Structure and activation of rhodopsin.
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ABSTRACT:
SUBMITTER: Zhou XE
PROVIDER: S-EPMC3677203 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Rhodopsin has served as the primary model for studying G protein-coupled receptors (GPCRs)-the largest group in the human genome, and consequently a primary target for pharmaceutical development. Understanding the functions and activation mechanisms of GPCRs has proven to be extraordinarily difficult, as they are part of a complex signaling cascade and reside within the cell membrane. Although X-ray crystallography has recently solved several GPCR structures that may resemble the activated conformation, the dynamics and mechanism of rhodopsin activation continue to remain elusive. Notably solid-state ((2))H NMR spectroscopy provides key information pertinent to how local dynamics of the retinal ligand change during rhodopsin activation. When combined with molecular mechanics simulations of proteolipid membranes, a new paradigm for the rhodopsin activation process emerges. Experiment and simulation both suggest that retinal isomerization initiates the rhodopsin photocascade to yield not a single activated structure, but rather an ensemble of activated conformational states. This article is part of a Special Issue entitled: Membrane protein structure and function.
| S-EPMC5270601 | biostudies-literature
Project description:Activation of class-A G-protein-coupled receptors (GPCRs) involves large-scale reorganization of the H3/H6 interhelical network. In rhodopsin (Rh), this process is coupled to a change in the protonation state of a key residue, E134, whose exact role in activation is not well understood. Capturing this millisecond pH-dependent process is a well-appreciated challenge. We have developed a scheme combining the harmonic Fourier beads (HFB) method and constant-pH molecular dynamics with pH-based replica exchange (pH-REX) to gain insight into the structural changes that occur along the activation pathway as a function of the protonation state of E134. Our results indicate that E134 is protonated as a consequence of tilting of H6 by ca. 4.0° with respect to its initial position and simultaneous rotation by ca. 23° along its principal axis. The movement of H6 is associated with breakage of the E247-R135 and R135-E134 salt bridges and concomitant release of the E134 side chain, which results in an increase in its pKa value above physiological pH. An increase in the hydrophobicity of the environment surrounding E134 leads to further tilting and rotation of H6 and upshift of the E134 pKa. Such atomic-level information, which is not accessible through experiments, refines the earlier proposed sequential model of Rh activation (see: Zaitseva, E.; et al. Sequential Rearrangement of Interhelical Networks Upon Rhodopsin Activation in Membranes: The Meta IIa Conformational Substate . J. Am. Chem. Soc. 2010, 132, 4815) and argues that the E134 protonation switch is both a cause and a consequence of the H6 motion.
| S-EPMC3774788 | biostudies-literature
Project description:Rhodopsin is a well-characterized structural model of a G protein-coupled receptor. Photoisomerization of the covalently bound retinal triggers activation. Surprisingly, the x-ray crystal structure of the active Meta-II state has a 180° rotation about the long-axis of the retinal polyene chain. Unbiased microsecond-timescale all-atom molecular dynamics simulations show that the retinal cofactor can flip back to the orientation observed in the inactive state of rhodopsin under conditions favoring the Meta-I state. Our results provide, to our knowledge, the first evidence from molecular dynamics simulations showing how rotation of the retinal ligand within its binding pocket can occur in the activation mechanism of rhodopsin.
| S-EPMC4472225 | biostudies-literature
Project description:Rhodopsin, the photoreceptor of rod cells, absorbs light to mediate the first step of vision by activating the G protein transducin (Gt). Several human diseases, such as retinitis pigmentosa or congenital night blindness, are linked to rhodopsin malfunctions. Most of the corresponding in vivo studies and structure-function analyses (e.g. based on protein x-ray crystallography or spectroscopy) have been carried out on murine or bovine rhodopsin. Because these rhodopsins differ at several amino acid positions from human rhodopsin, we conducted a comprehensive spectroscopic characterization of human rhodopsin in combination with molecular dynamics simulations. We show by FTIR and UV-visible difference spectroscopy that the light-induced transformations of the early photointermediates are very similar. Significant differences between the pigments appear with formation of the still inactive Meta I state and the transition to active Meta II. However, the conformation of Meta II and its activity toward the G protein are essentially the same, presumably reflecting the evolutionary pressure under which the active state has developed. Altogether, our results show that although the basic activation pathways of human and bovine rhodopsin are similar, structural deviations exist in the inactive conformation and during receptor activation, even between closely related rhodopsins. These differences between the well studied bovine or murine rhodopsins and human rhodopsin have to be taken into account when the influence of point mutations on the activation pathway of human rhodopsin are investigated using the bovine or murine rhodopsin template sequences.
| S-EPMC4536423 | biostudies-literature
Project description:The present study confirms our original assertion that peptides corresponding to the C-terminal sequence of rhodopsin are phosphorylated by rhodopsin kinase (RK), but only in the presence of photo-activated rhodopsin [Rho*, which is functionally equivalent to metarhodopsin II (Meta II)]. Under optimized conditions, the extent of peptide phosphorylation reached up to 60% that of Rho*. Rho* phosphorylation began to plateau within 15 min of the initiation of photolysis, whereas the peptide phosphorylation continued linearly for >60 min. This lack of co-ordination in the phosphorylation of the physiological (Rho*) and synthetic (peptide) substrates necessitated the present study, which showed that RK is activated for peptide phosphorylation not only by Meta II but also by Meta III, as well as by the phosphorylated derivatives of these species, but not by opsin. These results led to the conclusion that all the derivatives of opsin, which contain the Schiff base linkage with the all-trans-retinylidene moiety, retain the ability to activate RK.
| S-EPMC1222487 | biostudies-other
Project description:Conserved prolines in the transmembrane helices of G-protein-coupled receptors (GPCRs) are often considered to function as hinges that divide the helix into two segments capable of independent motion. Depending on their potential to hydrogen-bond, the free C=O groups associated with these prolines can facilitate conformational flexibility, conformational switching or stabilization of the receptor structure. To address the role of conserved prolines in family A GPCRs through solid-state NMR spectroscopy, we focus on bovine rhodopsin, a GPCR in the visual receptor subfamily. The free backbone C=O groups on helices H5 and H7 stabilize the inactive rhodopsin structure through hydrogen-bonds to residues on adjacent helices. In response to light-induced isomerization of the retinal chromophore, hydrogen-bonding interactions involving these C=O groups are released, thus facilitating repacking of H5 and H7 onto the transmembrane core of the receptor. These results provide insights into the multiple structural and functional roles of prolines in membrane proteins.
| S-EPMC4972713 | biostudies-literature
Project description:Activation of the G protein-coupled receptor (GPCR) rhodopsin is initiated by light-induced isomerization of the retinal ligand, which triggers 2 protonation switches in the conformational transition to the active receptor state Meta II. The first switch involves disruption of an interhelical salt bridge by internal proton transfer from the retinal protonated Schiff base (PSB) to its counterion, Glu-113, in the transmembrane domain. The second switch consists of uptake of a proton from the solvent by Glu-134 of the conserved E(D)RY motif at the cytoplasmic terminus of helix 3, leading to pH-dependent receptor activation. By using a combination of UV-visible and FTIR spectroscopy, we study the activation mechanism of rhodopsin in different membrane environments and show that these 2 protonation switches become partially uncoupled at physiological temperature. This partial uncoupling leads to approximately 50% population of an entropy-stabilized Meta II state in which the interhelical PSB salt bridge is broken and activating helix movements have taken place but in which Glu-134 remains unprotonated. This partial activation is converted to full activation only by coupling to the pH-dependent protonation of Glu-134 from the solvent, which stabilizes the active receptor conformation by lowering its enthalpy. In a membrane environment, protonation of Glu-134 is therefore a thermodynamic rather than a structural prerequisite for activating helix movements. In light of the conservation of the E(D)RY motif in rhodopsin-like GPCRs, protonation of this carboxylate also may serve a similar function in signal transduction of other members of this receptor family.
| S-EPMC2584695 | biostudies-literature
Project description:We present crystal structures of the Anabaena sensory rhodopsin transducer (ASRT), a soluble cytoplasmic protein that interacts with the first structurally characterized eubacterial retinylidene photoreceptor Anabaena sensory rhodopsin (ASR). Four crystal structures of ASRT from three different spacegroups were obtained, in all of which ASRT is present as a planar (C4) tetramer, consistent with our characterization of ASRT as a tetramer in solution. The ASRT tetramer is tightly packed, with large interfaces where the well-structured beta-sandwich portion of the monomers provides the bulk of the tetramer-forming interactions, and forms a flat, stable surface on one side of the tetramer (the beta-face). Only one of our four different ASRT crystals reveals a C-terminal alpha-helix in the otherwise all-beta protein, together with a large loop from each monomer on the opposite face of the tetramer (the alpha-face), which is flexible and largely disordered in the other three crystal forms. Gel-filtration chromatography demonstrated that ASRT forms stable tetramers in solution and isothermal microcalorimetry showed that the ASRT tetramer binds to ASR with a stoichiometry of one ASRT tetramer per one ASR photoreceptor with a K(d) of 8 microM in the highest affinity measurements. Possible mechanisms for the interaction of this transducer tetramer with the ASR photoreceptor via its flexible alpha-face to mediate transduction of the light signal are discussed.
| S-EPMC2041937 | biostudies-literature
Project description:Rhodopsins are the most universal biological light-energy transducers and abundant phototrophic mechanisms that evolved on Earth and have a remarkable diversity and potential for biotechnological applications. Recently, the first sodium-pumping rhodopsin KR2 from Krokinobacter eikastus was discovered and characterized. However, the existing structures of KR2 are contradictory, and the mechanism of Na+ pumping is not yet understood. Here, we present a structure of the cationic (non H+) light-driven pump at physiological pH in its pentameric form. We also present 13 atomic structures and functional data on the KR2 and its mutants, including potassium pumps, which show that oligomerization of the microbial rhodopsin is obligatory for its biological function. The studies reveal the structure of KR2 at nonphysiological low pH where it acts as a proton pump. The structure provides new insights into the mechanisms of microbial rhodopsins and opens the way to a rational design of novel cation pumps for optogenetics.
| S-EPMC6457933 | biostudies-literature
Project description:We determined the structure of the rhodopsin mutant N2C/D282C expressed in mammalian cells; the first structure of a recombinantly produced G protein-coupled receptor (GPCR). The mutant was designed to form a disulfide bond between the N terminus and loop E3, which allows handling of opsin in detergent solution and increases thermal stability of rhodopsin by 10 deg.C. It allowed us to crystallize a fully deglycosylated rhodopsin (N2C/N15D/D282C). N15 mutations are normally misfolding and cause retinitis pigmentosa in humans. Microcrystallographic techniques and a 5 microm X-ray beam were used to collect data along a single needle measuring 5 microm x 5 microm x 90 microm. The disulfide introduces only minor changes but fixes the N-terminal cap over the beta-sheet lid covering the ligand-binding site, a likely explanation for the increased stability. This work allows structural investigation of rhodopsin mutants and shows the problems encountered during structure determination of GPCRs and other mammalian membrane proteins.
| S-EPMC2258155 | biostudies-literature