Journal of Molecular Biology
Structure of Bovine Rhodopsin in a Trigonal Crystal Form
Introduction
G protein-coupled receptors (GPCRs) constitute the largest superfamily of transmembrane signalling proteins in the eukaryotic kingdom. They share a common core structure comprising seven transmembrane helices, as implied by a conserved pattern of amino acids at key positions along each of the seven hydrophobic sequence segments.1 These proteins capture external signals, including light, odorants, hormones and neurotransmitters, and transmit the stimuli across the plasma membrane by selectively activating heterotrimeric guanine nucleotide binding proteins (G proteins) on the cytoplasmic surface. Activation of G protein amplifies the signal and leads to activation of effector enzymes or molecules, which elicit the cell's signalling response indirectly by altering second messenger concentrations or by direct control of ion channel activities. Thus, the GPCRs play a central role in regulating many physiological processes. They are consequently major targets for drug design.
Rhodopsin, the photoreceptor protein in retina rod cells, is a prototypical GPCR. It contains a light-sensitive ligand, the 11-cis-retinal chromophore, bound covalently to the apoprotein opsin via a protonated Schiff base with Lys296 on helix 7. Absorption of a photon at a wavelength of about 500 nm isomerises the retinal to all-trans2 within picoseconds and with a quantum efficiency of 0.67. This event initiates the formation of a series of photointermediates3 with conformational changes in the opsin.4 The biochemically active conformation R* is attained within one millisecond at physiological temperature. It is spectroscopically identified with the metarhodopsin II (MII) intermediate, which has an absorption maximum at 380 nm due to deprotonation of the Schiff base of all-trans-retinal. R* binds and activates the heterotrimeric G-protein transducin (Gt) at the cytoplasmic surface, to catalyse GDP/GTP exchange on the Gt α-subunit and dissociation of the Gt heterotrimer. The GTP-bound α-subunit then activates the effector enzyme, phosphodiesterase (PDE). Hydrolysis of cyclic guanine monophosphate (cGMP), the second messenger, by PDE leads to closure of the cGMP-gated cation channel in the plasma membrane, causing hyperpolarisation and initiation of nerve impulse in the retina.5 In contrast to the efficient and rapid photoisomerisation and activation, the rate of thermal isomerisation is very low, about one per 400 years per rhodopsin molecule at 37 °C.6 The low thermal noise coupled with large amplification via transducin activation underpins the sensitivity of single photon detection operating in dim light vision.
An atomic structure of rhodopsin provides a model for all the visual pigments as well as the majority of the GPCRs. Crystallisation in two-dimensional (2D) lattices with endogenous membrane lipids, followed by electron cryo-microscopy (cryo-EM)7, 8, 9, 10, 11 at resolution limits up to 5 Å in the membrane plane and 13.5 Å normal to it, have produced images of bovine, frog and squid rhodopsins in a membrane-like environment, which are in essence similar. Using packing constraints derived by extensive sequence comparisons across the GPCR superfamily, the density peaks in cryo-EM maps were assigned to hydrophobic sequences, leading eventually to a Cα model for the seven transmembrane helices in the rhodopsin-like GPCR family.1, 12, 13 Remarkably, this model came within 2.3 Å rms deviation of Cα coordinates determined by X-ray crystallography subsequently14, 15 and it provided a framework for mutagenesis and biophysical studies in the absence of an atomic structure.
To determine the atomic structure of rhodopsin, we have obtained untwinned three-dimensional crystals of bovine rhodopsin in the trigonal space group P31 that diffract X-rays to 2.65 Å resolution, and prepared an ethylmercury derivative that showed anomalous scattering.16 However, significant non-isomorphism prevented experimental phasing. While this work was in progress, a structure of bovine rhodopsin in a tetragonal P41 space group at 2.8 Å resolution was reported.14, 17 Using these coordinates as search model, we have determined the structure of bovine rhodopsin to 2.65 Å resolution in the P31 lattice by molecular replacement followed by multiple crystal averaging among the non-isomorphous data sets. Here, we describe the refined structure of bovine rhodopsin in the trigonal crystal form, and compare it with previous structural reports of the tetragonal crystal form.14, 18, 19 In addition, we relate the crystal structures to the membrane environment by docking them into a cryo-EM map10 of 2D crystals. Structural implications for the stability of the “dark” state and the potential for light-induced conformational change, leading to transducin activation on the cytoplasmic surface, are discussed.
Section snippets
Structure determination
Bovine rhodopsin was crystallised in the trigonal space group P31 with two protein molecules per asymmetric unit, from a detergent mixture of C8E4 and LDAO as described in the accompanying paper.16 Data sets were obtained from native and mercury-derivatised crystals prepared and frozen under dim red light; however, they showed significant non-isomorphism with one another. Data collection and refinement statistics are given in Table 1.
Using the protein part only of the rhodopsin coordinates
Data collection and processing
Crystallisation and preparation of heavy atom derivatives are described in the accompanying paper.16 Complete data sets were obtained by combining wedges of 20–30° collected from different positions on one or more crystals. Data were processed using programs in the CCP4 suite.136 Intensities were integrated to anisotropic resolution limits using MOSFLM, and merged using SCALA. A consistent indexing regime was maintained, by re-indexing each wedge under the four indexing regimes of point group
Acknowledgements
We thank the ESRF, SPring-8, APS and Daresbury Laboratory for providing synchrotron radiation facilities and staff support during data collection, and in particular the ESRF for a long-term award to G.F.X. for use of the microfocus beamline ID13. We are grateful to Drs Richard Henderson, Paul Hargrave, Joyce Baldwin, Harry Powell and Alexei Murzin for helpful discussions; Luca Jovine for calculating the EDEN map from partially refined coordinates; Richard Henderson, Phil Evans, Andrew Leslie
References (148)
- et al.
Low resolution structure of bovine rhodopsin determined by electron cryo-microscopy
Biophys. J.
(1995) - et al.
The three-dimensional structure of bovine rhodopsin determined by electron cryomicroscopy
J. Biol. Chem.
(2003) - et al.
Three-dimensional structure of an invertebrate rhodopsin and basis for ordered alignment in the photoreceptor membrane
J. Mol. Biol.
(2001) - et al.
An alpha-carbon template for the transmembrane helices in the rhodopsin family of G-protein-coupled receptors
J. Mol. Biol.
(1997) - et al.
X-ray diffraction analysis of three-dimensional crystals of bovine rhodopsin obtained from mixed micelles
J. Struct. Biol.
(2000) - et al.
Photoactivation of rhodopsin and interaction with transducin in detergent micelles–effect of doping with steroid molecules
FEBS Letters
(1989) - et al.
Transducin-alpha C-terminal peptide binding site consists of C-D and E-F loops of rhodopsin
J. Biol. Chem.
(1997) - et al.
Rhodopsin mutants discriminate sites important for the activation of rhodopsin kinase and Gt
J. Biol. Chem.
(1995) - et al.
Transglutaminase modification of rhodopsin in retinal rod outer segment disk membranes
Arch. Biochem. Biophys.
(1986) - et al.
Rhodopsin determinants for transducin activation: a gain-of-function approach
J. Biol. Chem.
(2003)
Lipid bilayer thickness varies linearly with acyl chain length in fluid phosphatidylcholine vesicles
J. Mol. Biol.
Rhodopsin structure, dynamics, and activation: a perspective from crystallography, site-directed spin labeling, sulfhydryl reactivity, and disulfide cross-linking
Advan. Protein Chem.
Rhodopsin carbohydrate. Structure of small oligosaccharides attached at two sites near the NH2 terminus
J. Biol. Chem.
A rhodopsin mutant linked to autosomal dominant retinitis pigmentosa is prone to aggregate and interacts with the ubiquitin proteasome system
J. Biol. Chem.
Integrated methods for the construction of three dimensional models and computational probing of structure-function relations in G-protein coupled receptors
Methods Neurosci.
Interactions between conserved residues in transmembrane helices 1, 2, and 7 of the thyrotropin-releasing hormone receptor
J. Biol. Chem.
The functional microdomain in transmembrane helices 2 and 7 regulates expression, activation, and coupling pathways of the gonadotropin-releasing hormone receptor
J. Biol. Chem.
Related contribution of specific helix 2 and 7 residues to conformational activation of the serotonin 5-HT2A receptor
J. Biol. Chem.
Mapping of the amino acids in membrane-embedded helices that interact with the retinal chromophore in bovine rhodopsin
J. Biol. Chem.
Orientation of retinal in bovine rhodopsin determined by cross-linking using a photoactivatable analog of 11-cis-retinal
J. Biol. Chem.
The amino terminus of the fourth cytoplasmic loop of rhodopsin modulates rhodopsin–transducin interaction
J. Biol. Chem.
Mutation of the fourth cytoplasmic loop of rhodopsin affects binding of transducin and peptides derived from the carboxyl-terminal sequences of transducin alpha and gamma subunits
J. Biol. Chem.
Tyrosine structural changes detected during the photoactivation of rhodopsin
J. Biol. Chem.
Aromatic rings act as hydrogen bond acceptors
J. Mol. Biol.
Stability of dark-state rhodopsin is mediated by a conserved ion-pair in intradiscal loop E-2
J. Biol. Chem.
A comparison of the efficiency of G protein activation by ligand-free and light-activated forms of rhodopsin
Biophys. J.
Introduction of hydroxyl-bearing amino acids causes bathochromic spectral shifts in rhodopsin. Amino acid substitutions responsible for red-green color pigment spectral tuning
J. Biol. Chem.
Mechanisms of spectral tuning in blue cone visual pigments. Visible and Raman spectroscopy of blue-shifted rhodopsin mutants
J. Biol. Chem.
How color visual pigments are tuned
Trends Biochem. Sci.
pKa of the protonated Schiff base of bovine rhodopsin. A study with artificial pigments
Biophys. J.
The probable arrangement of the helices in G protein-coupled receptors
EMBO J.
The action of light on rhodopsin
Proc. Natl Acad. Sci. USA
Molecular basis of visual excitation
Science
Requirement of rigid-body motion of transmembrane helices for light activation of rhodopsin
Science
Rhodopsin: a prototypical G protein-coupled receptor
Prog. Nucl. Acid Res. Mol. Biol.
Two components of electrical dark noise in toad retinal rod outer segments
J. Physiol.
Projection structure of rhodopsin
Nature
Projection structure of frog rhodopsin in two crystal forms
Proc. Natl Acad. Sci. USA
Arrangement of rhodopsin transmembrane alpha-helices
Nature
Crystal structure of rhodopsin: a G protein-coupled receptor
Science
Structure. Rhodopsin sees the light
Science
Advances in determination of a high-resolution three-dimensional structure of rhodopsin, a model of G-protein-coupled receptors (GPCRs)
Biochemistry
Functional role of internal water molecules in rhodopsin revealed by X-ray crystallography
Proc. Natl Acad. Sci. USA
The crystal and molecular structure of 11-cis-retinal
Acta Crystallog. sect. B
Crystal structure of N-methyl-N-phenylretinal iminium perchlorate: a structural model for the bacteriorhodopsin chromophore
J. Am. Chem. Soc.
Crystallography & NMR system: a new software suite for macromolecular structure determination
Acta Crystallog. sect. D
Holographic methods in X-ray crystallography. II. Detailed theory and connections to other methods of crystallography
Acta Crystallog. sect. A
Improved Fourier coefficients for maps using phases from partial structures with errors
Acta Crystallog. sect. A
Rhodopsin mutants that bind but fail to activate transducin
Science
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