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

Abstract

In haloarchaea, light-driven ion transporters have been modified by evolution to produce sensory receptors that relay light signals to transducer proteins controlling motility behavior. The proton pump bacteriorhodopsin and the phototaxis receptor sensory rhodopsin II (SRII) differ by 74% of their residues, with nearly all conserved residues within the photoreactive retinal-binding pocket in the membrane-embedded center of the proteins. Here, we show that three residues in bacteriorhodopsin replaced by the corresponding residues in SRII enable bacteriorhodopsin to efficiently relay the retinal photoisomerization signal to the SRII integral membrane transducer (HtrII) and induce robust phototaxis responses. A single replacement (Ala-215-Thr), bridging the retinal and the membrane-embedded surface, confers weak phototaxis signaling activity, and the additional two (surface substitutions Pro-200 -Thr and Val-210 -Tyr), expected to align bacteriorhodopsin and HtrII in similar juxtaposition as SRII and HtrII, greatly enhance the signaling. In SRII, the three residues form a chain of hydrogen bonds from the retinal's photoisomerized C13AC14 double bond to residues in the membrane-embedded ␣-helices of HtrII. The results suggest a chemical mechanism for signaling that entails initial storage of energy of photoisomerization in SRII's hydrogen bond between Tyr-174, which is in contact with the retinal, and Thr-204, which borders residues on the SRII surface in contact with HtrII, followed by transfer of this chemical energy to drive structural transitions in the transducer helices. The results demonstrate that evolution accomplished an elegant but simple conversion: The essential differences between transport and signaling proteins in the rhodopsin family are far less than previously imagined.bacteriorhodopsin ͉ phototaxis ͉ retinal ͉ sensory rhodopsin ͉ transport M icrobial rhodopsins are photochemically reactive membrane-embedded proteins with seven transmembrane ␣-helices that form a pocket for the chromophore retinal. They are widespread in the microbial world in prokaryotes (bacteria and archaea) and in eukaryotes (fungi and algae) (1-3). A striking characteristic of these photoactive proteins is their wide range of seemingly dissimilar functions. Some are light-driven transporters, such as the proton pumps bacteriorhodopsin (BR) in haloarchaea (4) and proteorhodopsin in marine bacteria (5). Others are light sensors, such as the phototaxis receptors sensory rhodopsins (SR) I and II in haloarchaea (6-8). These sensory rhodopsins relay signals by protein-protein interaction to SR integral membrane transducer proteins (Htr) I and II, respectively. The signal relay mechanism from SR receptors to their cognate Htr transducers has become a focus of interest in part because of its importance to the general understanding of communication between integral membrane proteins, about which little is known.A close relationship between transport and sensory signaling activities was revealed when SRI was separated from its tight co...

Steric Constraint in the Primary Photoproduct of an Archaeal Rhodopsin from Regiospecific Perturbation of C−D Stretching Vibration of the Retinyl Chromophore

“…The wild-type and triple mutant [P200T/V210Y/A215T (BR-T)] proteins of BR were expressed in Halobacterium salinarum Pho81Wr -cells as described previously (13). The purified purple membrane suspension was used for the FTIR measurements of the unlabeled retinal (18).…”

“…Maximal stimuli from a 100 W Hg lamp through a heat filter and 40 nm band-pass interference filters delivered through microscope optics were as follows: 100 ms, 500 nm stimulus for wild-type SRII and SRII mutants; 100 ms, 580 nm stimulus for wild-type BR; and 100 ms, 550 nm stimulus for BR-T (13). The HtrII transducer protein that forms a complex with rhodopsin is from N. pharaonis in this study except for the measurements of BR and BR-T, where H. salinarum HtrII was used (13). The expression levels calculated from amplitudes of the pigments' main absorption bands in the visible spectrum for SRII-HtrII, BR-T-HtrII, T204A-HtrII, T204S-HtrII, T204C-HtrII, Y174F-HtrII, T79A-HtrII, N105D-HtrII, and BR-HtrII complexes in transformants used in this study were all similar, namely, 2.0 × 10 4 , 1.0 × 10 4 , 2.7 × 10 4 , 1.8 × 10 4 , 1.7 × 10 4 , 1.1 × 10 4 , 1.8 × 10 4 , 2.1 × 10 4 , and 3.5 × 10 4 molecules per cell, respectively.…”

“…C 14 -D Stretching and C 14 -HOOP Vibrations in BR-T K . BR-T activates HtrII, and the BR mutant gains light signaling activity (13). How do the vibrations related to the C 14 atom in BR-T differ from those of wild-type BR?…”

“…Therefore, BR-T was converted from the BR-type difference spectrum to the SRIItype difference spectrum by introduction of this steric constraint with the retinal chromophore (13). It should be noted that wild-type BR possesses the C 14 -HOOP mode at 811 and 803 cm -1 (part a of Figure 3B), but no C-D stretching vibrations (part a of Figure 3A), indicating that the C-D stretch is more specific to the structural change of the retinal chromophore.…”

“…Phototaxis function was lost in the mutants of Thr204 or Tyr174, suggesting the biological significance of the hydrogen bond between them (12). Sudo and Spudich successfully engineered BR into an SRII-like light sensor by only three residue replacements, threonine at SRII position 204 (Ala215 in BR) being the most critical (13). FTIR measurements of the triple mutant BR (BR-T) revealed a frequency shift only in the complex with the transducer protein, which can be ascribed to an O-H stretch of Thr215 which is the case for Thr204 of SRII (14).…”