Ocasio Victor J, Corrêa Fernando, Gardner Kevin H
Departments of Biophysics and Biochemistry, UT Southwestern Medical Center , Dallas, Texas 75390-8816, United States.
Biochemistry. 2015 Feb 17;54(6):1353-63. doi: 10.1021/bi501143b. Epub 2015 Feb 6.
To survive and adapt to environmental changes, bacteria commonly use two-component signaling systems. Minimally, these pathways use histidine kinases (HKs) to detect environmental signals, harnessing these to control phosphorylation levels of receiver (REC) domains of downstream response regulators that convert this signal into physiological responses. Studies of several prototypical REC domains suggest that phosphorylation shifts these proteins between inactive and active structures that are globally similar and well-folded. However, it is unclear how globally these findings hold within REC domains in general, particularly when they are considered within full-length proteins. Here, we present EL_LovR, a full-length REC-only protein that is phosphorylated in response to blue light in the marine α-proteobacterium, Erythrobacter litoralis HTCC2594. Notably, EL_LovR is similar to comparable REC-only proteins used in bacterial general stress responses, where genetic evidence suggests that their potent phosphatase activity is important to shut off such systems. Size exclusion chromatography, light scattering, and solution NMR experiments show that EL_LovR is monomeric and unfolded in solution under conditions routinely used for other REC structure determinations. Addition of Mg(2+) and phosphorylation induce progressively greater degrees of tertiary structure stabilization, with the solution structure of the fully activated EL_LovR adopting the canonical receiver domain fold. Parallel functional assays show that EL_LovR has a fast dephosphorylation rate, consistent with its proposed function as a phosphate sink that depletes the HK phosphoryl group, promoting the phosphatase activity of this enzyme. Our findings demonstrate that EL_LovR undergoes substantial ligand-dependent conformational changes that have not been reported for other RRs, expanding the scope of conformational changes and regulation used by REC domains, critical components of bacterial signaling systems.
为了生存并适应环境变化，细菌通常会使用双组分信号系统。这些信号通路至少会利用组氨酸激酶（HKs）来检测环境信号，并利用这些信号控制下游响应调节因子的接收（REC）结构域的磷酸化水平，从而将该信号转化为生理反应。对几种典型REC结构域的研究表明，磷酸化会使这些蛋白质在全局相似且折叠良好的无活性和活性结构之间转换。然而，目前尚不清楚这些发现总体上在REC结构域中具有多大的普遍性，尤其是当它们在全长蛋白质中被考虑时。在此，我们展示了EL_LovR，这是一种仅含REC结构域的全长蛋白质，在海洋α-变形菌——滨海红杆菌HTCC2594中，它会因蓝光照射而发生磷酸化。值得注意的是，EL_LovR与细菌一般应激反应中使用的类似的仅含REC结构域的蛋白质相似，遗传学证据表明，它们强大的磷酸酶活性对于关闭此类系统很重要。尺寸排阻色谱、光散射和溶液核磁共振实验表明，在用于其他REC结构测定的常规条件下，EL_LovR在溶液中呈单体状态且未折叠。添加Mg(2+)和磷酸化会逐渐诱导更高程度的三级结构稳定，完全激活的EL_LovR的溶液结构采用典型的接收结构域折叠。平行功能分析表明，EL_LovR具有快速的去磷酸化速率，这与其作为消耗HK磷酸基团的磷酸盐阱的假定功能一致，从而促进该酶的磷酸酶活性。我们的研究结果表明，EL_LovR经历了大量依赖配体的构象变化，这在其他RR中尚未有报道，扩展了REC结构域（细菌信号系统的关键组成部分）所使用的构象变化和调节的范围。
