Probing the Sensory Rhodopsin II Binding Domain of its Cognate Transducer by Calorimetry and Electrophysiology

Hippler-Mreyen, Silke; Klare, Johann P.; Wegener, Ansgar; Seidel, R.; Herrmann, Christian; Schmies, Georg; Nagel, Georg; Bamberg, Ernst; Engelhard, Martin

doi:10.1016/s0022-2836(03)00656-9

Cited by 58 publications

(85 citation statements)

References 47 publications

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“…4B), the K d was determined to be 0.35 M under our conditions. Similar measurement with HtrII-G83F produced a K d of 0.22 M. These values are close to the affinities measured in previous reports (8,21), and show that the G83F mutation does not significantly inhibit HtrII binding to SRII-D75N.…”

Section: Tryptophans Placed Within a Short Region In The Htrii Membrasupporting

confidence: 90%

“…The structure resolved extensive van der Waals and hydrogen-bonding contacts between HtrII TM2 (and to a lesser extent TM1) and SRII helices F and G within the membrane domain. The presence of the cytoplasmic membrane-proximal domain, comprised of 32 residues beyond Leu-82 at the membrane-cytoplasm interface, was necessary for high affinity binding of the HtrII fragment to SRII in detergent micelles used in the crystallization (7,8). The structure of the cytoplasmic extension was not resolved by the crystallographic refinement.…”

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confidence: 99%

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

Yang

Sineshchekov²,

Spudich

et al. 2004

Journal of Biological Chemistry

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The structures of the cytoplasmic loops of the phototaxis receptor sensory rhodopsin II (SRII) and the membrane-proximal cytoplasmic domain of its bound transducer HtrII were examined in the dark and in the lightactivated state by fluorescent probes and cysteine crosslinking. Light decreased the accessibility of E-F loop position 154 in the SRII-HtrII complex, but not in free SRII, consistent with HtrII proximity, which was confirmed by tryptophans placed within a 5-residue region identified in the HtrII membrane-proximal domain that exhibited Fö rster resonance energy transfer to a fluorescent probe at position 154 in SRII. The Fö rster resonance energy transfer was eliminated in the signaling deficient HtrII mutant G83F without loss of affinity for SRII. Finally, the presence of SRII and HtrII reciprocally inhibit homodimer disulfide cross-linking reactions in their membrane-proximal domains, showing that each interferes with the others self-interaction in this region. The results demonstrate close proximity between SRII-HtrII in the membrane-proximal domain, and in addition, light stimulation of the SRII inhibition of HtrII cross-linking was observed, indicating that the contact is enhanced in the photoactivated complex. A mechanism is proposed in which photoactivation alters the SRII-HtrII interaction in the membrane-proximal region during the signal relay process.Archaeal sensory rhodopsins I and II (SRI and SRII) 1 are membrane-embedded phototaxis receptors that modulate the motility apparatus of Halobacterium salinarum and related haloarchaeal species (1-3). The SRI and SRII proteins transmit signals to their cognate transducer proteins, HtrI and HtrII, respectively, and like their eubacterial counterparts in chemotaxis, the Htr transducers control a histidine kinase and phosphoregulator protein that modulates motor function.Biochemical and spectroscopic studies have shown that there is close interaction between the SR and Htr components of the signaling complex both in the light and in the dark (1); i.e. they are subunits of a molecular complex. Furthermore, chimera experiments demonstrated that the interaction specificities of SRI with HtrI and SRII with HtrII are determined by the transmembrane helices of the Htr subunits (4). Cubic lipid phase crystal x-ray structures of SRII have defined the membrane-embedded and membrane-external portions of the transmembrane helices and periplasmic and cytoplasmic loops of the protein (5, 6). Moreover, co-crystallization of SRII with an N-terminal fragment of HtrII containing its two transmembrane segments (TM1 and TM2) and a cytoplasmic extension of TM2 provided an atomic resolution structure by x-ray diffraction (7). The structure resolved extensive van der Waals and hydrogen-bonding contacts between HtrII TM2 (and to a lesser extent TM1) and SRII helices F and G within the membrane domain. The presence of the cytoplasmic membrane-proximal domain, comprised of 32 residues beyond Leu-82 at the membrane-cytoplasm interface, was necessary for high affinity binding o...

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Section: Tryptophans Placed Within a Short Region In The Htrii Membrasupporting

confidence: 90%

mentioning

confidence: 99%

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

Yang

Sineshchekov²,

Spudich

et al. 2004

Journal of Biological Chemistry

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“…Such a mechanism would require assuming that the relatively small differences in helical length between SRI-HtrI-(1-61) through SRI-HtrI-(1-66) would cause a significant alteration in the transmembrane structure of HtrI. This interpretation would be consistent with the observation that shortening of NpHtrII from 114 to 101 residues eliminates binding of NpHtrII truncation mutants to NpSRII in detergent micelles (22), although this phenomenon may be because of non-native folding by the shorter truncated NpHtrII. Considering all of these points we favor the direct interaction model as explaining the results most simply.…”

Section: Discussionmentioning

confidence: 75%

“…A 114-residue N-terminal Natronomonas pharaonis HtrII (NpHtrII) fragment containing the transmembrane domains (TM1 and TM2) and a 32-residue extension of TM2 into the cytoplasm (the "membrane-proximal domain") closes the channel of NpSRII in a micellar system and in Xenopus oocytes (22). X-ray structures of NpSRII are available both for the free receptor (13,14) and for receptor bound to this fragment (23).…”

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confidence: 99%

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

Chen¹,

Spudich²

2004

Journal of Biological Chemistry

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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) ...

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“…The first modification was the addition of a flexible linker of 11 residues connecting the cytoplasmic C terminus of BR and cytoplasmic N terminus of HtrII to ensure a 1:1 stoichiometry, as exists in the SRII-HtrII complex (24)(25)(26). The fusion of SRII and HtrII by their cytoplasmic ends in this manner permits normal phototaxis responses, indicating the linker does not influence signaling (see Fig.…”

Section: Resultsmentioning

confidence: 99%

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

Sudo

Spudich

2006

Proc. Natl. Acad. Sci. U.S.A.

102

125

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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...

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Probing the Sensory Rhodopsin II Binding Domain of its Cognate Transducer by Calorimetry and Electrophysiology

Cited by 58 publications

References 47 publications

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

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

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

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

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