Helix movement is coupled to displacement of the second extracellular loop in rhodopsin activation

Ahuja, Shivani; Horn̆ák, Viktor; Yan, Elsa C. Y.; Syrett, Natalie; Goncalves, Joseph A.; Hirshfeld, Amiram; Ziliox, Martine; Sakmar, Thomas P.; Sheves, Mordechai; Reeves, Philip J.; Smith, Steven O.; Eilers, Markus

doi:10.1038/nsmb.1549

Cited by 203 publications

(329 citation statements)

References 60 publications

(87 reference statements)

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“…It should be noted that position 4.66 is located at the junction between TMD4 and the second extracellular loop of AT 1 . Interestingly, the second extracellular loop of rhodopsin has recently been shown to move away from the binding pocket following activation, highlighting a possible role for this ECL in the GPCR activation process (49). However, for TMD1, we detected no such divergence in sensitivities between both receptor backgrounds, which indicates no major structural changes of TMD1 during activation.…”

Section: Discussioncontrasting

confidence: 41%

Analysis of Transmembrane Domains 1 and 4 of the Human Angiotensin II AT1 Receptor by Cysteine-scanning Mutagenesis

Yan

Holleran

Lavigne³

et al. 2010

Journal of Biological Chemistry

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The octapeptide hormone angiotensin II (AngII) exerts a wide variety of cardiovascular effects through the activation of the AT 1 receptor, which belongs to the G protein-coupled receptor superfamily. Like other G protein-coupled receptors, the AT 1 receptor possesses seven transmembrane domains that provide structural support for the formation of the ligand-binding pocket. Here, we investigated the role of the first and fourth transmembrane domains (TMDs) in the formation of the binding pocket of the human AT 1 receptor using the substituted-cysteine accessibility method. Each residue within the Phe-28( The octapeptide hormone angiotensin II (AngII) 4 is the active component of the renin-angiotensin system. It exerts a wide variety of physiological effects, including vascular contraction, aldosterone secretion, neuronal activation, and cardiovascular cell growth and proliferation (1, 2). Virtually all the known physiological effects of AngII are produced through the activation of the AT 1 receptor, which belongs to the G proteincoupled receptor (GPCR) superfamily (3, 4). The AT 1 receptor belongs to the rhodopsin-like family A of G protein-coupled receptors, which have a seven-transmembrane helix structure, an extracellular N-terminal tail, an intracellular C-terminal domain, and three extracellular and three intracellular loops.The seven transmembrane domains (TMDs) of GPCRs constitute structural support for signal transduction. Like other family A GPCRs such as rhodopsin and adrenergic receptors, the AT 1 receptor undergoes spontaneous isomerization between its inactive state and its active state (5). Movement of TMD helices through translational or rotational displacement is believed to be essential to achieve the active state (6 -8). These conformational changes would sustain GTP/GDP exchange on specific guanine nucleotide-binding proteins (G proteins) leading to activation of intracellular signaling cascades (5). For the AT 1 receptor, it has been proposed that TMD3, TMD5, TMD6, and TMD7 may participate in the activation process by providing a network of interactions throughout the AngII-binding pocket (9). The dynamics of this network would require that, following agonist binding, novel or existing interactions between the TMDs would either be created or broken, respectively.Based on homology with the recent high resolution structures of the ␤1 adrenergic, ␤2 adrenergic, and A 2A adenosine receptors (10 -12) it was expected that the binding site of the AT 1 receptor would be formed between its seven, mostly hydrophobic transmembrane domains and would be accessible to charged water-soluble agonists, like AngII. For this receptor, the binding site would thus be contained within a hydrophilic crevice, the binding pocket, extending from the extracellular surface of the receptor to the transmembrane portions. Because all crystallized structures to date suggest that TMD1 and TMD4 are somewhat removed from the binding pocket (13) The substituted-cysteine accessibility method (SCAM) (20 -22) is an ingenious ap...

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Section: Discussioncontrasting

confidence: 41%

Analysis of Transmembrane Domains 1 and 4 of the Human Angiotensin II AT1 Receptor by Cysteine-scanning Mutagenesis

Yan

Holleran

Lavigne³

et al. 2010

Journal of Biological Chemistry

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“…5). A key hydrogen-bonding network is disrupted about E2 connecting the extracellular ends of helices H4, H5, and H6 (26). At the opposite end of retinal, the C5-Me group of the β-ionone ring stays in close proximity to Glu 122 of TM helix H3 and Trp 265 of helix H6.…”

Section: Changes In Local Retinal Structure and Dynamics Initiate Colmentioning

confidence: 99%

“…However, the lightactivated Meta II state is transient and crystal deformation occurs giving low-resolution data (14). Crystal structures of ligand-free opsin (19) with a bound synthetic G t peptide (20) show an elongation of the protein (21, 22) due to helical displacements (19)-but opsin does not contain the activating all-trans-retinal ligand, and its activity does not match rhodopsin (20).Spectroscopic approaches including spin-label EPR (23), 13 C NMR (18,(24)(25)(26), and Fourier transform infrared (FTIR) (17, 27) studies are thus needed to establish the activation mechanism of the photoreceptor as it underlies the visual process. Solid-state NMR spectroscopy (28) is particularly important, as it gives knowledge of both protein structure and dynamics in a membrane lipid environment (29).…”

mentioning

confidence: 99%

“…Spectroscopic approaches including spin-label EPR (23), 13 C NMR (18,(24)(25)(26), and Fourier transform infrared (FTIR) (17, 27) studies are thus needed to establish the activation mechanism of the photoreceptor as it underlies the visual process. Solid-state NMR spectroscopy (28) is particularly important, as it gives knowledge of both protein structure and dynamics in a membrane lipid environment (29).…”

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confidence: 99%

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Solid-state ² H NMR relaxation illuminates functional dynamics of retinal cofactor in membrane activation of rhodopsin

Struts

Salgado

Brown³

2011

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

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Rhodopsin is a canonical member of the family of G proteincoupled receptors, which transmit signals across cellular membranes and are linked to many drug interventions in humans. Here we show that solid-state 2 H NMR relaxation allows investigation of light-induced changes in local ps-ns time scale motions of retinal bound to rhodopsin. Site-specific 2 H labels were introduced into methyl groups of the retinal ligand that are essential to the activation process. We conducted solid-state 2 H NMR relaxation (spinlattice, T 1Z , and quadrupolar-order, T 1Q ) experiments in the dark, Meta I, and Meta II states of the photoreceptor. Surprisingly, we find the retinylidene methyl groups exhibit site-specific differences in dynamics that change upon light excitation-even more striking, the C9-methyl group is a dynamical hotspot that corresponds to a crucial functional hotspot of rhodopsin. Following 11-cis to trans isomerization, the 2 H NMR data suggest the β-ionone ring remains in its hydrophobic binding pocket in all three states of the protein.We propose a multiscale activation mechanism with a complex energy landscape, whereby the photonic energy is directed against the E2 loop by the C13-methyl group, and toward helices H3 and H5 by the C5-methyl of the β-ionone ring. Changes in retinal structure and dynamics initiate activating fluctuations of transmembrane helices H5 and H6 in the Meta I-Meta II equilibrium of rhodopsin. Our proposals challenge the Standard Model whereby a single light-activated receptor conformation yields the visual response -rather an ensemble of substates is present, due to the entropy gain produced by photolysis of the inhibitory retinal lock.GPCR | solid-state NMR | generalized model-free analysis G protein-coupled receptors (GPCRs) (1-3) are the largest family of integral membrane proteins in the human genome, and they are the targets of about 30% of all human pharmaceuticals. At present the 3D structures of several GPCRs-including rhodopsin (4-8), the β 1 -and β 2 -adrenergic receptors (9, 10), and the adenosine A 2A receptor (11)-have been established in various functional states (2). Yet the mechanisms of GPCR activation remain elusive, as they are membrane-embedded molecules that are often recalcitrant to crystallization, and push the size limits of solution NMR spectroscopy. Rhodopsin is the quintessential prototype for Family A GPCRs, because previous X-ray (5,6) and electron diffraction (12) studies have yielded crystal structures of its dark state (4-6) and several photointermediates (13,14). Notably, the retinal ligand is buried deeply within a 7-transmembrane (TM) helical bundle, where it locks the receptor in the inactive state, with negligible basal (constitutive) activity (4). Upon light absorption, rhodopsin catalyzes the rapid and highly selective 11-cis to trans isomerization of retinal (15, 16) switching it from an inverse agonist to an agonist. In the Metarhodopsin I-Metarhodopsin II equilibrium, recognition sites are exposed for a heterotrimeric G protein (transducin or...

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“…High-resolution distance mapping with SDSL and double electron-electron resonance (DEER) in DDM micelles revealed the directions and magnitudes of the motions (19), and the structural changes were subsequently confirmed by crystal structures of the inactive and active states (20,21). At the cytoplasmic surface, crystal structures (21), solid-state NMR (22), SDSL (17), and infrared spectroscopy (23) found additional movements involving TM5. In the SDSL-EPR studies, TM5 motion was detected at site 231 in the aqueous extension of TM5, but not at site 225 in the bilayer domain (17); this is a site used in the present studies.…”

mentioning

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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Helix movement is coupled to displacement of the second extracellular loop in rhodopsin activation

Cited by 203 publications

References 60 publications

Analysis of Transmembrane Domains 1 and 4 of the Human Angiotensin II AT1 Receptor by Cysteine-scanning Mutagenesis

Analysis of Transmembrane Domains 1 and 4 of the Human Angiotensin II AT1 Receptor by Cysteine-scanning Mutagenesis

Solid-state ² H NMR relaxation illuminates functional dynamics of retinal cofactor in membrane activation of rhodopsin

Conformational equilibria of light-activated rhodopsin in nanodiscs

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Helix movement is coupled to displacement of the second extracellular loop in rhodopsin activation

Cited by 203 publications

References 60 publications

Analysis of Transmembrane Domains 1 and 4 of the Human Angiotensin II AT1 Receptor by Cysteine-scanning Mutagenesis

Analysis of Transmembrane Domains 1 and 4 of the Human Angiotensin II AT1 Receptor by Cysteine-scanning Mutagenesis

Solid-state 2 H NMR relaxation illuminates functional dynamics of retinal cofactor in membrane activation of rhodopsin

Conformational equilibria of light-activated rhodopsin in nanodiscs

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Solid-state ² H NMR relaxation illuminates functional dynamics of retinal cofactor in membrane activation of rhodopsin