Malla Tek Narsingh, Hernandez Carolina, Menendez David, Bizhga Dorina, Mendez Joshua H, Muniyappan Srinivasan, Schwander Peter, Stojković Emina A, Schmidt Marius
bioRxiv. 2023 Nov 9:2023.11.08.566274. doi: 10.1101/2023.11.08.566274.
Phytochromes are essential photoreceptor proteins in plants with homologs in bacteria and fungi that regulate a variety of important environmental responses. They display a reversible photocycle between two distinct states, the red-light absorbing Pr and the far-red light absorbing Pfr, each with its own structure. The reversible Pr to Pfr photoconversion requires covalently bound bilin chromophore and regulates the activity of a C-terminal enzymatic domain, which is usually a histidine kinase (HK). In plants, phytochromes translocate to nucleus where the C-terminal effector domain interacts with protein interaction factors (PIFs) to induce gene expression. In bacteria, the HK phosphorylates a response-regulator (RR) protein triggering downstream gene expression through a two-component signaling pathway. Although plant and bacterial phytochromes share similar structural composition, they have contrasting activity in the presence of light with most BphPs being active in the dark. The molecular mechanism that explains bacterial and plant phytochrome signaling has not been well understood due to limited structures of full-length phytochromes with enzymatic domain resolved at or near atomic resolution in both Pr and Pfr states. Here, we report the first Cryo-EM structures of a wild-type bacterial phytochrome with a HK enzymatic domain, determined in both Pr and Pfr states, between 3.75 and 4.13 Å resolution, respectively. Furthermore, we capture a distinct Pr/Pfr heterodimer of the same protein as potential signal transduction intermediate at 3.75 Å resolution. Our three Cryo-EM structures of the distinct signaling states of BphPs are further reinforced by Cryo-EM structures of the truncated PCM of the same protein determined for the Pr/Pfr heterodimer as well as Pfr state. These structures provide insight into the different light-signaling mechanisms that could explain how bacteria and plants see the light.
光敏色素是植物中必不可少的光受体蛋白，在细菌和真菌中也有同源物，可调节多种重要的环境反应。它们在两种不同状态之间呈现可逆的光循环，即吸收红光的Pr和吸收远红光的Pfr，每种状态都有其自身的结构。Pr到Pfr的可逆光转换需要共价结合的胆色素发色团，并调节C末端酶结构域的活性，该结构域通常是组氨酸激酶（HK）。在植物中，光敏色素转移到细胞核，C末端效应结构域与蛋白质相互作用因子（PIF）相互作用以诱导基因表达。在细菌中，HK使响应调节蛋白（RR）磷酸化，通过双组分信号通路触发下游基因表达。尽管植物和细菌的光敏色素具有相似的结构组成，但它们在光照下具有相反的活性，大多数细菌光敏色素在黑暗中具有活性。由于在Pr和Pfr状态下以原子分辨率或接近原子分辨率解析的具有酶结构域的全长光敏色素结构有限，解释细菌和植物光敏色素信号传导的分子机制尚未得到很好的理解。在这里，我们报告了野生型细菌光敏色素与HK酶结构域的首个低温电子显微镜结构，分别在Pr和Pfr状态下测定，分辨率在3.75至4.13 Å之间。此外，我们在3.75 Å分辨率下捕获了同一蛋白质的不同Pr/Pfr异二聚体作为潜在的信号转导中间体。我们对细菌光敏色素不同信号状态的三个低温电子显微镜结构通过对同一蛋白质的截短PCM的低温电子显微镜结构进一步加强，该结构针对Pr/Pfr异二聚体以及Pfr状态进行了测定。这些结构为不同的光信号传导机制提供了见解，这可以解释细菌和植物如何感知光。
