Transmembrane signaling proteins couple extracytosolic sensors to cytosolic effectors. Here, we examine how binding of Mg²⁺ to the sensor domain of an E. coli two component histidine kinase (HK), PhoQ, modulates its cytoplasmic kinase domain. We use cysteine-crosslinking and reporter-gene assays to simultaneously and independently probe the signaling state of PhoQ’s sensor and autokinase domains in a set of over 30 mutants. Strikingly, conservative single-site mutants distant from the sensor or catalytic site strongly influence PhoQ’s ligand-sensitivity as well as the magnitude and direction of the signal, endowing diverse signaling characteristics without need for epistasis. Data from 35 mutants are explained by a semi-empirical 3-domain model in which the sensor, intervening HAMP, and catalytic domains can adopt kinase-promoting or inhibiting conformations, that are in allosteric communication. The catalytic and sensor domains intrinsically favor a constitutively ‘kinase-on’ conformation, while the HAMP favors the ‘off’ state; when coupled, they create a bistable system responsive to physiological [Mg²⁺]. Mutants alter signaling by locally modulating these intrinsic equilibrium constants and couplings. Our model suggests signals transmit via interdomain allostery rather than propagation of a single concerted conformational change, explaining the diversity of signaling structural transitions observed in individual HK domains.

中文翻译：

---

双组分系统组氨酸激酶 PhoQ 信号转导的变构机制

跨膜信号蛋白将胞外传感器与胞质效应器偶联。在这里，我们研究了 Mg ²⁺如何与大肠杆菌的传感器结构域结合二组分组氨酸激酶 (HK) PhoQ 调节其细胞质激酶结构域。我们使用半胱氨酸交联和报告基因检测同时独立地探测 PhoQ 传感器和自激酶域在一组 30 多个突变体中的信号状态。引人注目的是，远离传感器或催化位点的保守单位点突变体强烈影响 PhoQ 的配体敏感性以及信号的大小和方向，赋予不同的信号特征而不需要上位。来自 35 个突变体的数据由半经验 3 域模型解释，其中传感器、干预 HAMP 和催化域可以采用激酶促进或抑制构象，这是变构通信。催化域和传感器域本质上有利于组成性的“激酶在”构象，而 HAMP 倾向于“关闭”状态；当耦合时，它们会创建一个对生理 [Mg²⁺ ]。突变体通过局部调节这些内在平衡常数和耦合来改变信号。我们的模型表明信号通过域间变构传递而不是单个协同构象变化的传播，解释了在单个 HK 域中观察到的信号结构转变的多样性。