Sudo Yuki, Yamabi Masaki, Kato Shinnosuke, Hasegawa Chisa, Iwamoto Masayuki, Shimono Kazumi, Kamo Naoki
Laboratory of Biophysical Chemistry, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan.
J Mol Biol. 2006 Apr 7;357(4):1274-82. doi: 10.1016/j.jmb.2006.01.061. Epub 2006 Feb 3.
Four rhodopsins, bacteriorhodopsin (bR), halorhodopsin (hR), sensory rhodopsin (sR) and phoborhodopsin (pR) exist in archaeal membranes. bR and hR work as a light-driven ion pump. sR and pR work as a photo-sensor of phototaxis, and form signaling complexes in membranes with their respective cognate transducer proteins HtrI (with sR) and HtrII (with pR), through which light signals are transmitted to the cytoplasm. What is the determining factor(s) of the specific binding to form the complex? Binding of the wild-type or mutated rhodopsins with HtrII was measured by isothermal titration calorimetric analysis (ITC). bR and hR could not bind with HtrII. On the other hand, sR could bind to HtrII, although the dissociation constant (K(D)) was about 100 times larger than that of pR. An X-ray crystallographic structure of the pR/HtrII complex revealed formation of two specific hydrogen bonds whose pairs are Tyr199(pR)/Asn74(HtrII) and Thr189(pR)/Glu43(HtrII)/Ser62(HtrII). To investigate the importance of these hydrogen bonds, the K(D) value for the binding of various mutants of bR, hR, sR and pR with HtrII was estimated by ITC. The K(D) value of T189V(pR)/Y199F(pR), double mutant/HtrII complex, was about 100-fold larger than that of the wild-type pR, whose K(D) value was 0.16 microM. On the other hand, bR and hR double mutants, P200T(bR)/V210Y(bR) and P240T(hR)/F250Y(hR), were able to bind with HtrII. The K(D) value of these complexes was estimated to be 60.1(+/-10.7) microM for bR and to be 29.1(+/-6.1) microM for hR, while the wild-type bR and hR did not bind with HtrII. We concluded that these two specific hydrogen bonds play important roles in the binding between the rhodopsins and transducer protein.
古菌膜中存在四种视紫红质，即细菌视紫红质（bR）、嗜盐视紫红质（hR）、感官视紫红质（sR）和避光视紫红质（pR）。bR和hR作为光驱动离子泵发挥作用。sR和pR作为趋光性的光传感器，并在膜中与它们各自的同源转导蛋白HtrI（与sR）和HtrII（与pR）形成信号复合物，光信号通过该复合物传递到细胞质中。形成复合物的特异性结合的决定因素是什么？通过等温滴定量热分析（ITC）测量野生型或突变视紫红质与HtrII的结合。bR和hR不能与HtrII结合。另一方面，sR可以与HtrII结合，尽管解离常数（K(D)）比pR的解离常数大约100倍。pR/HtrII复合物的X射线晶体结构显示形成了两个特定的氢键，其配对为Tyr199（pR）/Asn74（HtrII）和Thr189（pR）/Glu43（HtrII）/Ser62（HtrII）。为了研究这些氢键的重要性，通过ITC估计了bR、hR、sR和pR的各种突变体与HtrII结合的K(D)值。T189V（pR）/Y199F（pR）双突变体/HtrII复合物的K(D)值比野生型pR的K(D)值大约100倍，野生型pR的K(D)值为0.16 microM。另一方面，bR和hR双突变体P200T（bR）/V210Y（bR）和P240T（hR）/F250Y（hR）能够与HtrII结合。这些复合物的K(D)值对于bR估计为60.1（±10.7） microM，对于hR估计为29.1（±6.1） microM，而野生型bR和hR不与HtrII结合。我们得出结论，这两个特定的氢键在视紫红质与转导蛋白之间的结合中起重要作用。
