Importance of specific hydrogen bonds of archaeal rhodopsins for the binding to the transducer protein

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 μM. 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) μM for bR and to be 29.1(±6.1) μM 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.