Sudo Yuki, Okuda Hideyasu, Yamabi Masaki, Fukuzaki Yuta, Mishima Masaki, Kamo Naoki, Kojima Chojiro
Laboratory of Biophysical Chemistry, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan.
Biochemistry. 2005 Apr 26;44(16):6144-52. doi: 10.1021/bi047573z.
pHtrII, a pharaonis halobacterial transducer protein, possesses two transmembrane helices and forms a signaling complex with pharaonis phoborhodopsin (ppR, also called pharaonis sensory rhodopsin II, NpSRII) within the halobacterial membrane. This complex transmits a light signal to the sensory system located in the cytoplasm. It has been suggested that the linker region connecting the transmembrane region and the methylation region of pHtrII is important for binding to ppR and subsequent photosignal transduction. In this study, we present evidence to suggest that the linker region itself interacts directly with ppR in addition to the interaction in the membrane region. An in vitro pull-down assay revealed that the linker region bound to ppR, and its dissociation constant (K(D)) was estimated to be approximately 10 microM using isothermal titration calorimetry (ITC). Solution NMR analyses showed that ppR interacted with the linker region of pHtrII (pHtrII(G83)(-)(Q149)) and resulted in the broadening of many peaks, indicating structural changes within this region. These results suggest that the pHtrII linker region interacts directly with ppR. There was no demonstrable interaction between the C-terminal region of ppR (ppR(Gly224)(-)(His247)) and either the linker region (pHtrII(G83)(-)(Q149)) or the transmembrane region (pHtrII(M1)(-)(E114)) of pHtrII. On the basis of the NMR, CD, and photochemical data, we discuss the structural changes and role of the linker region of pHtrII in relation to photosignal transduction.
pHtrII是一种嗜盐菌转导蛋白，具有两个跨膜螺旋，并在嗜盐菌膜内与嗜盐菌视紫红质（ppR，也称为嗜盐菌感官视紫红质II，NpSRII）形成信号复合物。该复合物将光信号传递至位于细胞质中的传感系统。有人提出，连接pHtrII跨膜区和甲基化区的连接区对于与ppR结合及随后的光信号转导很重要。在本研究中，我们提供证据表明，除了在膜区的相互作用外，连接区本身还直接与ppR相互作用。体外下拉试验表明连接区与ppR结合，使用等温滴定量热法（ITC）估计其解离常数（K(D)）约为10微摩尔。溶液核磁共振分析表明，ppR与pHtrII的连接区（pHtrII(G83)(-)(Q149)）相互作用，导致许多峰变宽，表明该区域内的结构变化。这些结果表明pHtrII连接区直接与ppR相互作用。ppR的C末端区域（ppR(Gly224)(-)(His247)）与pHtrII的连接区（pHtrII(G83)(-)(Q149)）或跨膜区（pHtrII(M1)(-)(E114)）之间没有明显的相互作用。基于核磁共振、圆二色光谱和光化学数据，我们讨论了pHtrII连接区在光信号转导方面的结构变化和作用。
