Histidine kinases are key bacterial sensors that recognize diverse environmental stimuli. While mechanisms of phosphorylation and phosphotransfer by cytoplasmic kinase domains are relatively well-characterized, the ways in which extracytoplasmic sensor domains regulate activation remain mysterious. The Cpx envelope stress response is a conserved Gram-negative two-component system which is controlled by the sensor kinase CpxA. We report the structure of the CpxA sensor domain (CpxA-SD) as a globular Per-ARNT-Sim (PAS)-like fold highly similar to that of CpxA as determined by X-ray crystallography. Because sensor kinase dimerization is important for signaling, we used AlphaFold2 to model CpxA-SD in the context of its connected transmembrane domains, which yielded a novel dimer of PAS domains possessing a distinct dimer organization compared to previously characterized sensor domains. Gain of function ∗ alleles map to the dimer interface, and mutation of other residues in this region also leads to constitutive activation. CpxA activation can be suppressed by mutations that restore inter-monomer interactions, suggesting that inhibitory interactions between CpxA-SD monomers are the major point of control for CpxA activation and signaling. Searching through hundreds of structural homologs revealed the sensor domain of sensor kinase PfeS as the only PAS structure in the same novel dimer orientation as CpxA, suggesting that our dimer orientation may be utilized by other extracytoplasmic PAS domains. Overall, our findings provide insight into the diversity of the organization of PAS sensory domains and how they regulate sensor kinase activation.

中文翻译：

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细菌组氨酸激酶 CpxA 的传感器是胞质外 Per-ARNT-Sim 结构域的新型二聚体

组氨酸激酶是识别不同环境刺激的关键细菌传感器。虽然胞质激酶结构域的磷酸化和磷酸转移机制相对较好，但胞质外传感器结构域调节激活的方式仍然是个谜。 Cpx 包膜应激反应是一个保守的革兰氏阴性双组分系统，由传感器激酶 CpxA 控制。我们将 CpxA 传感器域 (CpxA-SD) 的结构报告为球状 Per-ARNT-Sim (PAS) 状折叠，与 X 射线晶体学确定的 CpxA 高度相似。由于传感器激酶二聚化对于信号传导非常重要，因此我们使用 AlphaFold2 在其连接的跨膜结构域的背景下对 CpxA-SD 进行建模，这产生了一种新型 PAS 结构域二聚体，与之前表征的传感器结构域相比，它具有独特的二聚体组织。功能增益*等位基因映射到二聚体界面，并且该区域中其他残基的突变也导致组成型激活。 CpxA 激活可以通过恢复单体间相互作用的突变来抑制，这表明 CpxA-SD 单体之间的抑制性相互作用是 CpxA 激活和信号传导的主要控制点。搜索数百个结构同源物揭示了传感器激酶 PfeS 的传感器结构域是与 CpxA 具有相同新型二聚体方向的唯一 PAS 结构，这表明我们的二聚体方向可能被其他胞质外 PAS 结构域利用。总的来说，我们的研究结果提供了对 PAS 感觉域组织的多样性以及它们如何调节传感器激酶激活的深入了解。