Time-resolved X-ray solution scattering observations of light-induced structural changes in sensory rhodopsin II.

Sensory rhodopsin II (SRII), a receptor for negative phototaxis in haloarchaea, transmits light signals through changes in protein-protein interaction with its transducer HtrII. Light-induced structural changes throughout the SRII-HtrII interface, which spans the periplasmic region, membrane-embedded domains, and cytoplasmic domains near the membrane, have been identified by several studies. Here we demonstrate by site-specific mutagenesis and analysis of phototaxis behavior that two residues in SRII near the membrane-embedded interface (Tyr174 on helix F and Thr204 on helix G) are essential for signaling by the SRII-HtrII complex. These residues, which are the first in SRII shown to be required for phototaxis function, provide biological significance to the previous observation that the hydrogen bond between them is strengthened upon the formation of the earliest SRII photointermediate (SRII(K)) only when SRII is complexed with HtrII. Here we report frequency changes of the S-H stretch of a cysteine substituted for SRII Thr204 in the signaling state intermediates of the SRII photocycle, as well as an influence of HtrII on the hydrogen bond strength, supporting a direct role of the hydrogen bond in SRII-HtrII signal relay chemistry. Our results suggest that the light signal is transmitted to HtrII from the energized interhelical hydrogen bond between Thr204 and Tyr174, which is located at both the retinal chromophore pocket and in helices F and G that form the membrane-embedded interaction surface to the signal-bearing second transmembrane helix of HtrII. The results argue for a critical process in signal relay occurring at this membrane interfacial region of the complex.

Recently, a large family of transducer proteins in the Archaeon Halobacterium salinarium was identified. On the basis of the comparison of the predicted structural domains of these transducers, three distinct subfamilies of transducers were proposed. Here we report isolation, complete gene sequences, and analysis of the encoded primary structures of transducer gene htrII, a member of family B, and its blue light receptor gene (sopII) of sensory rhodopsin II (SRII). The start codon ATG of the 714-bp sopII gene is one nucleotide beyond the termination codon TGA of the 2298-bp htrII gene. The deduced protein sequence of HtrII predicts a eubacterial chemotaxis transducer type with two hydrophobic membrane-spanning segments connecting sizable domains in the periplasm and cytoplasm. HtrII has a common feature with HtrI, the sensory rhodopsin I transducer; like HtrI, HtrII possesses a hydrophilic loop structure just after the second transmembrane segment. The C-terminal 299 residues (765 amino acid residues total) of HtrII show strong homology to the signaling and methylation domain of eubacterial transducer Tsr. The hydropathy plot of the primary structure of SRII indicates seven membrane-spanning alpha-helical segments, a characteristic feature of retinylidene proteins ("rhodopsins") from a widespread family of photoactive pigments. SRII shows high identity with SRI (42%), bacteriorhodopsin (BR) (32%), and halorhodopsin (24%). The crucial positions for retinal binding sites in these proteins are nearly identical, with the exception of Met-118 (numbering according to the mature BR sequence), which is replaced by Val in SRII. In BR, residues Asp-85 and Asp-96 are crucial in proton pumping. In SRII, the position corresponding to Asp-85 in BR is conserved, but the corresponding position of Asp-96 is replaced by an aromatic Tyr. Coexpression of the htrII and sopII genes restores SRII phototaxis to a mutant (Pho81) that contains a deletion in the htrI/sopI and insertion in htrII/sopII regions. This paper describes the first example that both HtrI and HtrII exist in the same halobacterial cell, confirming that different sensory rhodopsins SRI and SRII in the same organism have their own distinct transducers.

Sensory rhodopsin I (SRI) functions as a dual receptor regulating both negative and positive phototaxis. It transmits light signals through changes in protein-protein interactions with its transducer protein, HtrI. The phototaxis function of Halobacterium salinarum SRI (HsSRI) has been well characterized using genetic and molecular techniques, whereas that of Salinibacter ruber SRI (SrSRI) has not. SrSRI has the advantage of high protein stability compared with HsSRI and, therefore, provided new information about structural changes and Cl(-) binding of SRI. However, nothing is known about the functional role of SrSRI in phototaxis behavior. In this study, we expressed a SRI homologue from the archaeon Haloarcula vallismortis (HvSRI) as a recombinant protein which uses all-trans-retinal as a chromophore. Functionally important residues of HsSRI are completely conserved in HvSRI (unlike in SrSRI), and HvSRI is extremely stable in buffers without Cl(-). Taking advantage of the high stability, we characterized the photochemical properties of HvSRI under acidic and basic conditions and observed the effects of Cl(-) on the protein under both conditions. Fourier transform infrared results revealed that the structural changes in HvSRI were quite similar to those in HsSRI and SrSRI. Thus, HvSRI can become a useful protein model for improving our understanding of the molecular mechanism of the dual photosensing by SRI.

We have investigated the functional relationship between two proteins involved in the photosensory system of the archaeon Halobacterium salinarium: the photoreceptor sensory rhodopsin I (SRI) and the halobacterial transducer rhodopsin I (HtrI), which has been proposed to be the putative signal transducer of SRI, by genomic DNA analysis of two independent SRI negative mutants, Pho81 and D1. Southern and PCR analyses revealed that both strains bear alterations in the 5' flanking region of the gene encoding SRI, sopI. DNA sequence analysis confirmed the occurrence in this region of htrI, the gene encoding the putative transducer protein. PCR and Northern analyses have shown further that sopI and htrI are expressed as a single transcriptional unit, thus explaining the lack of SRI in mutants with a defective htrI. Expression of the cloned sopI under the control of a heterologous promoter did not restore the SRI-dependent photoresponse in the strain Pho81. Moreover, the photocycling rate of the expressed pigment was clearly lower than in wild type. HtrI is therefore essential for SRI function and most likely modulates the photochemical properties of the photoreceptor via direct physical interaction. Finally, reintroduction of both sopI and htrI into Pho81 and D1 restored the SRI photochemistry and its physiological function. Our results provide the first experimental evidence for the functional coupling between SRI and HtrI and corroborate the proposed model in which HtrI acts as the signal transducer of this archaeal seven-helix photoreceptor in a way analogous to the bacterial chemotaxis transducers.

Sensory rhodopsin I (SRI) functions in both positive and negative phototaxis in complex with halobacterial transducer protein I (HtrI). Orange light activation of SRI results in deprotonation of the retinylidene chromophore of SRI to produce the S 373 photocycle intermediate, the signaling state for positive phototaxis. In this study, we observed pH dependence on structural coupling between the two molecules upon the formation of the S 373 intermediate by means of Fourier transform infrared spectroscopy. At alkaline pH, where Asp76 (one of the counterions of the protonated retinylidene Schiff base) is deprotonated, HtrI-dependent alteration of the light-induced difference spectra is limited to reduction of amide I bands at 1661 (+)/ 1647 (-) cm (-1), and perturbation of one of the protonated carboxylic acid bands occurs at 1734 (-) cm (-1) (which appears to become ionized only when complexed with HtrI). However, at acidic pH, HtrI-complexed SRI exhibits not only light-induced reduction of the amide I changes but a wider range of spectral alterations including the appearance of several new amide I bands, perturbation of the chromophore-related vibrational modes, and other additional changes characteristic of tyrosine, glutamate, and aspartate residues. Since such pH dependence of structural changes was not observed in the complex of the D76N mutant of SRI, which behaves much like HtrI-complexed SRI in acidic conditions, we conclude that extensive orange light-induced conformational coupling between SRI and HtrI occurs only when Asp76 is neutralized.

The complexation between the photoreceptor sensory rhodopsin I (SRI) and its signal transducer protein HtrI was examined by assessing titration of the Schiff base chromophore of SRI with sodium hydroxide and reactivity with hydroxylamine in the presence or absence of HtrI. The apparent pKa of the protonated Schiff base of SRI is 12.2 in the presence and 9.5 in the absence of HtrI. Direct titration of the Schiff base proton was confirmed by titrating an artificial SRI reconstituted with a 14-fluororetinal which reduces the intrinsic pKa of the protonated Schiff base of the HtrI-complexed pigment from >12 to 9.0. The SRI chromophore exhibits high stability to hydroxylamine bleaching in the presence of HtrI; however, removal of HtrI accelerates the bleaching rate 2.4-fold. These results indicate that SRI is physically associated with HtrI in its unactivated (i.e., dark) state. In view of the previously identified association of the SRI signaling state (S373) with HtrI, we conclude that SRI transduces the signal to HtrI through its altered interaction with the prebound transducer protein. The effect of the altered SRI/HtrI interaction on resetting the signaling state of SRI was also examined. At neutral pH the decay of S373 is retarded by 20-fold when HtrI is absent. This effect was found to be due to a raised enthalpic barrier for the transition state during S373 decay. The energy barrier for S373 decay in this pigment can be lowered by providing extramembranous protons (lowering bulk pH). Therefore, light-induced alteration in SRI/HtrI interaction is important for reducing the energy barrier for S373 decay presumably by providing or assisting a proton supply for retinal Schiff base reprotonation.

Effects of modifications of the retinal beta-ionone ring on archaebacterial sensory rhodopsin I

We studied the role of added thylakoid lipids in the light-induced reversible structural changes in isolated macroaggregates of the main light-harvesting chlorophyll a/b complex of photosystem II (LHCII). Loosely stacked lamellar macroaggregates were earlier shown to undergo light-induced reversible structural changes and changes in the photophysical pathways, which resembled those in thylakoid membranes exposed to excess light [Barzda, V., et al. (1996) Biochemistry 35, 8981-8985]. This structural flexibility of LHCII depends critically on the lipid content of the preparations [Simidjiev, I., et al. (1997) Anal. Biochem. 250, 169-175]. It is now reported that lamellar aggregates of LHCII are capable of incorporating substantial amounts of different thylakoid lipids. The long-range order of the chromophores is retained, while the ultrastructure of the lipid-protein macroaggregates can be modified significantly. Addition of thylakoid lipids to the preparations significantly enhances the ability of the LHCII macroaggregates to undergo light-induced structural changes. The lipid environment of the LHCII complexes therefore plays a significant role in determining the structural flexibility of the macroaggregates. As concerns the mechanism of these changes, it is proposed that the absorption of light and the dissipation of its energy in the macrodomains induces thermal fluctuations which bring about changes in the shape or in the stacking interactions of the membranes, this in turn affecting the long-range order of the embedded chromophores. In thylakoids, a similar mechanism is likely to explain the light-induced structural changes which are largely independent of the photochemical activity of the membranes.

Sensory rhodopsin I (SRI) exists in the cell membranes of microorganisms such as the archaeon Halobacterium salinarum and is a photosensor responsible for positive and negative phototaxis. SRI forms a signaling complex with its cognate transducer protein, HtrI, in the membrane. That complex transmits light signals to the flagellar motor through changes in protein-protein interactions with the kinase CheA and the adaptor protein CheW, which controls the direction of the rotation of the flagellar motor. Recently, we cloned and characterized Salinibacter sensory rhodopsin I (SrSRI), which is the first SRI-like protein identified in eubacteria [Kitajima-Ihara, T., et al. (2008) J. Biol. Chem. 283, 23533-23541]. Here we cloned and expressed SrSRI with its full-length transducer protein, SrHtrI, as a fusion construct. We succeeded in producing the complex in Escherichia coli as a recombinant protein with high quality having all-trans-retinal as a chromophore for SRI, although the expression level was low (0.10 mg/L of culture). In addition, we report here the photochemical properties of the SrSRI-SrHtrI complex using time-resolved laser flash spectroscopy and other spectroscopic techniques and compare them to SrSRI without SrHtrI.

Recent progress is summarized on the mechanism of phototransduction by sensory rhodopsin I (SR-I), a phototaxis receptor in Halobacterium halobium. Two aspects are emphasized: (i) The coupling of retinal isomerization to protein conformational changes. Retinal analogs have been used to probe chromophore-apoprotein interactions during the receptor activation process. One of the most important results is the finding of a steric trigger deriving from the interaction of residues on the protein with a methyl group near the isomerizing bond of the retinal (at carbon 13). Recent work on molecular genetic methods to further probe structure/function includes the synthesis and expression of an SR-I apoprotein gene designed for residue replacements by cassette mutagenesis, and transformation of an H. halobium mutant lacking all retinylidene proteins known in this species to SR-I+ and bacteriorhodopsin (BR)+. (ii) The relay of the SR-I signal to a post-receptor component. A carboxylmethylated protein ("MPP-I") associated with SR-I and found in the H. halobium membrane exhibits homology with the signaling domain of eubacterial chemotaxis transducers (e.g., Escherichia coli Tar, Tsr, and Trg proteins), suggesting a model based on SR-I----MPP-I signal relay.

Sensory rhodopsin II (SRII), a repellent phototaxis receptor found in Halobacterium salinarum, has several homologous residues which have been found to be important for the proper functioning of bacteriorhodopsin (BR), a light-driven proton pump. These include Asp73, which in the case of bacteriorhodopsin (Asp85) functions as the Schiff base counterion and proton acceptor. We analyzed the photocycles of both wild-type SRII and the mutant D73E, both reconstituted in Halobacterium salinarum lipids, using FTIR difference spectroscopy under conditions that favor accumulation of the O-like, photocycle intermediate, SII540. At both room temperature and -20 degrees C, the difference spectrum of SRII is similar to the BR-->O640 difference spectrum of BR, especially in the configurationally sensitive retinal fingerprint region. This indicates that SII540 has an all-trans chromophore similar to the O640 intermediate in BR. A positive band at 1761 cm-1 downshifts 40 cm-1 in the mutant D73E, confirming that Asp73 undergoes a protonation reaction and functions in analogy to Asp85 in BR as a Schiff base proton acceptor. Several other bands in the C=O stretching regions are identified which reflect protonation or hydrogen bonding changes of additional Asp and/or Glu residues. Intense bands in the amide I region indicate that a protein conformational change occurs in the late SRII photocycle which may be similar to the conformational changes that occur in the late BR photocycle. However, unlike BR, this conformational change does not reverse during formation of the O-like intermediate, and the peptide groups giving rise to these bands are partially accessible for hydrogen/deuterium exchange. Implications of these findings for the mechanism of SRII signal transduction are discussed.

Sensory rhodopsin I (SRI) is one of the most interesting photosensory receptors because of its function in using the photochromic reaction to mediate opposing signals which depend on the color of light. It was initially thought that SRI exists only in the archaea, but we recently reported for the first time a newly functional SRI from a eubacterium, Salinibacter ruber (SrSRI). The amino acid sequence of SrSRI shows 43% identity with the well-known SRI (HsSRI) and contains most of the amino acid residues identified as necessary for SRI function. The photochemical properties of SrSRI are similar to those of HsSRI. In addition, SrSRI is a highly stable protein, even in dilute salt conditions. Thus, SrSRI could be a key protein for characterizing its association with the SrSRI transducer protein, SrHtrI, and for elucidating structural changes of SRI and HtrI that occur during their function. Recently, new approaches to manipulate cellular functions with rhodopsins have been established. SRI can activate and deactivate a kinase, CheA, by the photochromic reaction. Kinases are key molecules for signal transduction in various organisms, and their cellular functions could potentially be manipulated by SRI.

A Transporter Converted into a Sensor, a Phototaxis Signaling Mutant of Bacteriorhodopsin at 3.0 Å

Signal Transmission through the HtrII Transducer Alters the Interaction of Two α-Helices in the HAMP Domain

Bacteriorhodopsin (BR) and sensory rhodopsin II (SRII) function as a light-driven proton pump and a receptor for negative phototaxis in haloarchaeal membranes, respectively. SRII transmits light signals through changes in protein-protein interaction with its transducer HtrII. Recently, we converted BR by three mutations into a form capable of transmitting photosignals to HtrII to mediate phototaxis responses. The BR triple mutant (BR-T) provides an opportunity to identify structural changes necessary to activate HtrII by comparing light-induced infrared spectral changes of BR, BR-T, and SRII. The hydrogen out-of-plane (HOOP) vibrations of the BR-T were very similar to those of SRII, indicating that they are distributed more extensively along the retinal chromophore than in BR, as in SRII. On the other hand, the bands of the protein moiety in BR-T are similar to those of BR, indicating that they are not specific to photosensing. The alteration of the O-H stretching vibration of Thr-204 in SRII, which we had previously shown to be essential for signal relay to HtrII, occurs also in BR-T. In addition, 1670(+)/1664(-) cm(-1) bands attributable to a distorted alpha-helix were observed in BR-T in a HtrII-dependent manner, as is seen in SRII. Thus, we identified similarities and dissimilarities of BR-T to BR and SRII. The results suggest signaling function of the structural changes of the HOOP vibrations, the O-H stretching vibration of the Thr-215 residue, and a distorted alpha-helix for the signal generation. We also succeeded in measurements of L minus initial state spectra of BR-T, which are the first FTIR spectra of L intermediates among sensory rhodopsins.