Bacterial sensing mechanism revealed Escherichia coli use a transmembrane sensor protein to sense nitrate in their external environment and initiate a biochemical response. Gushchin et al. compared crystal structures of portions of the NarQ receptor that included the transmembrane helices in ligand-bound or unbound states. The structures suggest a signaling mechanism by which piston- and lever-like movements are transmitted to response regulator proteins within the cell. Such two-component systems are very common in bacteria and, if better understood, might provide targets for antimicrobial therapies. Science, this issue p. eaah6345 Crystal structures show how sensing of nitrate occurs in bacteria. INTRODUCTION Microorganisms obtain most of the information about their environments through membrane-associated signaling systems. One of the most abundant classes of membrane receptors, present in all domains of life, is sensor histidine kinases, members of two-component signaling systems (TCSs). Tens of thousands of TCSs are known. Many of these systems are essential for cell growth, survival, or pathogenicity and consequently can be targeted to reduce virulence. Several large families of transmembrane (TM) TCS receptors are known: (i) sensor kinases, which generally possess a periplasmic, membrane, or intracellular sensor module; a transmembrane domain; often one or more intracellular signal transduction domains such as HAMP, PAS, or GAF; and an intracellular autokinase module (DHp and CA domains), which phosphorylates the response regulator protein; (ii) chemoreceptors, which also possess the sensor module and the TM domain but lack the kinase domain and control a separate kinase protein (CheA) via a kinase control module; and (iii) phototaxis systems, which are similar to chemotaxis systems except that the sensor module—a light receptor sensory rhodopsin—is a separate protein. RATIONALE Despite the wealth of biochemical data, the structural mechanisms of transmembrane signaling by TCS sensors are poorly understood at the atomic level. In particular, high-resolution structures of the TM segments connected to the adjacent domains are lacking. Deciphering of the signaling-associated conformational changes would shed light on the details of long-range transmembrane signal transduction and might help in the development of novel classes of antimicrobials targeting TCSs. RESULTS We used the in meso crystallization approach and single-wavelength anomalous dispersion to determine the crystal structures, at resolutions of up to 1.9 Å, of a fragment of Escherichia coli nitrate/nitrite sensor histidine kinase NarQ that contains the sensor, TM, and HAMP domains in a symmetric ligand-free apo state and in symmetric and asymmetric ligand-bound holo-S and holo-A states. In all of the structures, the TM domain is an antiparallel four-stranded coiled coil (CC) consisting of nine CC layers. The sensor domain is connected to the TM domain through continuous α-helical linkers that are partially disrupted in the holo state. The intracellular HAMP domain is connected to the TM helices via flexible proline junctions and robust hydrogen bonds conserved in all signaling states. The structures reveal the mechanism of transmembrane signal transduction in NarQ and show that binding of ligand induces displacement of the sensor domain helices by ~0.5 to 1 Å. This displacement translates into rearrangements and ~2.5 Å pistonlike shifts of transmembrane helices and is later converted, via leverlike motions of the HAMP domain protomers, into 7 Å shifts of the output helices and changes of the CC helical phase. The structures also demonstrate that the signaling-associated conformational changes in the TM domain do not need to be symmetric. CONCLUSION The determined structures of the transmembrane and membrane-proximal domains of the nitrate/nitrite receptor NarQ in ligand-free and ligand-bound forms present a template for studies of other TCS receptors, establish the importance of the pistonlike displacements of the TM helices for TM signal transduction, and highlight the role of the HAMP domain as an amplifier and converter of a piston-like displacement into helical rotation. Overall, the results show how a mechanistic signal is generated and amplified while being transduced through the protein over distances of 100 Å or more. Because membrane-associated TCSs are ubiquitous in microorganisms and are central for bacterial sensing, we believe that our results will help to elucidate a broad range of cellular processes such as basic metabolism, sporulation, quorum sensing, and virulence. They may also provide insights useful for the development of novel antimicrobial treatments targeting TCSs. The structures of histidine kinase NarQ in ligand-free and ligand-bound forms. The structures reveal rearrangement of transmembrane α helices during signal transduction and show that pistonlike shifts of the transmembrane helices result in leverlike motions of the HAMP domain protomers. One of the major and essential classes of transmembrane (TM) receptors, present in all domains of life, is sensor histidine kinases, parts of two-component signaling systems (TCSs). The structural mechanisms of TM signaling by these sensors are poorly understood. We present crystal structures of the periplasmic sensor domain, the TM domain, and the cytoplasmic HAMP domain of the Escherichia coli nitrate/nitrite sensor histidine kinase NarQ in the ligand-bound and mutated ligand-free states. The structures reveal that the ligand binding induces rearrangements and pistonlike shifts of TM helices. The HAMP domain protomers undergo leverlike motions and convert these pistonlike motions into helical rotations. Our findings provide the structural framework for complete understanding of TM TCS signaling and for development of antimicrobial treatments targeting TCSs.

中文翻译：

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传感器组氨酸激酶的跨膜信号传导机制

细菌传感机制揭示了大肠杆菌使用跨膜传感器蛋白来感知外部环境中的硝酸盐并启动生化反应。古什钦等人。比较了包括配体结合或未结合状态的跨膜螺旋的 NarQ 受体部分的晶体结构。这些结构表明了一种信号机制，通过这种机制，活塞和杠杆状运动被传递到细胞内的响应调节蛋白。这种双组分系统在细菌中非常常见，如果更好地理解，可能会为抗菌疗法提供靶点。科学，这个问题 p。eaah6345 晶体结构显示了细菌如何感知硝酸盐。引言 微生物通过膜相关信号系统获得有关其环境的大部分信息。存在于所有生命领域中的最丰富的膜受体类别之一是传感器组氨酸激酶，它是双组分信号系统 (TCS) 的成员。已知数以万计的 TCS。许多这些系统对于细胞生长、存活或致病性是必不可少的，因此可以作为降低毒力的目标。已知有几个大家族的跨膜 (TM) TCS 受体： (i) 传感器激酶，通常具有周质、膜或细胞内传感器模块；跨膜结构域；通常有一个或多个细胞内信号转导结构域，例如 HAMP、PAS 或 GAF；和细胞内自激酶模块（DHp 和 CA 结构域），它使反应调节蛋白磷酸化；(ii) 化学感受器，它还具有传感器模块和 TM 结构域，但缺少激酶结构域，并通过激酶控制模块控制单独的激酶蛋白 (CheA)；(iii) 趋光性系统，除了传感器模块——光受体感觉视紫质——是一种单独的蛋白质外，它们与趋化性系统相似。基本原理尽管有丰富的生化数据，但在原子水平上对 TCS 传感器跨膜信号传导的结构机制知之甚少。特别是，缺乏连接到相邻域的 TM 片段的高分辨率结构。破译与信号相关的构象变化将揭示长程跨膜信号转导的细节，并可能有助于开发针对 TCS 的新型抗菌药物。结果我们使用细观结晶方法和单波长异常色散来确定包含传感器、TM 和 HAMP 的大肠杆菌硝酸盐/亚硝酸盐传感器组氨酸激酶 NarQ 片段的晶体结构，分辨率高达 1.9 Å域处于对称无配体 apo 状态以及对称和不对称配体结合的全息-S 和全息-A 状态。在所有结构中，TM 域是由九个 CC 层组成的反平行四股卷曲线圈 (CC)。传感器域通过在全息状态下部分中断的连续 α-螺旋接头连接到 TM 域。细胞内 HAMP 结构域通过灵活的脯氨酸连接和在所有信号状态中保守的强大氢键连接到 TM 螺旋。这些结构揭示了 NarQ 中跨膜信号转导的机制，并表明配体的结合诱导传感器结构域螺旋位移约 0.5 到 1 Å。这种位移转化为跨膜螺旋的重排和 ~2.5 Å 活塞状位移，然后通过 HAMP 域原体的杠杆状运动转化为输出螺旋的 7 Å 位移和 CC 螺旋相的变化。这些结构还表明 TM 域中与信号相关的构象变化不需要对称。结论 硝酸盐/亚硝酸盐受体 NarQ 在无配体和配体结合形式中的跨膜和近膜结构域的确定结构为研究其他 TCS 受体提供了模板，确定 TM 螺旋的活塞状位移对 TM 信号转导的重要性，并强调 HAMP 域作为放大器和将活塞状位移转化为螺旋旋转的转换器的作用。总体而言，结果显示了机械信号是如何在通过蛋白质转导 100 Å 或更长距离时产生和放大的。由于膜相关的 TCS 在微生物中无处不在，并且是细菌感知的核心，我们相信我们的结果将有助于阐明广泛的细胞过程，如基本代谢、孢子形成、群体感应和毒力。它们还可以为开发针对 TCS 的新型抗菌治疗提供有用的见解。无配体和结合配体形式的组氨酸激酶 NarQ 的结构。这些结构揭示了信号转导过程中跨膜 α 螺旋的重排，并表明跨膜螺旋的活塞式移动导致 HAMP 结构域原体的杠杆式运动。存在于所有生命领域的跨膜 (TM) 受体的主要和必需类别之一是传感器组氨酸激酶，它是双组分信号系统 (TCS) 的一部分。这些传感器的 TM 信号的结构机制知之甚少。我们展示了大肠杆菌硝酸盐/亚硝酸盐传感器组氨酸激酶 NarQ 的周质传感器结构域、TM 结构域和细胞质 HAMP 结构域的晶体结构，处于配体结合状态和突变的无配体状态。该结构表明，配体结合诱导了 TM 螺旋的重排和活塞状位移。HAMP 域原体经历杠杆式运动并将这些活塞式运动转化为螺旋旋转。我们的研究结果为完全理解 TM TCS 信号传导和开发针对 TCS 的抗菌治疗提供了结构框架。