Journal of Molecular Biology
Volume 343, Issue 5, 5 November 2004, Pages 1409-1438
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Structure of Bovine Rhodopsin in a Trigonal Crystal Form

https://doi.org/10.1016/j.jmb.2004.08.090Get rights and content

We have determined the structure of bovine rhodopsin at 2.65 Å resolution using untwinned native crystals in the space group P31, by molecular replacement from the 2.8 Å model (1F88) solved in space group P41. The new structure reveals mechanistically important details unresolved previously, which are considered in the membrane context by docking the structure into a cryo-electron microscopy map of 2D crystals.

Kinks in the transmembrane helices facilitate inter-helical polar interactions. Ordered water molecules extend the hydrogen bonding networks, linking Trp265 in the retinal binding pocket to the NPxxY motif near the cytoplasmic boundary, and the Glu113 counterion for the protonated Schiff base to the extracellular surface. Glu113 forms a complex with a water molecule hydrogen bonded between its main chain and side-chain oxygen atoms. This can be expected to stabilise the salt-bridge with the protonated Schiff base linking the 11-cis-retinal to Lys296.

The cytoplasmic ends of helices H5 and H6 have been extended by one turn. The G-protein interaction sites mapped to the cytoplasmic ends of H5 and H6 and a spiral extension of H5 are elevated above the bilayer. There is a surface cavity next to the conserved Glu134-Arg135 ion pair. The cytoplasmic loops have the highest temperature factors in the structure, indicative of their flexibility when not interacting with G protein or regulatory proteins. An ordered detergent molecule is seen wrapped around the kink in H6, stabilising the structure around the potential hinge in H6.

These findings provide further explanation for the stability of the dark state structure. They support a mechanism for the activation, initiated by photo-isomerisation of the chromophore to its all-trans form, that involves pivoting movements of kinked helices, which, while maintaining hydrophobic contacts in the membrane interior, can be coupled to amplified translation of the helix ends near the membrane surfaces.

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

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