Insights into histidine kinase activation mechanisms from the monomeric blue light sensor EL346
Insights into histidine kinase activation mechanisms from the monomeric blue light sensor EL346
复制标题
DOI：
10.1073/pnas.1813586116
复制
发表时间：
2019-03-12
影响因子：
11.1
通讯作者：
Gardner, Kevin H.
中科院分区：
文献类型：
--
作者：
Dikiy, Igor;Edupuganti, Uthama R.;Gardner, Kevin H.
将环境信号转化为细胞行为是所有生命形式的必要过程。在细菌中，该过程通常涉及双组分系统，其中传感器组氨酸激酶（HK）在响应刺激时自磷酸化，随后将磷酸化基团转移到控制下游效应物的反应调节因子。由于这些大型多结构域蛋白的多种信号状态相关的并发症，HK活化的分子机制的许多细节仍不清楚。为了解决这些挑战，我们结合了互补溶液生物物理方法来研究来自litoralis红杆菌HTCC2594， EL346的最小蓝光感应组氨酸激酶激活后的构象变化。我们的数据表明，溶液中EL346的暗态中存在多种构象共存，这可能解释了酶的残余暗态活性。我们还观察到，激活包括磷酸化受体组氨酸所在的二聚化和组氨酸磷酸转移样结构域的螺旋的不稳定，以及它们与催化结构域的相互作用。类似的光诱导变化在某种程度上甚至发生在构成活性或非活性突变体中，这表明光感可以与激酶活性的激活分离。这些结构变化反映了通过比较非活性和活性HK片段的x射线晶体结构推断出的结果，表明它们是导致HK活化的构象变化的核心。更广泛地说，我们的发现揭示了这个简单系统的惊人复杂性，并使我们能够概述HK激活的多个步骤的机制。
Translation of environmental cues into cellular behavior is a necessary process in all forms of life. In bacteria, this process frequently involves two-component systems in which a sensor histidine kinase (HK) autophosphorylates in response to a stimulus before subsequently transferring the phosphoryl group to a response regulator that controls downstream effectors. Many details of the molecular mechanisms of HK activation are still unclear due to complications associated with the multiple signaling states of these large, multidomain proteins. To address these challenges, we combined complementary solution biophysical approaches to examine the conformational changes upon activation of a minimal, blue-light-sensing histidine kinase from Erythrobacter litoralis HTCC2594, EL346. Our data show that multiple conformations coexist in the dark state of EL346 in solution, which may explain the enzyme's residual dark-state activity. We also observe that activation involves destabilization of the helices in the dimerization and histidine phosphotransfer-like domain, where the phosphoacceptor histidine resides, and their interactions with the catalytic domain. Similar light-induced changes occur to some extent even in constitutively active or inactive mutants, showing that light sensing can be decoupled from activation of kinase activity. These structural changes mirror those inferred by comparing X-ray crystal structures of inactive and active HK fragments, suggesting that they are at the core of conformational changes leading to HK activation. More broadly, our findings uncover surprising complexity in this simple system and allow us to outline a mechanism of the multiple steps of HK activation.