Rhodopsin Activation Switches in a Native Membrane Environment<sup>†</sup>

Lüdeke, Steffen; Mahalingam, Mohana; Vogel, Reiner

doi:10.1111/j.1751-1097.2008.00490.x

Cited by 21 publications

(29 citation statements)

References 34 publications

(51 reference statements)

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“…2 B and C) suggests that protonation also may regulate GPCR structure and function. Although speculative, this idea is consistent with evidence that light-activated (3,(33)(34)(35) and ligand-activated (36-38) GPCR complexes are regulated directly by pH.…”

Section: Significancesupporting

confidence: 69%

Buried ionizable networks are an ancient hallmark of G protein-coupled receptor activation

Isom¹,

Dohlman

2015

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

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Seven-transmembrane receptors (7TMRs) have evolved in prokaryotes and eukaryotes over hundreds of millions of years. Comparative structural analysis suggests that these receptors may share a remote evolutionary origin, despite their lack of sequence similarity. Here we used structure-based computations to compare 221 7TMRs from all domains of life. Unexpectedly, we discovered that these receptors contain spatially conserved networks of buried ionizable groups. In microbial 7TMRs these networks are used to pump ions across the cell membrane in response to light. In animal 7TMRs, which include light-and ligand-activated G protein-coupled receptors (GPCRs), homologous networks were found to be characteristic of activated receptor conformations. These networks are likely relevant to receptor function because they connect the ligandbinding pocket of the receptor to the nucleotide-binding pocket of the G protein. We propose that agonist and G protein binding facilitate the formation of these electrostatic networks and promote important structural rearrangements such as the displacement of transmembrane helix-6. We anticipate that robust classification of activated GPCR structures will aid the identification of ligands that target activated GPCR structural states.7-transmembrane receptor | G protein-coupled receptor | buried charge | structural bioinformatics | molecular evolution S even-transmembrane receptors (7TMRs) are present in all domains of life. In archaea and bacteria these receptors convert light energy into transmembrane ion gradients or intracellular signaling cascades. In humans 7TMRs comprise a family of more than 800 G protein-coupled receptors (GPCRs) that detect extracellular signals such as odorants, taste, light, hormones, and neurotransmitters. Although microbial and eukaryotic 7TMRs lack sequence similarity, their 3D folds share a degree of similarity that often is observed in remote evolutionary relationships identified by structure comparison algorithms (1).Recent breakthroughs in membrane protein crystallography have advanced our understanding of GPCR function by providing models of unactivated and activated receptor conformations (2-8). However, structure-based approaches for systematically quantifying differences between GPCR activation states are lacking. Here we introduce a computational approach that correlates GPCR activation with networks of electrostatic interactions in the receptor core. We show that membrane-spanning networks of ionizable residues, which we find are a common feature of microbial 7TMRs, also represent a unique signature of GPCR activation that is likely to be essential to receptor function.The molecular changes that accompany 7TMR activation were studied initially in bacteriorhodopsin and rhodopsin, the first microbial 7TMR and GPCR to be crystallized (9-11). More recently, a rapid expansion of GPCR structural information has revealed several molecular switches and structural features that are thought to be indicative of receptor activation. These include the ionic loc...

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Section: Significancesupporting

confidence: 69%

Buried ionizable networks are an ancient hallmark of G protein-coupled receptor activation

Isom¹,

Dohlman

2015

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

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“…2B shows the corresponding UV-visible absorption spectra that document the nearly quantitative conversion of the inactive state (500 nm absorbance) to the MII species (380 nm). In DDM and in native disk membranes at pH 6.0, the functionally active MIIbH + species is essentially quantitatively populated (8,35,36).…”

Section: Resultsmentioning

confidence: 99%

“…The situation in nanodiscs is strikingly different. At pH 6.0, where MIIbH + is reported to be the dominant population in a membrane environment (8,35,36), the distance distribution for TM6 has three resolved components of comparable populations at positions corresponding to the populations in the inactive state in both DDM and nanodiscs. The populations at 29 Å and 34 Å apparently correspond to the inactive and active state crystal structures.…”

Section: Resultsmentioning

confidence: 99%

Conformational equilibria of light-activated rhodopsin in nanodiscs

Eps

Lydia

Morizumi

et al. 2017

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

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Conformational equilibria of G-protein-coupled receptors (GPCRs) are intimately involved in intracellular signaling. Here conformational substates of the GPCR rhodopsin are investigated in micelles of dodecyl maltoside (DDM) and in phospholipid nanodiscs by monitoring the spatial positions of transmembrane helices 6 and 7 at the cytoplasmic surface using site-directed spin labeling and double electron-electron resonance spectroscopy. The photoactivated receptor in DDM is dominated by one conformation with weak pH dependence. In nanodiscs, however, an ensemble of pH-dependent conformational substates is observed, even at pH 6.0 where the MIIbH + form defined by proton uptake and optical spectroscopic methods is reported to be the sole species present in native disk membranes. In nanodiscs, the ensemble of substates in the photoactivated receptor spontaneously decays to that characteristic of the inactive state with a lifetime of ∼16 min at 20°C. Importantly, transducin binding to the activated receptor selects a subset of the ensemble in which multiple substates are apparently retained. The results indicate that in a native-like lipid environment rhodopsin activation is not analogous to a simple binary switch between two defined conformations, but the activated receptor is in equilibrium between multiple conformers that in principle could recognize different binding partners.G -protein-coupled receptors (GPCRs) are central components of protein-protein interaction networks and can serve as hubs that link the extracellular environment of cells to intracellular signaling events (1). As such, they interact with several intracellular proteins (i.e., arrestins, G proteins, kinases). To obtain such diversity in molecular interaction, GPCRs are thought to be in equilibrium between multiple conformations at the cytoplasmic surface (2-4), enabling conformation-dependent interactions with different partners.The rod photoreceptor rhodopsin has served as a model for members in the class A family of GPCRs. In native membranes (5-7) and reconstituted liposomes (8, 9), photoactivated rhodopsin within milliseconds reaches a pH-dependent quasi-equilibrium between states designated MI and MII, which are distinguished by their optical absorbance maxima and signature absorbances in the infrared (10-12). The optically identified MII state has been found to consist of isochromic substates, namely MIIa, MIIb, and MIIbH + (13-15) (Fig. 1A). MIIbH + , populated at pH 6.0, is thought to be the functionally active substate that recognizes the cognate G-protein transducin (8,15,16).Early data relating global protein structural changes to activation in a GPCR were provided by continuous wave (CW) sitedirected spin labeling (SDSL)-Electron Paramagnetic Resonance (EPR) studies in rhodopsin, where it was shown that a generally outward motion of TM6 and a smaller inward motion of TM7 at the cytoplasmic surface were hallmarks of activation in dodecyl maltoside (DDM) micelles (17, 18). High-resolution distance mapping with SDSL and double electron...

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“…The MII b state takes up a proton at Glu-134 (14) in the class-conserved D(E)RY motif at the C-terminal end of helix-3 (H3) leading to the MII b H ϩ intermediate (15,16), which activates transducin (G t ), the G protein of the photoreceptor cell. Glu-134 regulates the pH sensitivity of receptor signaling (17) in membranes as reviewed previously (18), and in complex with G t the protonated state of the carboxyl group becomes stabilized (19). This charge alteration is linked to the release of an "ionic lock," originally described for the ␤ 2 -adrenergic receptor (20), which also in rhodopsin stabilizes the inactive state (16) through interactions between the cytosolic ends of H3 and H6 (21).…”

Section: G Protein-coupled Receptors (Gpcrs)mentioning

confidence: 99%

Lipid Protein Interactions Couple Protonation to Conformation in a Conserved Cytosolic Domain of G Protein-coupled Receptors

Madathil

Fahmy

2009

Journal of Biological Chemistry

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The visual photoreceptor rhodopsin is a prototypical class I (rhodopsin-like) G protein-coupled receptor. Photoisomerization of the covalently bound ligand 11-cis-retinal leads to restructuring of the cytosolic face of rhodopsin. The ensuing protonation of Glu-134 in the class-conserved D(E)RY motif at the C-terminal end of transmembrane helix-3 promotes the formation of the G protein-activating state. Using transmembrane segments derived from helix-3 of bovine rhodopsin, we show that lipid protein interactions play a key role in this cytosolic "proton switch." Infrared and fluorescence spectroscopic pK a determinations reveal that the D(E)RY motif is an autonomous functional module coupling side chain neutralization to conformation and helix positioning as evidenced by side chain to lipid headgroup Foerster resonance energy transfer. The free enthalpies of helix stabilization and hydrophobic burial of the neutral carboxyl shift the side chain pK a into the range typical of Glu-134 in photoactivated rhodopsin. The lipid-mediated coupling mechanism is independent of interhelical contacts allowing its conservation without interference with the diversity of ligandspecific interactions in class I G protein-coupled receptors. G protein-coupled receptors (GPCRs)2 are hepta-helical membrane proteins that couple a large variety of extracellular signals to cell-specific responses via activation of G proteins. In the visual photoreceptor rhodopsin, a prototypical class I GPCR (1, 2), molecular activation processes can be monitored in real time by spectroscopic assays and analyzed in the context of several crystal structures (3-8). The primary signal for rhodopsin is the 11-cis to all-trans photoisomerization of retinal covalently bound to the apoprotein opsin through a protonated Schiff base to Lys 296 . Current models converge toward a picture in which "microdomains" act as conformational switches that are coupled to different degrees to the primary activation process. Two activating "proton switches" have been identified (9) as follows: breakage of an intramolecular salt bridge (10) by transfer of the Schiff base proton to its counter ion Glu-113 (11) is followed by movement of helix-6 (H6) (12, 13) in the metarhodopsin II a (MII a ) to MII b transition. The MII b state takes up a proton at in the class-conserved D(E)RY motif at the C-terminal end of helix-3 (H3) leading to the MII b H ϩ intermediate (15, 16), which activates transducin (G t ), the G protein of the photoreceptor cell. Glu-134 regulates the pH sensitivity of receptor signaling (17) in membranes as reviewed previously (18), and in complex with G t the protonated state of the carboxyl group becomes stabilized (19). This charge alteration is linked to the release of an "ionic lock," originally described for the ␤ 2 -adrenergic receptor (20), which also in rhodopsin stabilizes the inactive state (16) through interactions between the cytosolic ends of H3 and H6 (21).In the absence of a lipidic bilayer, proton uptake and H6 movement become uncoupled (15). Lip...

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Rhodopsin Activation Switches in a Native Membrane Environment^†

Cited by 21 publications

References 34 publications

Buried ionizable networks are an ancient hallmark of G protein-coupled receptor activation

Buried ionizable networks are an ancient hallmark of G protein-coupled receptor activation

Conformational equilibria of light-activated rhodopsin in nanodiscs

Lipid Protein Interactions Couple Protonation to Conformation in a Conserved Cytosolic Domain of G Protein-coupled Receptors

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Rhodopsin Activation Switches in a Native Membrane Environment†

Cited by 21 publications

References 34 publications

Buried ionizable networks are an ancient hallmark of G protein-coupled receptor activation

Buried ionizable networks are an ancient hallmark of G protein-coupled receptor activation

Conformational equilibria of light-activated rhodopsin in nanodiscs

Lipid Protein Interactions Couple Protonation to Conformation in a Conserved Cytosolic Domain of G Protein-coupled Receptors

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Rhodopsin Activation Switches in a Native Membrane Environment^†