotameric flip of GLUex results in an open, or near-open, channel pore. The equilibrium population of this state is low (∼1%), consistent with the low open probability of CLC-2 observed experimentally in the absence of a membrane potential stimulus (0 mV). In the second additional state, GLUex rotates to occlude the channel pore. This state, which has a low equilibrium population (∼1%), is only accessible when GLUex is protonated. Together, these pathways model the opening of both an inner and outer gate within the CLC-2 selectivity filter, as a function of GLUex protonation. Collectively, our findings are consistent with published experimental analyses of CLC-2 gating and provide a high-resolution structural model to guide future investigations.
这项工作报道了基于 600 微秒分子动力学 (MD) 模拟的 CLC-2“快速”（孔）门控的动态 Markov 状态模型。在我们的 CLC-2 模型的起始构象中，内外通道门均关闭。我们数据集中的第一个构象变化涉及内门骨架沿残基 S168-G169-I170 的旋转。这种变化与牛 CLC-K 通道的冷冻电镜结构中观察到的变化非常相似，尽管我们模型中离子传导途径的细胞内（内）区域的体积进一步扩大。从这个状态（内门打开，外门关闭），观察到另外两个状态，每个状态都涉及外门残基 GLUex 的独特构象翻转。这两种额外的状态都涉及到将 GLUex 构象改变为远离离子传导途径的细胞外（外）区域的构象变化。在第一个额外状态中，GLUex 的构象翻转导致通道孔打开或接近打开。该状态的平衡种群较低（约 1%），与实验中在没有膜电位刺激（0 mV）时观察到的 CLC-2 低开放概率一致。在第二个额外状态中，GLUex 旋转以阻塞通道孔。该状态的平衡种群较低（约 1%），仅在 GLUex 质子化时才可以进入。这两种途径共同模拟了 CLC-2 选择性过滤器中内门和外门的打开，这是 GLUex 质子化的函数。总的来说，我们的发现与 CLC-2 门控的已发表实验分析一致，并提供了一个高分辨率的结构模型来指导未来的研究。
