Kamo Takanari, Matsushita Takaaki, Hamada Masako, Fujisawa Tomotsumi, Eki Toshihiko, Unno Masashi, Hirose Yuu
Department of Applied Chemistry and Life Science, Toyohashi University of Technology, 1-1 Hibarigaoka, Tempaku, Toyohashi, Aichi 441-8580, Japan.
Department of Chemistry and Applied Chemistry, Faculty of Science and Engineering, Saga University, 1 Honjocho, Saga 840-8502, Japan.
Plant Cell Physiol. 2025 Feb 28;66(2):181-192. doi: 10.1093/pcp/pcae085.
Cyanobacteriochromes (CBCRs) are members of the phytochrome superfamily of photosensor proteins that bind a bilin chromophore. CBCRs exhibit substantial diversity in their absorption wavelengths through a variety of bilin-protein interactions. RcaE is the first discovered CBCR as a regulator of chromatic acclimation, where cyanobacteria optimize the absorption wavelength of their photosynthetic antenna. RcaE undergoes a reversible photoconversion between green-absorbing (Pg) and red-absorbing (Pr) states, where the bilin chromophore adopts a deprotonated C15-Z,anti and a protonated C15-E,syn structures, respectively. This photocycle is designated as the 'protochromic photocycle' as the change in the bilin protonation state is responsible for the large absorption shift. With the guidance of recently determined Pg and Pr structures of RcaE, in this study, we investigated bilin-protein interaction by site-directed mutagenesis on three key residues referred to as a protochromic triad and also other conserved residues interacting with the bilin. Among the protochromic triad residues, Glu217 and Lys261 are critical for the formation of the Pr state, while Leu249 is critical for the formation of both Pg and Pr states. Substitution in other conserved residues, including Val218, Phe219 and Pro220 in the wind-up helix and Phe252, Phe214 and Leu209 in a part of the bilin-binding pocket, had less substantial effects on the spectral sensitivity in RcaE. These data provide insights into our understanding of the bilin-protein interaction in the protochromic photocycle and also its evolution in the CBCRs.
蓝藻光色素（CBCRs）是光传感器蛋白的植物色素超家族成员，可结合一个胆色素发色团。通过多种胆色素 - 蛋白质相互作用，CBCRs在吸收波长上表现出显著的多样性。RcaE是首个被发现作为色适应调节因子的CBCR，在此过程中蓝藻优化其光合天线的吸收波长。RcaE在吸收绿光的（Pg）和吸收红光的（Pr）状态之间经历可逆的光转换，其中胆色素发色团分别采用去质子化的C15 - Z，反式和顺式质子化的C15 - E结构。这种光循环被称为“原色素光循环”，因为胆色素质子化状态的变化导致了大的吸收峰位移。在最近确定的RcaE的Pg和Pr结构的指导下，在本研究中，我们通过对三个关键残基（称为原色素三联体）以及其他与胆色素相互作用的保守残基进行定点诱变，研究了胆色素 - 蛋白质相互作用。在原色素三联体残基中，Glu217和Lys261对Pr状态的形成至关重要，而Leu249对Pg和Pr状态的形成都至关重要。在其他保守残基中的取代，包括在卷曲螺旋中的Val218、Phe219和Pro220以及在胆色素结合口袋一部分中的Phe252、Phe214和Leu209，对RcaE的光谱敏感性影响较小。这些数据为我们理解原色素光循环中的胆色素 - 蛋白质相互作用及其在CBCRs中的进化提供了见解。
