Five Residues in the HtrI Transducer Membrane-proximal Domain Close the Cytoplasmic Proton-conducting Channel of Sensory Rhodopsin I

Abstract

Transducer-free sensory rhodopsins carry out lightdriven proton transport in Halobacterium salinarum membranes. Transducer binding converts the proton pumps to signal-relay devices in which the transport is inhibited. In sensory rhodopsin I (SRI) binding of its cognate transducer HtrI inhibits transport by closing a cytoplasmic proton-conducting channel necessary for proton uptake during the SRI photochemical reaction cycle. To investigate the channel closure, a series of HtrI mutants truncated in the membrane-proximal cytoplasmic portion of an SRI-HtrI fusion were constructed and expressed in H. salinarum membranes. We found that binding of the membrane-embedded portion of HtrI is insufficient for channel closure, whereas cytoplasmic extension of the second HtrI transmembrane helix by 13 residues blocks proton conduction through the channel as well as full-length HtrI. Specifically the closure activity is localized in this 13-residue membrane-proximal cytoplasmic domain to the 5 final residues, each of which incrementally contributes to reduction of proton conductivity. Moreover, these same residues in the dark incrementally and proportionally increase the pK a of the Asp-76 counterion to the protonated Schiff base chromophore in the membrane-embedded photoactive site. We conclude that this critical region of Halobacterium salinarum membranes contain 4 structurally similar seven-helix retinylidene proteins: bacteriorhodopsin (BR), 1 halorhodopsin (HR), sensory rhodopsin I (SRI), and sensory rhodopsin II (SRII). SRI and SRII, together with their bound transducers HtrI and HtrII, respectively, mediate phototaxis responses (1-3). BR and HR are transport rhodopsins that carry out electrogenic outward proton and inward chloride translocation (4, 5). Without their transducers bound, the SRs also exhibit electrogenic proton pumping activity (6 -9). This result and the similar atomic structures of BR (10, 11), HR (12), and NpSRII (13, 14) suggest a common mechanism for haloarchaeal rhodopsin transport and signaling (15). In particular, in BR, a light-induced outward tilting of helices (primarily helix F and to a smaller extent helix G) opens a cytoplasmic side channel important for proton uptake in its pumping cycle (16). Flash photolysis, proton flux measurements, and mutant data (15) and site-directed spin labeling (17) provide compelling evidence that a similar conformational change opens cytoplasmic proton-conducting channels in transducer-free SRs during their photocycles. Transducer binding inhibits the light-induced cytoplasmic side proton-conductivity increase and therefore either prevents the channel opening or blocks the channels (7, 18 -20). Channel closure by the transducers appears to be a key part of the mechanism for converting SRs from proton pumps to sensory receptors (21) and understanding its structural basis is expected to provide insight into the SR-Htr signal-relay mechanism.A 114-residue N-terminal Natronomonas pharaonis HtrII (NpHtrII) fragment containing the transmembrane domains (TM1 and TM2) ...

The Cytoplasmic Membrane-proximal Domain of the HtrII Transducer Interacts with the E-F Loop of Photoactivated Natronomonas pharaonis Sensory Rhodopsin II

“…Aligning HtrI and HtrII shows that positions 62-66 in HtrI correspond to positions 91-95 in HtrII, the interacting 5-residue region revealed in our FRET analysis of the SRII-HtrII complex. The evidence for receptor interaction in the corresponding 5-residue stretch in HtrI obtained by a completely different method in the accompanying article (13) further strengthens the conclusions here, and argues for a similar coupling between SRI and HtrI as we observe for SRII and HtrII.…”

“…A stretch of 5 residues on the HtrI membrane-proximal domain (positions 62-66) blocks or prevents opening of the cytoplasmic proton-conducting channel during the photocycle of transducer-free SRI (13). It is very likely that SRI undergoes a similar conformational movement of helix F and the E-F loop as do BR and SRII.…”

“…Photocycle Measurements-Nd-YAG laser flash absorbance changes were acquired on a laboratory constructed flash spectrometer as described in the accompanying article (13).…”

Three strategically placed hydrogen-bonding residues convert a proton pump into a sensory receptor

“…Flash-induced absorption changes in vesicle suspensions in the millisecond to seconds time window were acquired on a personal computer with a digital oscilloscope program (ClampX 8.2) by following a Nd-YAG laser flash (Continuum, Santa Clara, CA; Surelight I; 532 nm, 6 ns, 40 mJ) in a lab-constructed flash photolysis system as described in ref. 48. Monitoring wavelengths are shown in Fig.…”

A Novel Six-Rhodopsin System in a Single Archaeon

“…Nd-YAG laser (532 nm, 6-ns pulse, 40 mJ) flash-absorbance changes were measured in the laboratory of John Spudich by employing a laboratory-constructed laser cross-beam flash spectrometer as previously described (6). The purified proteins were dissolved in buffer E to reach a concentration of 0.3 at a maximum-wavelength ( max ) OD (OD max ), and transient-absorbance changes were recorded at their corresponding groundstate max .…”