Structure of the Light-Driven Chloride Pump Halorhodopsin at 1.8 Å Resolution

Kolbe, Michael; Besir, Hüseyin; Essen, Lars‐Oliver; Oesterhelt, Dieter

doi:10.1126/science.288.5470.1390

Cited by 525 publications

(517 citation statements)

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“…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).…”

mentioning

confidence: 66%

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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mentioning

confidence: 66%

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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“…One is on the extracellular side of the central cluster, near HR-T111. This chloride is found in the crystal structure (Cl -501 in 1E12 and 2JAG) (41,42), and in earlier MD simulations (33). It is near the position of the carboxylate side chain of D85 in BR.…”

Section: Resultsmentioning

confidence: 93%

Halorhodopsin pumps Cl^–and bacteriorhodopsin pumps protons by a common mechanism that uses conserved electrostatic interactions

Song¹,

Gunner

2014

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

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Key mutations differentiate the functions of homologous proteins. One example compares the inward ion pump halorhodopsin (HR) and the outward proton pump bacteriorhodopsin (BR). Of the nine essential buried ionizable residues in BR, six are conserved in HR. However, HR changes three BR acids, D85 in a central cluster of ionizable residues, D96, nearer the intracellular, and E204, nearer the extracellular side of the membrane to the small, neutral amino acids T111, V122, and T230, respectively. In BR, acidic amino acids are stationary anions whose proton affinity is modulated by conformational changes, establishing a sequence of directed binding and release of protons. Multiconformation continuum electrostatics calculations of chloride affinity and residue protonation show that, in reaction intermediates where an acid is ionized in BR, a Cl -is bound to HR in a position near the deleted acid. In the HR ground state, Cl -binds tightly to the central cluster T111 site and weakly to the extracellular T230 site, recovering the charges on ionized BR-D85 and neutral E204 in BR. Imposing key conformational changes from the BR M intermediate into the HR structure results in the loss of Cl -from the central T111 site and the tight binding of Cl -to the extracellular T230 site, mirroring the changes that protonate BR-D85 and ionize E204 in BR. The use of a mobile chloride in place of D85 and E204 makes HR more susceptible to the environmental pH and salt concentrations than BR. These studies shed light on how ion transfer mechanisms are controlled through the interplay of protein and ion electrostatics.

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“…Buried water molecules have been observed in the interiors of several other high resolution structures of helical transmembrane proteins, including visual rhodopsin (26), a glutamate transporter (27), halorhodopsin (28), and sensory rhodopsin II (29). These water molecules bond almost exclusively to the side chains or backbone within α-helix regions.…”

Section: S = [U]/[f] and K S ' = [Uh]/[fh]mentioning

confidence: 99%

Transmembrane Helix−Helix Association: Relative Stabilities at Low pH

Valluru¹,

Silva²,

Dhage³

et al. 2006

Biochemistry

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We have previously studied the unfolding equilibrium of bacterio-opsin in a single phase solvent, using Förster mechanism fluorescence resonance energy transfer (FRET) as a probe, from tryptophan donors to a dansyl acceptor. We observed an apparent unfolding transition in bacterio-opsin perturbed by increasing ethanol concentrations [Nannepaga et al.(2004) Biochemistry 43, 50-59]. We have further investigated this transition and find the unfolding is pH-dependent. We have now measured the apparent pK of acid-induced unfolding of bacterio-opsin in 90% ethanol. When the acceptor is on helix B (Lys 41), the apparent pK for unfolding is 4.75; on the EF connecting loop (Cys 163), 5.15; and on helix G (Cys 222), 5.75. Five-helix proteolytic fragments are less stable. The apparent unfolding pKs are, for residues 72-248 (Cys 163), 5.46; and 1-166 (Lys 41), 7.36. When interpreted in terms of a simple equilibrium model for unfolding, the apparent pKs give relative free energies of unfolding, in the range of −0.54 to −3.5 kcal/mol. The results suggest that the C-terminal helix of bacterio-opsin is less stably folded than the N-terminal helices. We analyzed the pair-wise helixhelix interaction surfaces of bacteriorhodopsin and three other 7-transmembrane helix proteins, based on crystal structures. The results show that the interaction surfaces are smoother and the helix axis separations are closer in the amino-terminal two-thirds of the proteins compared with the carboxyl terminal one-third. However, the F helix is important in stabilizing the folded structure, as shown by the instability of the 1-166 fragment. Considering the high resolution crystal structure of bacteriorhodopsin, there are no obvious helix-helix interactions involving protein side chains which would be destabilized by protonation at the estimated pH of the unfolding transitions. However, a number of helix-bridging water molecules could become protonated, thereby weakening the helixhelix interactions.In comparison with water-soluble proteins, integral membrane protein folding mechanisms are poorly understood. Results of several folding studies, both in vivo (1) and in vitro (2-6), have been published on membrane proteins containing transmembrane helices. In some in vitro experiments, the membrane proteins were solubilized in detergent micelles, and unfolding was induced by an anionic amphiphile perturbant, docecyl sulfate. In order to obtain free energies of unfolding, it is necessary to assume that the free energy changes are linear with perturbant concentration (7). Due to non-ideal mixing when charged amphiphiles are added to neutral micelles, the assumption of linearity does not necessarily hold (8). An alternative approach would be to examine unfolding in a single phase solvent system. We have been studying the † Supported by a grant from the National Institutes of Health (GM 08194) *To whom to address correspondence at Dept. unfolding equilibrium of bacterio-opsin (BO) 1 in ethanol/water mixtures. We found an apparent unfolding transition in...

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Structure of the Light-Driven Chloride Pump Halorhodopsin at 1.8 Å Resolution

Cited by 525 publications

References 42 publications

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

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

Halorhodopsin pumps Cl^–and bacteriorhodopsin pumps protons by a common mechanism that uses conserved electrostatic interactions

Transmembrane Helix−Helix Association: Relative Stabilities at Low pH

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Structure of the Light-Driven Chloride Pump Halorhodopsin at 1.8 Å Resolution

Cited by 525 publications

References 42 publications

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

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

Halorhodopsin pumps Cl–and bacteriorhodopsin pumps protons by a common mechanism that uses conserved electrostatic interactions

Transmembrane Helix−Helix Association: Relative Stabilities at Low pH

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Product

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Halorhodopsin pumps Cl^–and bacteriorhodopsin pumps protons by a common mechanism that uses conserved electrostatic interactions