| Bacteriorhodopsin (BR) and proteorhodopsin (PR) are light-driven ion pumps, found in the domains Archaea and Bacteria, respectively, and sensory rhodopsin II (SRII), also found in Archaea, acts as a photoreceptor that transmits a light-induced conformational change to its transducer HtrII. Despite these distinctly different functions between BR and SRII, a single residue substitution, Ala215 to Thr215 in the BR, enables its photochemical reactions to transmit signals to HtrII and mediate phototaxis. In order to determine the structural changes caused by the A215T mutation, we solved a crystal structure of the signaling BR mutant (BR_A215T) to 3.0 Å resolution. The analysis was complemented by molecular dynamics simulation of BR mutated in silico. The main conclusions emerging from the comparison of BR_A215T, BR, and SRII structures are: (i) the Thr215 residue in signaling BR is positioned nearly identically with respect to retinal as in SRII, consistent with its role in producing a steric conflict with the retinal C14 group during photoisomerization, proposed earlier to be essential for SRII signaling; (ii) Tyr174–Thr204 hydrogen bonding, critical in SRII signaling and mimicked in signaling BR, is likely auxiliary, for example, to maintain Thr204 in the proper position for the steric trigger to occur; and (iii) the primary role of Arg72 in SRII is spectral tuning and not signaling.;PRs comprise the largest family of microbial rhodopsins, yet very little structural information is available. We report here the first crystals structures of two blue light-absorbing proteorhodopsins (BPR): native Med12BPR to 2.31 Å resolution, and the 2.70 Å and 2.60 Å structures of D97N and D97N/Q105L HOT75BPR mutants, respectively. Six molecules of Med12BPR form a doughnut-shaped hexamer, while HOT75BPR assembles into a pentamer. The structures display similar features to BR, but also reveal significant differences that may be unique to proteorhodopsins. The retinal Schiff base deviates from other rhodopsins by an overall shift of atom C14 about 1.1 Å towards helix C, and the putative proton-release region in BPR differs significantly from those of BR and xanthorhodopsin (XR). The most striking feature of proteorhodopsin is the position of His75, also found in XR, which interacts with the proton acceptor, Asp97, and also forms a hydrogen bond with Trp34 of a neighboring promoter. Trp34 may function by stabilizing His75 in a conformation that favors a deprotonated Asp97 in the dark state, and suggests cooperative behavior between protomers when the protein is in an oligomeric state. |
