Environmental awareness is an essential attribute of all organisms. The homeoviscous adaptation system of Bacillus subtilis provides a powerful experimental model for the investigation of stimulus detection and signaling mechanisms at the molecular level. These bacteria sense the order of membrane lipids with the transmembrane (TM) protein DesK, which has an N-terminal sensor domain and an intracellular catalytic effector domain. DesK exhibits autokinase activity as well as phosphotransferase and phosphatase activities toward a cognate response regulator, DesR, that controls the expression of an enzyme that remodels membrane fluidity when the temperature drops below ∼30°C. Membrane fluidity signals are transmitted from the DesK sensor domain to the effector domain via rotational movements of a connecting 2-helix coiled coil (2-HCC). Previous molecular dynamic simulations suggested important roles for TM prolines in transducing the initial signals of membrane fluidity status to the 2-HCC. Here, we report that individual replacement of prolines in DesKs TM1 and TM5 helices by alanine (DesKPA) locked DesK in a phosphatase-ON state, abrogating membrane fluidity responses. An unbiased mutagenic screen identified the L174P replacement in the internal side of the repeated heptad of the 2-HCC structure that alleviated the signaling defects of every transmembrane DesKPA substitution. Moreover, substitutions by proline in other internal positions of the 2-HCC reestablished the kinase-ON state of the DesKPA mutants. These results imply that TM prolines are essential for finely tuned signal generation by the N-terminal sensor helices, facilitating a conformational control by the metastable 2-HCC domain of the DesK signaling state.IMPORTANCE Signal sensing and transduction is an essential biological process for cell adaptation and survival. Histidine kinases (HK) are the sensory proteins of two-component systems that control many bacterial responses to different stimuli, like environmental changes. Here, we focused on the HK DesK from Bacillus subtilis, a paradigmatic example of a transmembrane thermosensor suited to remodel membrane fluidity when the temperature drops below 30°C. DesK provides a tractable system for investigating the mechanism of transmembrane signaling, one of the majors interrogates in biology to date. Our studies demonstrate that transmembrane proline residues modulate the conformational switch of a 2-helix coiled-coil (2-HCC) structural motif that controls input-output in a variety of HK. Our results highlight the relevance of proline residues within sensor domains and could inspire investigations of their role in different signaling proteins.

中文翻译：

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跨膜脯氨酸介导枯草芽孢杆菌DesK组氨酸激酶中的信号传感和解码。

环保意识是所有生物的基本属性。枯草芽孢杆菌的homeoviscous适应系统为分子水平上的刺激检测和信号传导机制的研究提供了强大的实验模型。这些细菌利用跨膜（TM）蛋白DesK感测膜脂的顺序，该蛋白具有N端传感器结构域和细胞内催化效应器结构域。DesK对关联反应调节剂DesR表现出自身激酶活性以及磷酸转移酶和磷酸酶活性，该调节剂控制温度降低到约30°C以下时可重塑膜流动性的酶的表达。膜流动性信号通过连接的2螺旋线圈（2-HCC）的旋转运动从DesK传感器域传输到效应器域。先前的分子动力学模拟表明，TM脯氨酸在将膜流动性状态的初始信号转导至2-HCC中起着重要作用。在这里，我们报道丙氨酸（DesKPA）单独取代DesKs TM1和TM5螺旋中的脯氨酸，将DesK锁定在磷酸酶开启状态，从而消除了膜的流动性响应。一个无偏见的诱变筛选在2-HCC结构的重复七肽的内侧鉴定了L174P置换，该置换减轻了每个跨膜DesKPA置换的信号缺陷。此外，在2-HCC的其他内部位置被脯氨酸取代，重新建立了DesKPA突变体的激酶-ON状态。这些结果表明，TM脯氨酸对于N端传感器螺旋产生的微调信号产生至关重要，DesK信号状态的亚稳态2-HCC结构域有助于构象控制。重要信息信号传感和转导是细胞适应和存活的重要生物学过程。组氨酸激酶（HK）是两组分系统的感觉蛋白，可控制许多细菌对不同刺激（例如环境变化）的反应。在这里，我们重点研究枯草芽孢杆菌的HK DesK，这是跨膜热传感器的一个典型示例，适用于在温度降至30°C以下时重塑膜的流动性。DesK为研究跨膜信号传导的机制提供了一个易于处理的系统，而跨膜信号传导是迄今为止生物学研究的主要内容之一。我们的研究表明，跨膜脯氨酸残基可调节2螺旋卷曲螺旋（2-HCC）结构基序的构象转换，从而控制各种HK中的输入输出。我们的结果突出了脯氨酸残基在传感器域内的相关性，并可能激发人们对其在不同信号蛋白中的作用进行研究。