Phosphorylation of Escherichia coli CheY protein transduces chemoreceptor stimulation to a highly cooperative flagellar motor response. CheY binds to the N-terminal peptide of the FliM motor protein (FliMN). Constitutively active D13K-Y106W CheY has been an important tool for motor physiology. The crystal structures of CheY and CheY ⋅ FliMN with and without D13K-Y106W have shown FliMN-bound CheY contains features of both active and inactive states. We used molecular dynamics (MD) simulations to characterize the CheY conformational landscape accessed by FliMN and D13K-Y106W. Mutual information measures identified the central features of the long-range CheY allosteric network between D57 phosphorylation site and the FliMN interface, namely the closure of the α4-β4 hinge and inward rotation of Y- or W106 with W58. We used hydroxy-radical foot printing with mass spectroscopy (XFMS) to track the solvent accessibility of these and other side chains. The solution XFMS oxidation rate correlated with the solvent-accessible area of the crystal structures. The protection of allosteric relay side chains reported by XFMS confirmed the intermediate conformation of the native CheY ⋅ FliMN complex, the inactive state of free D13K-Y106W CheY, and the MD-based network architecture. We extended the MD analysis to determine temporal coupling and energetics during activation. Coupled aromatic residue rotation was a graded rather than a binary switch, with Y- or W106 side-chain burial correlated with increased FliMN affinity. Activation entrained CheY fold stabilization to FliMN affinity. The CheY network could be partitioned into four dynamically coordinated sectors. Residue substitutions mapped to sectors around D57 or the FliMN interface according to phenotype. FliMN increased sector size and interactions. These sectors fused between the substituted K13-W106 residues to organize a tightly packed core and novel surfaces that may bind additional sites to explain the cooperative motor response. The community maps provide a more complete description of CheY priming than proposed thus far.

中文翻译：

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鞭毛运动蛋白 FliM 对大肠杆菌 CheY 的变构启动

大肠杆菌 CheY 蛋白的磷酸化将化学感受器刺激转导为高度协作的鞭毛运动反应。CheY 与 FliM 运动蛋白 (FliMN) 的 N 端肽结合。组成型活性 D13K-Y106W CheY 一直是运动生理学的重要工具。CheY 和 CheY · FliMN 的晶体结构有和没有 D13K-Y106W 表明 FliMN 结合的 CheY 包含活跃和非活跃状态的特征。我们使用分子动力学 (MD) 模拟来表征 FliMN 和 D13K-Y106W 访问的 CheY 构象景观。互信息测量确定了 D57 磷酸化位点和 FliMN 界面之间的远程 CheY 变构网络的中心特征，即 α4-β4 铰链的闭合和 Y- 或 W106 与 W58 的向内旋转。我们使用带有质谱 (XFMS) 的羟基自由基足印来跟踪这些和其他侧链的溶剂可及性。溶液 XFMS 氧化速率与晶体结构的溶剂可及区域相关。XFMS报告的变构中继侧链保护证实了天然CheY·FliMN复合物的中间构象，游离D13K-Y106W CheY的非活性状态，以及基于MD的网络架构。我们扩展了 MD 分析以确定激活过程中的时间耦合和能量学。耦合的芳香族残基旋转是分级而不是二元开关，Y 或 W106 侧链埋藏与增加的 FliMN 亲和力相关。激活夹带 CheY 折叠对 FliMN 亲和力的稳定性。CheY 网络可以划分为四个动态协调的扇区。根据表型，残基替换映射到 D57 或 FliMN 界面周围的扇区。FliMN 增加了部门规模和互动。这些扇区在被取代的 K13-W106 残基之间融合以组织紧密堆积的核心和新的表面，这些表面可能会结合其他位点来解释协作运动反应。社区地图提供了比迄今为止提出的更完整的 CheY 启动描述。