As the first potassium channel with a 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 NMR (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.

中文翻译：

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借助分子动力学模拟为NMR实验提供信息，以表征KcsA离子通道的主要活化态

作为确定的第一个具有X射线结构的钾通道，并使其与真核通道具有同源性，已对pH门控的原核通道KcsA进行了广泛的研究。然而，特别是与其闸门之间的变构偶联有关的问题仍然存在。许多当前可用的X射线晶体学结构似乎对应于活化和失活的各个阶段，从而为这些机制的分子基础提供了见识。由于这些研究需要突变，与抗体的复合以及替代脂质的去污剂的替代，因此需要在更天然的条件下检查通道。固态NMR（SSNMR）可用于研究活化条件（低pH），室温和拟细菌脂质体中的野生型蛋白。在这项工作中，我们试图在结构上分配存在于SSNMR实验中的活化态。我们结合使用了分子动力学（MD）模拟，化学位移预测算法和贝叶斯推断技术，以确定迄今解析出的最合理的X射线结构中哪一个最能代表SSNMR中捕获的活化态。我们首先通过模拟NMR化学位移确定了特定的核，当比较MD模拟的部分开放和完全开放的集合体时，它们具有明显的差异。然后将这些特定核的模拟NMR化学位移与实验值进行了比较，揭示了部分开放状态的模拟与SSNMR数据非常吻合。