Chromophore channeling in the G-protein coupled receptor rhodopsin.
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ABSTRACT:
SUBMITTER: Wang T
PROVIDER: S-EPMC2819262 | biostudies-literature | 2007 Jun
REPOSITORIES: biostudies-literature
Publications
Chromophore channeling in the G-protein coupled receptor rhodopsin.
Journal of the American Chemical Society 20070515 22
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Project description:The rhodopsin crystal structure provides a structural basis for understanding the function of this and other G protein-coupled receptors (GPCRs). The major structural motifs observed for rhodopsin are expected to carry over to other GPCRs, and the mechanism of transformation of the receptor from inactive to active forms is thus likely conserved. Moreover, the high expression level of rhodopsin in the retina, its specific localization in the internal disks of the photoreceptor structures [termed rod outer segments (ROS)], and the lack of other highly abundant membrane proteins allow rhodopsin to be examined in the native disk membranes by a number of methods. The results of these investigations provide evidence of the propensity of rhodopsin and, most likely, other GPCRs to dimerize, a property that may be pertinent to their function.
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Project description:Rhodopsin is a retinal photoreceptor protein of bipartite structure consisting of the transmembrane protein opsin and a light-sensitive chromophore 11-cis-retinal, linked to opsin via a protonated Schiff base. Studies on rhodopsin have unveiled many structural and functional features that are common to a large and pharmacologically important group of proteins from the G protein-coupled receptor (GPCR) superfamily, of which rhodopsin is the best-studied member. In this work, we focus on structural features of rhodopsin as revealed by many biochemical and structural investigations. In particular, the high-resolution structure of bovine rhodopsin provides a template for understanding how GPCRs work. We describe the sensitivity and complexity of rhodopsin that lead to its important role in vision.
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Project description:G protein-coupled receptor kinases (GRKs) play pivotal roles in desensitizing GPCR signaling but little is known about how GRKs recognize and phosphorylate GPCRs due to the technical difficulties in detecting the highly dynamic GPCR/GRK interaction. By combining a genetic approach with multiple biochemical assays, we identified the key determinants for the assembly of the prototypical GPCR rhodopsin with its kinase GRK1. Our work reveals that the regulatory G-protein signaling homology (RH) domain of GRKs is the primary binding site to GPCRs and an active conformation of the GRK1 kinase domain is required for efficient interaction with rhodopsin. In addition, we provide a mechanistic solution for the longstanding puzzle about the gain-of-function Q41L mutation in GRK5. This mutation is in the RH domain and increases the capacity of the GRK mutant to interact with and to desensitize GPCRs. Finally we present the principal architecture of a rhodopsin/GRK complex through negative stain electron microscopy reconstruction. Together, these data define the key components for the rhodopsin/GRK1 interaction and provide a framework for understanding GRK-mediated desensitization of GPCRs.
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Project description:G-protein-coupled receptor (GPCR) kinases (GRKs) selectively phosphorylate activated GPCRs, thereby priming them for desensitization1. Although it is unclear how GRKs recognize these receptors2-4, a conserved region at the GRK N terminus is essential for this process5-8. Here we report a series of cryo-electron microscopy single-particle reconstructions of light-activated rhodopsin (Rho*) bound to rhodopsin kinase (GRK1), wherein the N terminus of GRK1 forms a helix that docks into the open cytoplasmic cleft of Rho*. The helix also packs against the GRK1 kinase domain and stabilizes it in an active configuration. The complex is further stabilized by electrostatic interactions between basic residues that are conserved in most GPCRs and acidic residues that are conserved in GRKs. We did not observe any density for the regulator of G-protein signalling homology domain of GRK1 or the C terminus of rhodopsin. Crosslinking with mass spectrometry analysis confirmed these results and revealed dynamic behaviour in receptor-bound GRK1 that would allow the phosphorylation of multiple sites in the receptor tail. We have identified GRK1 residues whose mutation augments kinase activity and crosslinking with Rho*, as well as residues that are involved in activation by acidic phospholipids. From these data, we present a general model for how a small family of protein kinases can recognize and be activated by hundreds of different GPCRs.
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Project description:Rhodopsin, the membrane protein responsible for dim-light vision, until recently was the only G-protein-coupled receptor (GPCR) with a known crystal structure. As a result, there is enormous interest in studying its structure, dynamics, and function. Here we report the results of three all-atom molecular dynamics simulations, each at least 1.5 micros, which predict that substantial changes in internal hydration play a functional role in rhodopsin activation. We confirm with (1)H magic angle spinning NMR that the increased hydration is specific to the metarhodopsin-I intermediate. The internal water molecules interact with several conserved residues, suggesting that changes in internal hydration may be important during the activation of other GPCRs. The results serve to illustrate the synergism of long-time-scale molecular dynamics simulations and NMR in enhancing our understanding of GPCR function.
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Project description:G protein-coupled receptors are cell-surface seven-helical membrane proteins that undergo conformational changes on activation. The mammalian photoreceptor, rhodopsin, is the best-studied member of this superfamily. Here, we provide the first evidence that activation in rhodopsin may involve differential dynamic properties of side-chain versus backbone atoms. High-resolution NMR studies of alpha-(15)N-labeled receptor revealed large backbone motions in the inactive dark state. In contrast, indole side-chain (15)N groups of tryptophans showed well resolved, equally intense NMR signals, suggesting restriction to a single specific conformation.
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Project description:Membrane protein stability is a key parameter with important physiological and practical implications. Inorganic salts affect protein stability, but the mechanisms of their interactions with membrane proteins are not completely understood. We have undertaken the study of a prototypical G-protein-coupled receptor, the α-helical membrane protein rhodopsin from vertebrate retina, and explored the effects of inorganic salts on the thermal decay properties of both its inactive and photoactivated states. Under high salt concentrations, rhodopsin significantly increased its activation enthalpy change for thermal bleaching, whereas acid denaturation affected the formation of a denatured loose-bundle state for both the active and inactive conformations. This behavior seems to correlate with changes in protonated Schiff-base hydrolysis. However, chromophore regeneration with the 11-cis-retinal chromophore and MetarhodopsinII decay kinetics were slower only in the presence of sodium chloride, suggesting that in this case, the underlying phenomenon may be linked to the activation of rhodopsin and the retinal release processes. Furthermore, the melting temperature, determined by means of circular dichroism and differential scanning calorimetry measurements, was increased in the presence of high salt concentrations. The observed effects on rhodopsin could indicate that salts favor electrostatic interactions in the retinal binding pocket and indirectly favor hydrophobic interactions at the membrane protein receptor core. These effects can be exploited in applications where the stability of membrane proteins in solution is highly desirable.
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Project description:The photoreceptor rhodopsin (Rho) becomes active when a tethered inverse agonist ligand (11CR) is photoconverted to an agonist (ATR). The ligand-binding pocket of inactive rhodopsin is completely enclosed, whereas active rhodopsin displays pores accessible from the lipid bilayer. Stabilization of active rhodopsin impedes 11CR binding and photoreceptor dark adaptation. Here, we used genetic code expansion and bioorthogonal labeling to engineer Rho mutants that serve as FRET sensors for measuring 11CR binding kinetics and energetics. We found that mutations that alter a channel between transmembrane helices 5 and 6 (TM5/6) dramatically affect 11CR binding kinetics but not agonist release kinetics. Our data provide direct experimental evidence for 11CR entry between TM5/6 in Rho that involves dynamic allosteric control of the ligand entry channel. Our findings provide a conceptual framework for understanding the function of G protein-coupled receptors with hydrophobic ligands that are hypothesized to enter their binding pockets through transmembrane pores.
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Project description:Rhodopsin is the only member of the pharmacologically important superfamily of G-protein-coupled receptors with a known structure at atomic resolution. A molecular dynamics model of rhodopsin in a POPC phospholipid bilayer was simulated for 15 ns, revealing a conformation significantly different from the recent crystal structures. The structure of the bilayer compared with a protein-free POPC control indicated hydrophobic matching with the nonpolar interface of the receptor, in agreement with deuterium NMR experiments. A new generalized molecular surface method, based on a three-dimensional Voronoi cell construction for atoms with different radii, was developed to quantify cross-sectional area profiles for the protein, lipid acyl chains and headgroups, and water. Thus, it was possible to investigate the bilayer deformation due to curvature of the individual lipid monolayers. Moreover, the generalized molecular surface derived hydrophobic interface allowed benchmarking of the hydropathy sequence analysis, an important structural genomics tool. Five water molecules diffused into internal hydration sites during the simulation, yielding a total of 12 internal waters. The cytoplasmic loops and the C-terminal tail, containing the G-protein recognition and protein sorting sequences, exhibited a high mobility, in marked contrast to the extracellular and transmembrane domains. The proposed functional coupling of the highly conserved ERY motif to the lipid-water interface via the cytoplasmic loops provides insight into lipid effects on G-protein-coupled receptor activation in terms of a flexible surface model, involving the spontaneous monolayer curvature.
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Project description:Deactivation of light-activated rhodopsin (metarhodopsin II) involves, after rhodopsin kinase and arrestin interactions, the hydrolysis of the covalent bond of all-trans-retinal to the apoprotein. Although the long-lived storage form metarhodopsin III is transiently formed, all-trans-retinal is eventually released from the active site. Here we address the question of whether the release results in a retinal that is freely diffusible in the lipid phase of the photoreceptor membrane. The release reaction is accompanied by an increase in intrinsic protein fluorescence (release signal), which arises from the relief of the fluorescence quenching imposed by the retinal in the active site. An analogous fluorescence decrease (uptake signal) was evoked by exogenous retinoids when they non-covalently bound to native opsin membranes. Uptake of 11-cis-retinal was faster than formation of the retinylidene linkage to the apoprotein. Endogenous all-trans-retinal released from the active site during metarhodopsin II decay did not generate the uptake signal. The data show that in addition to the retinylidene pocket (site I) there are two other retinoidbinding sites within opsin. Site II involved in the uptake signal is an entrance site, while the exit site (site III) is occupied when retinal remains bound after its release from site I. Support for a retinal channeling mechanism comes from the rhodopsin crystal structure, which unveiled two putative hydrophobic binding sites. This mechanism enables a unidirectional process for the release of photoisomerized chromophore and the uptake of newly synthesized 11-cis-retinal for the regeneration of rhodopsin.
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