Informing NMR experiments with molecular dynamics simulations to characterize the dominant activated state of the KcsA ion channel.
Informing NMR experiments with molecular dynamics simulations to characterize the dominant activated state of the KcsA ion channel.
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通过分子动力学模拟为NMR实验提供信息，以表征KcsA离子通道的主要活化状态。
DOI：
10.1063/5.0040649
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发表时间：
2021-04-28
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--
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作为第一个钾离子通道的X射线结构确定，并考虑到其同源性的真核细胞通道，pH门控原核细胞通道KcsA已被广泛研究。然而，特别是与其门之间的变构偶联相关的问题仍然是开放的。许多目前可用的X射线晶体学结构似乎对应于激活和失活的各个阶段，提供了对这些机制的分子基础的见解。由于这些研究需要突变、与抗体复合以及用去污剂代替脂质，因此需要在更天然的条件下检查通道。固态核磁共振（SSNMR）可用于研究野生型蛋白在活化条件下（低pH值），在室温下，在拟细菌脂质体。在这项工作中，我们试图从结构上分配SSNMR实验中存在的活化态。我们使用了分子动力学（MD）模拟，化学位移预测算法和贝叶斯推理技术的组合，以确定最合理的X射线结构解决最好的代表在SSNMR中捕获的激活状态。我们首先确定了特定的核与模拟的NMR化学位移显着不同时，比较部分开放与完全开放的合奏从MD模拟。然后将这些特定核的模拟NMR化学位移与实验结果进行比较，发现部分开放态的模拟与SSNMR数据吻合得很好。有效区分部分和完全开放状态的核属于分布在序列上的残基，并提供构象变化的分子水平描述。
As the first potassium channel with an x-ray structure determined, and given its homology to eukaryotic channels, the pH-gated prokaryotic channel KcsA has been extensively studied. Nevertheless, questions related, in particular, to the allosteric coupling between its gates remain open. The many currently available x-ray crystallography structures appear to correspond to various stages of activation and inactivation, offering insights into the molecular basis of these mechanisms. Since these studies have required mutations, complexation with antibodies, and substitution of detergents in place of lipids, examining the channel under more native conditions is desirable. Solid-state nuclear magnetic resonance (SSNMR) can be used to study the wild-type protein under activating conditions (low pH), at room temperature, and in bacteriomimetic liposomes. In this work, we sought to structurally assign the activated state present in SSNMR experiments. We used a combination of molecular dynamics (MD) simulations, chemical shift prediction algorithms, and Bayesian inference techniques to determine which of the most plausible x-ray structures resolved to date best represents the activated state captured in SSNMR. We first identified specific nuclei with simulated NMR chemical shifts that differed significantly when comparing partially open vs fully open ensembles from MD simulations. The simulated NMR chemical shifts for those specific nuclei were then compared to experimental ones, revealing that the simulation of the partially open state was in good agreement with the SSNMR data. Nuclei that discriminate effectively between partially and fully open states belong to residues spread over the sequence and provide a molecular level description of the conformational change.
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影响因子：
8.4
作者：
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