Functional evaluation of a CMT2DD-causing ATP1A1 variant
Functional evaluation of a CMT2DD-causing ATP1A1 variant
复制标题
引起 CMT2DD 的 ATP1A1 变异的功能评估
DOI：
10.1016/j.bpj.2023.11.2441
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发表时间：
2024
影响因子：
3.4
通讯作者：
Artigas, Pablo
中科院分区：
文献类型：
--
作者：
Spontarelli, Kerri;Scherer, Steven S.;Bird, Shawn J.;McCray, Brett;Artigas, Pablo
红细胞最丰富的膜蛋白带3通过其Cl-和碳酸氢根离子的双向交换对调节酸碱平衡和呼吸起关键作用。在运输周期期间，带3采用交替存取机构，在面向外（OF）和面向内（IF）状态之间转换。利用我们最近的高分辨率cryo-EM结构解决这两个国家，我们采用平衡和非平衡分子动力学模拟表征自发阴离子结合的蛋白质和构象变化的OF IF状态在运输过程中。一组扩展的多个ms长的平衡模拟显示经常性的阴离子结合事件，无论是Cl-和碳酸氢盐，在特定的网站允许识别的带3的阴离子位点，在IF和OF状态。接下来，我们应用增强的采样方法来诱导从OF到IF状态的转变。在类似升降机的机制中，运输域相对于固定支架域经历组合的平移/旋转运动，如由模拟中的特定集体变量引起的。得到的过渡路径随后放松和细化使用字符串方法与成群的轨迹。然后，一维偏置交换，伞形采样被用来计算自由能沿沿着细化途径。对载脂蛋白和底物结合状态以及实验结构中识别的结合PIP 2已被去除的系统进行自由能分析。自由能曲线清楚地证实了实验报告的脂质对带3的抑制作用。这种全面的计算方法研究的机制带3揭示了结构动力学和自由能之间的复杂关系，在一个重要的阴离子运输过程。
The red blood cell’s most abundant membrane protein, band 3, is pivotal for regulating the acid-base balance and respiration through its bidirectional exchange of Cl À and bicarbonate ions. During the transport cycle, band 3 employs the alternating access mechanism, transitioning between outward facing (OF) and inward facing (IF) states. Leveraging our recent high-resolution cryo-EM structure resolved in both states, we employed equilibrium and nonequilibrium molecular dynamic simulations to characterize spontaneous anion binding to the protein and the conformational change from the OF to the IF state during transport. An extended set of multiple ms-long equilibrium simulations revealed recurrent anion binding events, both for Cl À and bicarbonate, at specific sites allowing for identification of the anion sites of band 3, in both IF and OF states. Next, we applied enhanced sampling methods to induce the transition from the OF to the IF state. In an elevator-like mechanism, the transport domain undergoes a combined translational/rotational motion relative to the stationary scaffold domain, as induced by specific collective variables in the simulation. The obtained transition pathway was subsequently relaxed and refined using the string method with swarms of trajectories. Then, 1D-bias-exchange, umbrella sampling was used to calculate the free energy along the refined pathway. The free energy analysis was performed on both apo and substrate-bound states, as well as on a system in which bound PIP2s identified in the experimental structure had been removed. The free energy profiles clearly substantiate the experimentally reported inhibitory effect of the lipid on band 3. This comprehensive computational approach studying the mechanism of band 3 sheds light on the intricate relationship between structural dynamics and free energy in a vital anion transport process.