Structure of Bovine Rhodopsin in a Trigonal Crystal Form

doi:l 0.1016/j.jmb.2004.08.090 J. MoL BioL (2004) 343, 1409-1438

Available online at www.sciencedirect.com jib SCIENCE ~DIRI=CT • ELSEVIER

Jade Li 1., Patricia C. Edwards 1, Manfred Burghammer 2, Claudio Villa 1 and Gebhard F. X. Schertler 1.

1Medical Research Council We have determined the structure of bovine rhodopsin at 2.65 A resolution Laboratory of Molecular Biology using untwinned native crystals in the space group P31, by molecular Hills Road replacement from the 2.8 A model (1F88) solved in space group P41. The Cambridge CB2 2QH, UK new structure reveals mechanistically important details unresolved 2European Synchrotron previously, which are considered in the membrane context by docking the structure into a cryo-electron microscopy map of 2D crystals. Radiation Facility, BP 220 Kinks in the transmembrane helices facilitate inter-helical polar 8043 Grenoble, France 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. © 2004 Elsevier Ltd. All rights reserved. Keywords: G protein-coupled receptor; G protein activation; ligand binding *Corresponding authors pocket; membrane protein structure; visual pigments

Abbreviations used: C8E4, n-octyltetraoxyethylene; C1, cytoplasmic loop connecting helices 1 and 2; C2, cytoplasmic loop connecting helices 3 and 4; C3, cytoplasmic loop connecting helices 5 and 6; cGMP, 3~,5~-cyclic guanosine monophosphate; cryo-EM, electron cryomicroscopy; El, extracellular loop connecting helices 2 and 3; E2, extracellular loop connecting helices 4 and 5; E3, extracellular loop connecting helices 6 and 7; EM, electron microscopy; EMTS, ethyl mercurithiosalicylate; FTIR, Fourier transform infrared spectroscopy; GPCR, G protein-coupled receptor; G-protein, heterotrimeric guanine nucleotide binding protein; Gt, transducin; Gtc~, transducin ~-subunit; Gt~ 340-350, C-terminal peptide from transducin ~z-subunit; LDAO, N,N-dimethyldodecylamine-N-oxide; MI and MII, metarhodopsin I and II; NCS, non-crystallographic symmetry; PDE, phosphodiesterase. E-mail addresses of the corresponding authors: [email protected]; [email protected]

Introduction and 13.5 A normal to it, have produced images of bovine, frog and squid rhodopsins in a membrane- G protein-coupled receptors (GPCRs) constitute like environment, which are in essence similar. Using the largest superfamily of transmembrane signal- packing constraints derived by extensive sequence ling proteins in the eukaryotic kingdom. They share comparisons across the GPCR superfamily, the a common core structure comprising seven trans- density peaks in cryo-EM maps were assigned to membrane helices, as implied by a conserved hydrophobic sequences, leading eventually to a C a pattern of amino acids at key positions along each model for the seven transmembrane helices in the of the seven hydrophobic sequence segments. 1 rhodopsin-like GPCR fairly. 1"12'13 Remarkably, this These proteins capture external signals, including model came within 2.3 A rms deviation of C a light, odorants, hormones and neurotransmitters, coordinates determined by X-ray crystallography and transmit the stimuli across the plasma subsequently14"15 and it provided a framework for membrane by selectively activating heterotrimeric mutagenesis and biophysical studies in the absence of guanine nucleotide binding proteins (G proteins) on an atomic structure. the cytoplasmic surface. Activation of G protein To determine the atomic structure of rhodopsin, amplifies the signal and leads to activation of we have obtained untwinned three-dimensional effector enzymes or molecules, which elicit the crystals of bovine rhodopsin in the trigonal space cell's signalling response indirectly by altering group P31 that diffract X-rays to 2.65 A resolution, second messenger concentrations or by direct and prepared an ethylmercury derivative that control of ion channel activities. Thus, the GPCRs showed anomalous scattering. 16 However, signifi- play a central role in regulating many physiological cant non-isomorphism prevented experimental processes. They are consequently major targets for phasing. While this work was in progress, a drug design. structure of bovine rhodopsin in a tetragonal P41 Rhodopsin, the photoreceptor protein in retina space group at 2.8 A resolution was reported. 14'17 rod cells, is a prototypical GPCR. It contains a light- Using these coordinates as search model, we have sensitive ligand, the 11-cis-retinal chromophore, determined the structure of bovine rhodopsin to bound covalently to the apoprotein opsin via a 2.65 A resolution in the P31 lattice by molecular protonated Schiff base with Lys296 on helix 7. replacement followed by multiple crystal averaging Absorption of a photon at a wavelength of about among the non-isomorphous data sets. Here, we 500 nm isomerises the retinal to all-trans 2 within describe the refined structure of bovine rhodopsin picoseconds and with a quantum efficiency of 0.67. in the trigonal crystal form, and compare it with This event initiates the formation of a series of previous structural reports of the tetragonal crystal photointermediates3 with conformational changes form. 14"18"19 In addit'ion, we relate the crystal in the opsin. 4 The biochemically active confor- structures to the membrane environment by mation R* is attained within one millisecond at docking them into a cryo-EM map 1° of 2D crystals. physiological temperature. It is spectroscopically Structural implications for the stability of the identified with the metarhodopsin II (MII) inter- "dark" state and the potential for light-induced mediate, which has an absorption maximum at conformational change, leading to transducin 380 nm due to deprotonation of the Schiff base of activation on the cytoplasmic surface, are discussed. all-trans-retinal. R binds and activates the heterotrimeric G-protein transducin (Gt) at the cytoplasmic surface, to catalyse GDP/GTP exchange Results and Discussion on the Gt ~-subunit and dissociation of the Gt heterotrimer. The GTP-bound ~-subunit then acti- Structure determination vates the effector enzyme, phosphodiesterase (PDE). Hydrolysis of cyclic guanine mono- Bovine rhodopsin was crystallised in the trigonal phosphate (cGMP), the second messenger, by PDE space group P31 with two protein molecules per leads to closure of the cGMP-gated cation channel asymmetric unit, from a detergent mixture of C8E4 in the plasma membrane, causing hyperpolaris- and LDAO as described in the accompanying ation and initiation of nerve impulse in the retina. 5 paper. 16 Data sets were obtained from native and In contrast to the efficient and rapid photoisomer- mercury-derivatised crystals prepared and frozen isation and activation, the rate of thermal isomer- under dim red light; however, they showed isation is very low, about one per 400 years per significant non-isomorphism with one another. rhodopsin molecule at 37 °C. 6 The low thermal Data collection and refinement statistics are given noise coupled with large amplification via trans- in Table 1. ducin activation underpins the sensitivity of single Using the protein part only of the rhodopsin photon detection operating in dim light vision. coordinates solved in a tetragonal P41 space An atomic structure of rhodopsin provides a model group (PDB code 1F88) 14'17 at 2.8 A resolution as for all the visual pigments as well as the majority of search model, molecular replacement solutions were the GPCRs. Crystallisation in two-dimensional (2D) calculated in parallel for three sets of amplitudes lattices with endogenous membrane lipids, followed (Native-2, EMTS-1 and EMTS-2). The resulting by electron cryo-microscopy (cryo-EM) 7-11 at 2Fo--F~ maps showed no density in three cyto- resolution limits up to 5 A in the membrane plane plasmic regions of 1F88 coordinates, and the Rhodopsin Structure in Trigonal Crystals 1411

Table 1. Data collection and final refinement statistics: space group P31

A. Data collection Native-2a EMTS-1 EMTS-2 Native-3

X-ray source o ESRF ID13 ESRF ID13 ESRF ID14-4 ESRF ID13 Wavelength (A) 0.782 1.005 1.0065 0.782 Unit cell a, c (A) 104.2, 77.1 113.9, 78.4 109.3, 77.6 103.8, 76.6 No. of crystals 2 1 1 4 Mosaicity (deg.) 1.0 1.1 1.1 0.75 Twin fraction 0 0 0.31 0 Resolution (A) 3.2 (3.37-3.20) 3.6 (3.71-3.60) 3.4 (3.45-3.40) 2.65 (2.79-2.65) amergeb 0.127 (0.322) 0.169 (0.464) 0.139 (0.426) 0.119 (0.434) I/o 7.5 (2.3) 7.6 (2.2) 17.2 (4.7) 11.0 (1.4) Unique reflections 15087 11481 13320 26026 Completeness (%) 0.982 (0.894) 0.868 (0.180) 0.834 (0.172) 0.970 (0.861) Multiplicity 3.1 (1.6) 5.3 (2.9) 11.3 (9.5) 4.4 (1.6) Wilson B-factor (~2) 84.6 59.7 54.2 58.2

B. Refinement against Native-3

Reflections in working set 24704 (2165) Protein chains 2 Reflections in test set 1322 (130) Protein residues 652 Resolution range (A) 46-2.65 (2.74-2.65) Palmitoyl chains 4 Rc~ys~ 0.202 (0.312) N-linked carbohydrate chains 4 R free 0.235 (0.315) Carbohydrate residues 12 Luzzati coordinate error (5.0-2.65 ~) 0.31 A LDAO 2 SigmaA coordinate error (5.0-2.65 A) 0.42 ,~ C8E4 12 tins-deviation from ideal geometry Phospholipid 2 Bond lengths (A) 0.008 Water 40 Bond angles (deg.) 1.293 Ions 2 Dihedral angles (deg.) 18.7 Improper rotations (deg.) 0.876 Ramachandran plot Residues in most favoured regions (%) 90.6 Residues in additional allowed regions (%) 7.1 Residues in generously allowed regions (%) 2.4 Residues in disallowed regions (%) 0 Average B-factor (~2) 56.0 B rmsd for bonded main-chain atoms ~2) 1.501 B rmsd for bonded side-chain atoms IA2) 1.996 B rmsd for angle main-chain atoms ~A2) 2.624 B rmsd for angle side-chain atoms (A2) 3.134 Values in parentheses apply to the outer resolution shell. a Names of data sets conform to Table I of the accompanying paper. 16 Native-1 is not referred to here, because it was not used in the structure determination. b Rmerge= Y'~hkt Y'~i ]Ii --(I)]/~hkl Y'~i Ii, where Ii is the intensity of an individual reflection and is the mean intensity obtained from multiple observations of symmetry-related reflections taken from one or more crystals. c Rcrysty~hkl(]Fo(hkl) _ Fc(hkl)D/y~hkl(]Fo(hkl)])" calculated for reflections in the working set. a Rfre e y~hk1(iFo(hki) _Fc(hkl)[)/y~hkl(iFo(hkl)b, calculated for a random 5% of the data placed in the test set. corresponding Fo-Fc maps showed negative peaks residue 247 in the 1F88 coordinates to residue 244. in the same regions. These regions were: the C2 loop The middle section of the C3 loop (residues 233-239 connecting transmembrane helices H3 and H4; the in molecule A and 233-237 in molecule B) and the C C3 loop connecting H5 and H6; and the C-terminal terminus still had no interpretable densities, so they tail of residues 334-348. By contrast, density was were left out of the model. The Schiff base linked present in the 2Fo-Fc maps over positions of the 11-cis-retinal chromophore was built from the small retinal chromophore, even though, as a control, we molecule crystal structures 2°'21 by adjusting torsion had omitted the retinal from the 1F88 coordinates angles along the polyene according to the averaged used for molecular replacement. No negative peaks density. were seen in the retinal-binding pocket. Therefore A new native data set (Native-3) was then our amplitude data disagreed with the starting collected from four untwinned crystals to 2.65 model in the cytoplasmic regions. Exploiting the resolution, and used to refine the atomic model, large non-isomorphism, we carried out cross- using CNS 22 under 2-fold non-crystallographic crystal averaging among these data sets. The symmetry restraints with simulated annealing and averaged map for Native-2 showed densities for restrained individual B-factors. Bound water the C2 and C3 loops at positions different from the molecules were located using the water_pick task starting model, enabling them to be rebuilt and the of CNS 22 and confirmed in maps calculated using cytoplasmic end of helix H6 to be extended from EDEN. 23 Tightly bound phospholipids and 1412 Rhodopsin Structure in Trigonal Crystals detergent molecules were added. Including the (x--0.333, y= 0.167). Both helix bundles are tilted by bound water and amphiphile molecules in the 108 ° from c and make antiparallel contacts along the refinement further improved the map, so that the entire length of helix 5. The antiparallel dimers are cytoplasmic ends of both H5 and H6 were extended stacked according to the 31 screw symmetry, and the C3 loop was completed. The most signifi- forming two protein columns in the unit cell, cant differences between our structure and all the which are centred on two of the three 3-fold published coordinates from the P41 crystal are positions of the trigonal lattice, and leaving a found in this region. solvent channel centred on the remaining 3-fold position. Between layers of stacked dimers the Quality of the structure contacts involve mainly the sides of helices I and 4. The other helices participate in crystal contacts to a The final crystallographic R-factor is 0.202 with a lesser extent or indirectly via interleaved palmitoy- free-R-factor of 0.235, and there is close agreement late chains or the C8E4 molecule (see subsection with ideal geometry (Table 1B). The Ramachandran titled Bound lipid and detergent molecules). Thus, plot contains 90.6% of the protein residues in the uncommonly among crystals of membrane pro- most favoured regions, compared with values of teins, the inter-molecular contacts in the P31 form 80.9-82.5% for the published rhodopsin structures are predominantly hydrophobic. The hydrophilic in the tetragonal crystal form. The ~A-weighted 24 extra-membrane segments point into the solvent 2Fo-F~ map showed an average real-space corre- channel and are not responsible for ordered crystal lation of 0.95 for all residues including the non- contacts (Figure l(b)). Consequently, the P31 crystal protein residues. These statistics demonstrate that packing has retained an amphipathic molecular the structure was well determined, and bias toward environment characteristic of the native membrane, the starting model was minimal. permitting the hydrophilic segments to display The refined model (Table 1B) comprises amino conformations present in a membrane environment acid residues 1-326 in both molecules, compared unhindered by packing interactions. with the complete sequence of 348 residues. Post-translational modifications have been defined on both molecules, including N-acetylation of Met1, Coordinate differences between NCS copies and N-glycosylation of Asn2 and Asn15, palmitoylation between crystal forms of Cys322 and Cys323, and the Schiff base linkage of 11-cis-retinal to Lys296. In addition, there are 20 The 1L9H coordinates 19 at 2.6 .~ resolution are ordered water molecules bound to each protein used to represent the P41 form. Here, the rms C = chain and a number of tightly bound lipid and distance between NCS copies is 0.470 A for 301 detergent molecules. The covalent structure is matched residues, and the rms B-factor difference is shown as a ribbon diagram in Figure l(a). The 9.530 .~2 with molecule A being the more ordered residue ranges of the seven transmembrane helices copy. In the P31 form with closer NCS, the two are: H1, 34-64; H2, 71-100; H3, 106-140; H4, molecules show rms differences of only 0.13 A and 150-173; H5, 200-230; H6, 241-276 and H7, 2.216/~2 for 326 matched residues. Therefore, 286-309. Residues in the peripheral helix H8 are molecules A from the 1L9H coordinates and the 311-321, and those making up the B-strands are: [31, present structure are used to compare the crystal 4-6; B2, 9-11; ~3, 177-180 and f~4, 187-190. Portions forms. of the transmembrane helices, H4 between residues Figure 2(a) shows the coordinate difference per 170 and 173 and H7 between residues 296 and 299, residue as a function of sequence position, between are in the 310 conformation. NCS copies in each crystal form and between the molecules A of different crystal forms. Both crystal Comparison with the tetragonal crystal form forms showed significant main chain NCS differences, above 1 A, in the C2 and C3 loops and the C-terminal tail following helix 8. These regions also Packing differences have the highest B-factors and disorder in each Both the P3l and P41 crystal forms contain two crystal form (Figure 2(b)). Part of the C3 loop is rhodopsin molecules per asymmetric unit. In the unresolved in both molecules of the P41 form. The P41 form, they are related by a non-crystallographic C-terminal tail in the P31 form is located in the rotation of 172.5 ° about an axis nearest to a and are solvent channel and disordered, while in the P41 oriented obliquely from the P41 axis. They make form it is partially resolved only in molecule A, contacts in the middle of helix 1 but mainly over where it is stabilised through contacts with the the hydrophilic extra-membrane segments. In extracellular domain of a neighbouring molecule. particular, the C terminus of molecule A from the Finding significant NCS differences in the same cytoplasmic domain forms a contact with the surface segments indicates an intrinsic confor- extracellular domain of molecule A in another mational variability or flexibility of those segments, asymmetric unit. and the high B-factors of the segments support this. In the P31 form, the non-crystallographic These segments are known to contain residues that symmetry (NCS) is a nearly perfect 2-fold about interact with transducin (Gt) and the regulatory an axis parallel with the 31 axis and passing through proteins rhodopsin kinase and arrestin; 2~31 their Rhodopsin Structure in Trigonal Crystals 1413

(a) (b)

Surf~ resic~ crystal ~itherc lia ord~ and d~ mol~

N-linked ~~ oligosaccharides

Figure 1. The bovine rhodopsin molecule in the transmembrane orientation with cytoplasmic surface at the top and extracellular surface at the bottom. (a) Ribbon representation. The seven transmembrane and one peripheral helices are coloured in the rainbow order: H1 (residues 34-64), dark blue; H2 (71-100), light blue; H3 (106-140), blue-green; H4 (150-173), yellow-green; H5 (200-230), yellow; H6 (241-276), orange; H7 (286-309), red; H8 (311-321), magenta. The 13- strands are coloured in cyan. They are arranged in the order of 131 (4-6), 132 (9-11), f~3 (177-180), and J~4 (187-190), from the extracellular surface into the retinal-binding pocket. The transmembrane helices are kinked around the 11-cis-retinal to form the binding pocket. Also shown are the N-linked oligosaccharides attached to Asn2 (near 131)and Asn15, and the palmitoylate chains attached to Cys322 and Cys323 after H8. This colour scheme for the secondary structures is used throughout. (b) Space filling representation. Residues coloured in yellow form the inter-molecular contacts in the P31 crystal form, directly or via ordered lipid and detergent molecules. They are all located in the transmembrane helices. The oligosaccharides are coloured pink, and the C = trace is shown in grey for comparison with (a). Figures 1, 2(b), 4, 5, 6, 7(b) and 9(a) were prepared using MOLSCRIPT; 146 Figures 1, 2(b) and 9(a) also using Raster3D. 147 flexibility in the absence of the binding partners more flexible. Figure 2(a) shows that, for the C2 may have a functional role. loop, the difference between crystal forms is on the If main-chain atoms in the molecules A of the two same scale as the NCS difference within the P41 crystal forms are superimposed by aligning the form, so they are accountable by local flexibility and more ordered regions selected by a B-factor cut-off packing contacts. However, around the C3 loop the of 90,~ 2, then the rms coordinate difference is differences between the two crystal forms are much 0.36 A, which is slightly less than the mean larger than the NCS differences in either form. coordinate error for all atoms in either structure. Therefore, they indicate statistically significant Therefore, the two structures are in good agreement disagreement. in the ordered core of the molecule, which comprised residues 1-137, 154-223 and 250-321, Location of major coordinate differences between and excluded only the neighbourhood of the C2 and crystal forms C3 loops, and the C-terminal tail. The backbone differences are concentrated in the same regions Figure 2(b) shows the C ~ trace of the molecules A shown by the NCS comparisons to be intrinsically from the two crystal forms side-by-side, coloured 1414 Rhodopsin Structure in Trigonal Crystals

(a) 20

P3(1) vs P4(I), main chain A P3(I) vs P4(1), side chain

"" 16 JI, P3(I) ncs, main chain o= I P4(1) ncs, main chain ==C .=:_= 12 "0 --N c2t~-

e" • m 8 "0 L 0 0 0

0 1 51 101 151 201 251 301 Sequence position (b) C-terminal fragment P41

250 ~O /O ~ 1 IU I.SU 13U

B-factor scale (~)

Figure 2. (legend opposite) Rhodopsin Structure in Trigonal Crystals 1415 by the mean B-factor in each residue. The colour inter-helical H-bonding network (see subsection scheme shows that the new structure from the P31 titled Transmembrane domain). The N-linked crystal form has the lower B-factors everywhere carbohydrate chains do not form ordered crystal except in the C3 loop, which is absent in the other contacts in the P31 crystals, unlike in the P41 crystals structure, and the major differences lie in the (see subsection titled Extracellular domain). regions of higher B-factors. The C2 loop has a similar "L-shape "14 in both Placing the X-ray structure in the membrane structures but with a different orientation relative to context the helix bundle. The difference may be described as a hinge movement about the junctions with the Before coordinates from the tetragonal 3D cytoplasmic ends of H3 and H4. crystals became available, we were able to deter- The C3 loop and the cytoplasmic ends of H5 and mine the orientation of rhodopsin molecules in the H6 show the most striking difference between the P31 crystals by rotation search at 5 A resolution, 16 two structures. In molecule A of the P41 form, H5 using as search model the density for a single terminates at residue 226, following which the molecule masked from a cryo-EM map of 2D polypeptide chain re-enters the bilayer ~nd appears crystals 1° (see Methods). Conversely, by docking disordered between residues 236 and 240, while H6 the refined X-ray coordinates into this cryo-EM terminates at residue 244. In the P31 form, H5 map, 1° we placed the atomic structure in the extends to residue 230, then the C a trace continues membrane context of the 2D crystal. The height of in a helix-like spiral path away from the membrane rhodopsin relative to the bilayer was therefore to residue 236, where it changes direction to run ascertained, since the p22121 symmetry of those 2D parallel with the membrane and joins the cyto- crystals requires symmetrical insertion of molecules plasmic end of H6 at residue 241. The cytoplasmic from both faces of the bilayer and therefore the tip of H6 is slightly bent at residue 244 towards H5. plane defined by c = 0 is equivalent to the midplane The C-terminal tail between residues 334-348 in of the bilayer. the P41 structure adopts an extended conformation Figure 3(a) shows the mean residue height in the supported by contacts with a neighbouring bilayer as a function of sequence position, overlaid molecule. In the P31 structure this segment is by the mean residue B-factors of the refined P31 disordered, except for a di-peptide suggested by structure. The sequence ranges of the ~ and the density in contact with the cytoplasmic loop 1 secondary structure elements are shown as vertical (loop C1). The disorder can be expected, since the bars. Small-angle X-ray and neutron scattering have fragment, without disulphide bridges, is too small shown that phosphate peaks in the retinal disc to form an independently folded domain, and it is membranes are separated by 40 A across the located in a solvent channel in this crystal form. bilayer, 4°'41 and lipid head groups in model The structure of the cytoplasmic domain of the membranes occupy a layer about a 10 A thick P31 form is discussed at greater length in the context centred on the phosphate peaks. 42 Therefore in of explaining the surface accessibility and mobility Figure 3 the hydrophobic zone extends from observed in site-specific spin labelling, chemical 32 35" approximately - 15 A to + 15 A, and the lipid head labelling and cross-linking studies, - as well as group° layers extend from there to approximately the relationship to G protein activation, 36-39 in the -25 A and + 25 A. On this scale, the 33 N-terminal section Cytoplasmic domain. residues preceding H1 fall entirely outside the hydrophobic zone. They form the extracellular domain. However only the residues 1-5 and 13-21, Other differences between the crystal forms which carry the N-linked carbohydrate chains The P31 structure shows mechanistically impor- on Asn2 and Asn15, reach into the intra-discal tant differences from the 1L9H coordinates in the space. conformation of the covalently linked 11-cis-retinal On the extracellular face all transmembrane chromophore (see subsection titled Conformation helices terminate in the lipid head group layer, and environment of the chromophore). All the except H4, which terminates within the hydro- internal water molecules have now been located, phobic zone. The strand fk3 lies at the interface thus leaving no cavity TM in the protein. The eight between hydrophobic zone and head group layer, newly identified water molecules include one in while ~4 is contained within the hydrophobic zone. the vicinity of Trp265, and they augment the On the cytoplasmic face, helices H1, H2, H4 and H7

Figure 2. Comparison between the P31 and P41 crystal forms. (a) Coordinate difference as a function of sequence position. The main chain and side-chain differences between the molecules A from different crystal forms are superimposed on the main chain difference between NCS copies within each crystal form. Significant differences exist only around the C2 loop (residues 141-149) connecting H3 and H4 and the C3 loop (residues 231-240) connecting H5 and H6, and in the C-terminal tail following HS. (b) C ~ trace of the molecules A from the two crystal forms coloured by their mean residue B-factors. The two structures differ in the orientation of the C2 loop, the lengths of H5 and H6, the conformation of the C3 loop and the C-terminal fragment. Regions of maximal structural differences also show the highest B-factors. 141 6 Rhodopsin Structure in Trigonal Crystals

(a) Cytoplasmic 180 40

160 30

A 140 20 '~

O,-.= .0_ 120 lO "~ 0 13_ fie 100 0 ~

.Q -I0 6 80 t- d~ I--- 60 -20

40 -30

20 -40 51 101 151 201 251 301 ExtraceUular Residue Number

Iml~ Helices Y~II: Strands -- B-factor --~ Trans-bilayerPosiffon I

(b)

200

160 &-. % (D 120 o9 .'.'. rv0

O. • o °. 80

°. ,0 m .° ":: "'.i... ..- ~. :..;'•~ ;:. ::.i .o • . • o" • . ~. ,.....,,... .~ • .'. t.: : 40 • # "'~ l" "

0 -40 -30 -20 -10 10 20 30 40 Mean residue distance from mid-plane of bilayer (A) Figure 3. Relation of the P31 crystal structure to the membrane environment• (a) Mean residue height relative to the bilayer derived by docking the X-ray coordinates in the cryo-EM map of 2D crystals, with mean residue B-factors superimposed• The mid-plane of the bilayer is defined by the c = 0 plane of the p22121 2D lattice. The hydrophobic zone is estimated to lie in a 30 A band about this plane, and the lipid headgroups in two 10 A wide layers outside that. The secondary structure assignments are represented by vertical bars, with notches in the bars for H4 and H7 indicating the location of 310 segments. The curve of residue heights follows a straight path over sections of H4 and H6 that are perpendicular to the bilayer, but shows a zigzag pattern for the tiffed sections. Surperimposed on this curve is the mean B-factor per residue as a function of the sequence. (b) Mean residue B-factor as a function of transmembrane position. The B-factors are well clustered between the lipid phosphate positions at __ 20 A. They increase from the membrane interior towards the surface, but the lowest B-factors are found just to the extracellular side of the bilayer mid-plane, at the depth where the retinal is bound. The sharp rise of B-factors on the cytoplasmic surface is due to conformational variability in several regions. Rhodopsin Structure in Trigonal Crystals 1417

Table 2. Tilt and kinks in the transmernbrane helices Tilt from membrane Azimuthalorientation c Kink angle Helix Straight segmentsa Residuerange a normalb (deg.) (deg.)

H1 N-segment 35-54 23 13 9 C-segment 55-65 30 2 H2 N-segment 70-87 -24 -85 33 C-segment 88-100 -33 -14 H3 N-segment 106-113 34 55 12 Midsegment 113-127 32 77 8 C-segment 127-139 24 80 H4 N-segment 150-169 -4 67 35 C-segment 169-173 -33 1 H5 N-segment 200-211 20 8 13 C-segment 211-231 26 36 H6 N-segment 244-265 --3 9 35 C-segment 265-279 -34 -53 H7 N-segment 286-296 25 62 27 Midsegment 297-302 4 13 14 C-segment 303-309 18 4 H8 311-321 -86 89 a Residue range of the straight segments were determined by fitting cylinders of 2.8 A radius over the C a positions. All segments have a-helical conformation, except for the C-segment of helix 4 and residues 296-299 within the mid-segment of helix 7, which have the 310 conformation. b The membrane normal was taken from the c-axis of the 2D crystal in the two-sided plane group p22121. Positive tilt angle means the helix runs from the extracellular side towards the cytoplasmic side as the sequence number increases. c Angle from the a-axis of the p22121 lattice to the projection of helix segments in the plane of the 2D crystals, by a right-handed rotation about the membrane nornial. terminate in the head group layer, whereas H3, H5 the transmembrane domain (Figure l(a)) has been and H6 reach into the cytoplasmic space. Residues described as forming a "plug" for the chromophore- 66, 138-141, 144-147 and 229-248 from the three binding pocket. 14 The two B-hairpins are cross- cytoplasmic loops are well exposed above the lipid linked by water-mediated H-bonds between main- head group layer (Figure 3(a)) and available for chain atoms of Tyrl0 in 132 and Pro180 in ~3 interactions with cytoplasmic proteins. 3°'38'39 The (Figure 4). Three water molecules (3, 5 and 13) are peripheral helix H8 is partitioned between the head responsible for this, of which one (13) was identified group layer and the cytoplasm and is slightly in the P41 structure. 19 These water molecules form inclined with its N terminus more exposed to the additional H-bonds to Gly182 and Gln184 in the cytoplasm than its C terminus. The polypeptide ~3-f~4 hairpin and to Tyr192 near 134, further linking following H8 turns to the cytoplasm, but the the 131-132 hairpin to the transmembrane domain palmitoylate chains on Cys322 and Cys323 are and securing the plug. Between Pro12 at the end of inserted into the hydrophobic zone. 132 and Pro23, the polypeptide forms an outward Transmembrane helices in rhodopsin are kinked "horn" (Figures l(a) and 4), which is rigid with a (see subsection Transmembrane domain), and only turn (residues 15-18) at the apex and several side- the N-terminal segments of H4 and H6 are oriented chain to main-chain H-bonds. The horn was roughly normal to the membrane (Table 2). suggested to act as a spacer between opposite Figure 3(b) shows that the mean residue B-factors faces of the retinal disc, 43 but the N-linked are strongly correlated with the depth in the bilayer, oligosaccharides on Asn2 and Asn15 may also irrespective of the helix tilt angles. The lowest contribute in this role (Figure l(a)). In our structure B-factors, on average, are found not at the midplane there is density for only three residues in each of the of the bilayer midplane but in a 4 A band to the oligosaccharide chains on Asn2 and Asn15:44 extracellular side of it. Therefore the dark state GlcNAc-(131,4)-GlcNAc-(~l,4)-mannose. The distal rhodopsin structure is most ordered at that depth in carbohydrate residues cannot be resolved, probably the membrane, which coincides with where the because they adopt heterogeneous orientations ionone ring of the 11-cis-retinal chromophore within the solvent channel. The orientations of the is located. Beyond the phosphate head group oligosaccharide chains relative to the protein differ positions (+20 A), the B-factors rise distinctly from those in the P41 structure, in which they more rapidly on the cytoplasmic surface than in were found to form intermolecular contacts. The the extracellular domain, which is due to confor- remainder of this domain contains two turns mational variability in several regions. (residues 22-25, 28-32) in the lipid head group layer adjacent to the transmembrane domain. Extracellular domain Many H-bonds and van der Waals interactions exist between the extracellular domain and the base The extraceUular domain includes the short ~1-B2 of the transmembrane domain, lending credence to hairpin, which together with the ~3-B4 hairpin in the view that the N-terminal domain may fold first 1418 Rhodopsin Structure in Trigonal Crystals

H3 #4 1-/6

;. / YI02

G280 F9

GIcNAc

Figure 4. Interface between the extracellular and transmembrane domains. Loops in the two domains are coloured blue and yellow, respectively. Note the water-mediated H-bonds linking 132 and 133, the stacking of Pro12 with Pro285 and Pro27 with Tyrl02, and the environment of Pro23. Amino acids are designated by the one-letter code for clarity in Figures 4-10. and provide a template to assist the assembly of the invertebrate opsins show few insertions (GPCRDB helical domain. 45 Figure 4 shows that Asn2 is in data baser). The longest insertion, of 13 residues, is contact with Gly280 and Asp282 in the E3 (H6-H7) located in the turn of the 131-132 hairpin; the others, loop, and Pro12 is stacked with Pro285 in the same of two, one and five residues each, are found after loop. Pro27 is stacked with Tyrl02 in the E1 (H2-H3) residue 332. Within the 131 and 132 strands visual loop, which is completely conserved among pigments share around 30% sequence identity. At vertebrate opsins. Pro23 and Gin28 form main- this level of similarity the invertebrate opsins can be chain H-bonds with Glyl01 and Tyrl02. Pro23, the expected 47 to contain a similar B-hairpin and most conserved (99%) residue in the extracellular accommodate the insertion on the extracellular domain of vertebrate opsins, is in a buried turn and surface (Figure 3(a)). in contact with Phe9 and Va111 in 132, and Gln28, as well as Tyrl02 and Phel03 in the E1 loop and Pro180 Transmembrane domain and Gln184 in the E2 loop. Mutations of Pro23 can prevent correct folding of both domains. Pro23His, Fitting cylinders of 2.8 A radius to the C ~ trace in the most frequent mutation in North American 0 48 divided the transmembrane helices into two or patients with retinitis pigmentosa, has been shown three straight segments each, with kink angles of 8 ° to cause aggregation of misfolded rhodopsin, which to 36 ° in-between (Table 2). Having kinks allows the is targeted for degradation by the ubiquitin proteo- tilted transmembrane helices to pack more inti- some pathway in cultured cells but also impairs the mately than otherwise possible and to enfold the degradation pathway, and that impairment may chromophore in a tight binding pocket. The kinks in underlie the neurodegenerative phenotype. 46 H1, H4, H5, H6 and H7 are caused by proline Sequences of visual pigments are highly conserved and, relative to vertebrate opsins, the t http://www.gpcr.org/7tm/ Rhodopsin Structure in Trigonal Crystals 1419 residues at positions 53, 170 171, 215, 267 and 302, Asp83 and Asn302 was required for activation of respectively. Except for Pro53 these proline residues G proteins, but their positions can be interchanged are among the most conserved residues in without loss of this activity. 57-59 The examples rhodopsin-like GPCRs, 1"49 so this set of kinks is suggest that different H-bonding interactions of expected at equivalent sequence positions through- these residues are required for stabilising the dark out this family, although the kink angles may vary state and for attaining the activated state. in a sequence-dependent manner. On the other In bovine rhodopsin Asp83 is not in contact with hand, H2 is distorted at the consecutive glycine the chromophore (Figure 5), but its replacement by residues Gly89-Gly90, which allows the side-chain Asn causes a slight blue shift of the absorption of Thr92 to H-bond to the main chain at residue 88. maximum, indicating an alteration of the Schiff base In H3 the kinks occur at Glu113, the counterion 5°-s2 environment. 6° Low-temperature Fourier transform for the protonated Schiff base in the dark state, and infrared (FTIR) difference spectroscopy showed next to the ionone ring of the chromophore. In H7 a structural changes in two internal water molecules second kink occurs near Lys296, which is at the start and peptide groups during the conversion to of the 3w segment and the site of chromophore bathorhodopsin, 61 the first intermediate state attachment. These kinks in H2, H3 and H7 are in following photo-isomerisation of the retinal, contact with the chromophore and would be shared which would have been reached within pico- by the visual pigments. Helix kinks have been noted seconds of light absorption had it occurred at in rhodopsin structures determined in the tetra- physiological temperature. 3"62"63 The spectral gonal form of 3D crystals and in the p22121 form of change due to one of the water molecules is 2D crystals; w'ls our definition (Table 2) differs abolished when the Glu113 counterion is replaced slightly from earlier descriptions regarding location by Gin, but the change due to the second water and angle of the kinks. molecule persists with a frequency shift. 61 The latter The kinks are also important as sites of lateral was thought to be vibrationally linked to the Schiff polar interactions, where rotation of the peptide base region through the peptide backbone and at a planes enables formation of inter-helical H-bonds location affected by replacement of Asp83 and and water-mediated H-bonds. Thus the helices Gly120. 61'64 This interpretation is borne out by the are linked by three-dimensional H-bonding net- water-mediated H-bonds between Asp83 and the works, ls'19 which in our refined structure are more peptide backbone of H3 and H7 (Figure 5), and extensive, laterally and perpendicular to the mem- Wat12 can be identified as that second water brane, than previously reported. molecule. Other FTIR studies showed that Asp83 Figure 5 shows one of the H-bonding networks is protonated in the dark state and remains so in the that involves Asn55 at the kink in H1, and also biochemically active metarhodopsin II (MII) state, Asp83 in H2, Gly120 in H3, Met257 and Trp265 in but it undergoes a change or increase of H-bonding H6, Ser298, Ala299, Tyr301 and Asn302 in H7, and on entering MII. 60 "65 Note that the internal water four water molecules including the hitherto un- molecules in the surrounding of Asp83 (Figure 5) identified Watl0. This network extends from Trp265 could, by small movements, facilitate rapid in the chromophore binding pocket via Watl0 all the exchange of H-bonds without requiring concerted way to the highly conserved NPxxY motif s3 movement of the protein. (Asn302-Tyr306 in bovine rhodopsin) in H7 close Trp265 in this H-bonding network was one of to the cytoplasmic limit of the hydrophobic zone the first chromophore contacts identified. 66'67 Its (see Figure 3(a)). Asn55 and Asp83 in this network side-chain is in a U-shaped bend formed by the are respectively the most conserved residues in H1 11-cis-retinal and the side-chain of Lys296, with the and H2 of rhodopsin-like GPCRs. 54 Trp265, Ser298, indole plane perpendicular to the membrane and in Ala299 and Asn302 are all widely conserved across contact with the ionone ring (Figures 5 and 6). this family, 1 and Met257 is conserved in vertebrate Resonance Raman data suggest that its micro- opsins. The proximity and interactions of these environment becomes less hydrophobic within residues are critical to the stability and function of picoseconds of photon absorption, which was GPCRs and of rhodopsin in particular. attributed to changing interaction with the ionone The H-bonds between Asn55, Asp83, between ring. 6s On MII formation, UV absorbance changes Asn55 and the peptide O of Ala299 in the kink of H7 also indicate a weakening of the indole H-bond in at the C terminus of its 310 segment, and the water- Trp265 and Trp126. 69 In the dark state structure the mediated H-bonds between Asp83 and Asn302 on side-chain of Trp265 is oriented with the NH group the other side of this H7 kink can be expected to pointing to the cytoplasmic side and forming an stabilise the packing of H1, H2 and H7 in the dark H-bond with Watl0 (Figure 5), which is located state. H-bonding between these conserved residues in-line with the NH group. The UV difference in MII underlies the apparent interaction amon~ corres- might indicate a net displacement of the Trp265 ponding residues in related GPCRs. 5s-59 For side-chain towards the extracellular side. example, the thyrotropin-releasing hormone The newly identified water molecule Watl0 is in receptor was completely inactivated by mutation close contact with the side-chain of Ala124 in H3 to a non-H-bonding residue at the position of Asp83 (Figure 5). In rhodopsin-like GPCRs the residue at or at both positions of Asn55 and Asn302.55- In position 124 is highly conserved, but the predomi- i several receptors an Asp-Asn pair at positions of nant amino acid is Ser. From this we infer that 1420 Rhodopsin Structure in Trigonal Crystals

El.?4

T62 114253 L72 H3

L 13 H7

L76 M257

H1 ,o303 L79 ~: 'i: ~,~ H6

e Y3oi w9 [953 N55

"... 1124

_ W265 A299 /,#,- ,$298 Gf20 ~ "....

0 G89 K296 G90

Figure 5. A transmembrane slice showing H-bonding networks and hydrophobic contacts between the retinal-binding pocket and the cytoplasmic surface.

Watl0 is a surrogate for the Ser OH in providing an Asp83 and Trp265 are both linked by H-bonds H-bond to the indole NH of Trp265, and further- through ordered water molecules to Asn302 and more that H-bonding between this pair of side- Met257. This extended H-bonding network is chains in H3 and H6 is a conserved feature of GPCR sealed from the cytoplasmic compartment by a structure in the inactive state. hydrophobic barrier made of six residues from The side-chain of Ser298 is adjacent to Trp265, but helices H2 (Leu76, Leu79), H3 (Leu128, Leu131) and it is H-bonded instead to the main chain O at H6 (Met253, Met257). Five of those residues are residue 295 (Figure 5). This side-chain to main- highly conserved in rhodopsin-like GPCRs, 1 and chain H-bond compensates for the elongation of the sixth, Met253, is 100% conserved in vertebrate helix pitch at the start of the 310 segment (residues visual pigments. 49 They are arranged with meth- 296-299). In rhodopsin-like GPCRs, position 298 is ionine residues on one side and leucine residues on highly conserved as Ser or Asn," both being the other (Figure 5) and could facilitate the relative residues capable of forming side-chain to main movement of H3 and H64 following photoactiva- chain H-bonds. Therefore it is likely, that H7 in the tion while retaining van der Waals contacts. wider family also contains a 310 segment at this level Replacement of Met257 by all other amino acids in the membrane. except Leu caused bovine opsin to activate Gt Rhodopsin Structure in Trigonal Crystals 1421

~~ H6

Figure 6. Environment of the 11-cis-refinal. (a) Environment of the protonated Schiff base and its counterion Glu113. (b) Environment of the ionone ring and the kinks in H6 and H7 cross-linked by a water molecule. constitutively. The level of constitutive activity is The kinks in H3 and H5 are intimately associated well correlated with the affinity of mutant opsins with the lining of the chromophore pocket. At for all-trans-retinal, suggesting that the mutations Glu113 in H3, a water molecule (Wat16) is disposed the apoprotein towards the active H-bonded between the peptide carbonyl and side- conformation normally attained only following chain carboxyl oxygen atoms. Consequently, the light-induced chromophore isomerisation. 7° By peptide oxygen atom of Glu113 is not H-bonded implication, Met257 in wild-type rhodopsin con- along the helix axis, hence the kink (Figure 6(a)). tributes a critical packing interaction. It is at the Wat16 is most likely the water molecule responsible junction between the H-bonding network and the for that low-temperature FTIR difference feature hydrophobic barrier, is in contact with Asn302 of between the dark and batho states, that was the NPxxY motif in H7, and prevents the helix abolished when Glu113 was replaced by Gin. 61 movement until this is triggered by chromophore OE1 of Glu113 is H-bonded to the peptide N of isomerisation. Cys187 in ~4 (Figure 6(a)), so the Schiff base Tyr306, at the cytoplasmic end of the NPxxY counterion is in contact with an H-bonding network motif in H7, is at the height of the lipid headgroup that leads from the chromophore-binding pocket, layer (Figure 3(a)) where it is H-bonded via Wat7 to through the plug, to the extracellular surface. This Thr62 in H1 and directly to Asn73 in H2 (Figure 5), network encompasses Tyr268 in H6, and residues two residues which are conserved in vertebrate Glu181, Gly182, Gln184, Tyr191 and Tyr192 from the visual pigments and rhodopsin-like GPCRs, [33-134 and [34-H5 loops (Figure 6(a)), as well as Tyrl0 respectively. Tyr306 is stacked against Phe313 in from ~2 and five water molecules. The H-bonds that H8, and similarly Ile307 in H7 is in contact with cross-link the ~1-B2 and ~3-[34 hairpins (Figure 4) Met317. Such interactions can relay movements of belong to this network. Tyr268, whose OH group is transmembrane helices to parts of the cytoplasmic 3.6 A from the 11,12-cis bond of the retinal in the surface, for example realigning H8 after the dark state (see Table 4 below) was implicated by photoisomerisation. Spin-label data on Tyr306, FTIR and Raman studies to participate in the initial Phe313 and Cys316 showed light-dark structural conformational change at the batho stage and in the differences, 71 and double spin labelling showed transition to the MII state. 68"78'79 movements by 2-4 A of the cytoplasmic end of H7 The second kink in H3 is in contact with the relative to that of H1, 72 and of H2 relative to H8. 73 ionone ring of the chromophore, where H3 and H5 The proper realignment of H8 appears to are linked due to a triangular set of H-bonds on be important in the interaction between the cyto- the side of the ionone ring away from Trp265 plasmic surface of rhodopsin and the C-terminal (Figure 6(b)). The Trp126 indole NH forms a pair of peptide of Gta, because the inhibitory mutations bifurcated H-bonds with OE1 of Glu122 and ND1 of either map to the structurally sensitive connecting His211, while OE2 of Glu122 is H-bonded to the region between the transmembrane H7 and backbone O of His211, which is so oriented because peripheral H8 (Asn310, Lys311 and Gln312), or of the kink in H5 at His211 due to Pro215. Glu122 is form buried contacts between H7 and H8 (Tyr306, known to be protonated (neutral) in the dark state Ile307, Phe313 and Met317). 39"74-77 and in MII. 6° These interactions account for the 1422 Rhodopsin Structure in Trigonal Crystals

(a) (b)

Figure 7. The non-conventional H-bond between a ring hydrogen of Phe203 in H5 and the backbone O atom in H4. (a) Electron density map around Phe203. (b) Hydrogen bonds between residues in H4, H5 and the several loops connecting these helices and the B3, f~4 strands. Four newly identified water molecules are located in this region. Besides Wat8 and Wat11 shown here, Wat14 is H-bonded to Pro194 O, Glu196 OE1 and Glu201 N, and Wat17 is H-bonded to Tyr192 O and to Gly280 N in the H6-H7 loop. For clarity, the side-chains of Tyr191 and Tyr192 have been omitted. Figures 7(a), 8(a) and 10 were prepared using 0. 48

FTIR observation that mutations of His211 influence Tyr185-Pr0186 in helix F of bacteriorhodopsin. 82 Glu122 through a third residue, which is itself That role is to provide a pivot for the outward tilt of affected by mutation of Glu122 to Ala but not to the cytoplasmic segment of H6 during the transition Gin. s° Figure 6(b) suggests that Trp126 is the third to MII as indicated by spin-label studies.4 The residue. These residues can be expected to show structures of dark-state bacteriorhodopsin and a sensitivity to movements of the ionone ring, with triple mutant mimic of its M-state (analogous to the which Glu122 is in direct contact. FTIR difference MII state in rhodopsin) were shown to differ mainly suggested stronger H-bonding of Glu122 on tran- by an outward tilt of the mutant's helix F by a sition to MI,80 '81 whereas UV absorption difference maximum of 3.5 ,~ at the cytoplasmic end, which indicated reduced hydrophobicity of the micro- can be described as a hinge movement extrapolat- environment for Trp126 as well as weaker ing to a pivot around the Tyr-Pro pair. s2 If a similar H-bonding of its indole NH caused by a general movement occurs in the transition of rhodopsin to conformational change in the formation of MII. 69 its MII state, then the water molecule between the The kinks in H6 and H7 are only 4.8 A apart H6 and H7 kinks would appear to be securing the between C ~ atoms of Tyr268 and Pro291. A water potential pivot in the configuration of the dark state. molecule (Watl), H-bonded to the backbone at At the kink in H4 near its extracellular end, the Tyr268, Pro291 and Ala295, bridges the kinks 2Fo-Fc map (Figure 7(a)) reveals a non-conven- (Figure 6(b)). H6 shows the most pronounced kink tional H-bond, with an aromatic ring hydrogen among the rhodopsin helices (Table 2) and contains atom of Phe203 in H5 acting as donor s3 to the the conserved Pro267-Tyr268 pair at this kink. carbonyl O atom of Cys167 as acceptoro(Figure 7(b)). Similarity in the light versus dark FTIR difference The donor-acceptor distance of 3.10 A indicates a between [2H]tyrosine-labelled rhodopsin and relatively strong H-bond. Residue 203 is Tyr in the bacteriorhodopsin has led to the proposal 79 that consensus sequence of rhodopsin-like GPCRs, and during photoactivation Pro267-Tyr268 in H6 it is Tyr in 76% of the visual pigment sequences but of rhodopsin may play an analogous role to Phe in the rest s4 such as bovine rhodopsin. While Rhodopsin Structure in Trigonal Crystals 1423

Table 3. Dihedral angles along the conjugated double bond system of the ll-cis-refinal chromophore

Observed values (deg.) Refinement restraints

Dihedral angle Molecule A Molecule B Average (error a) (deg.) Target (deg.) Weight (kcal/mol)

C4, C5, C6, C7 -179 177 179 (2) 180 100 C5, C6, C7, C8 -57 -53 -55 (2) None none

C6, C7, C8, C9 - 179 - 176 - 178 (2) 180 100 C7, C8, C9, C10 - 178 - 178 - 178 (0) 180 100 C8, C9, C10, Cll 179 179 179 (0) 180 100 C9, C10, Cll, C12 176 173 175 (2) 180 100 C10, Cll, C12, C13 - 10 - 15 - 13 (2) 0 50 Cll, C12, C13, C14 173 170 172 (2) 180 50 C12, C13, C14, C15 176 176 176 (0) 180 100 C13, C14, C15, N~ 177 178 178 (1) 180 100 C14, C15, N~, Ca 179 -179 179 (0) 180 100 a Error: deviation of observed values from their mean in the absence of noncrystallographic symmetry restraints on the retinal moiety.

Tyr can be expected to form an H-bond at this replacement model, we excluded the retinal as a test position, our observation demonstrates that in the against model bias, and we have built the initial low dielectric medium of the membrane interior, model of the chromophore from the small molecule Phe can replace Tyr in donating a stabilising crystal structure of 11-cis-retinal 2° by adjusting its H-bond to the conserved kink in H4. torsion angles to fit the cross-crystal averaged From the Pro170 at the kink in H4 to Asp190 at electron density map (see Methods), which was the end of the 133-134 hairpin, visual pigments consistent with three planar segments. Refinement contain nine highly conserved residues, attesting was carried out with planarity restraints imposed to the importance of this region to the structure and on the chromophore, as was appropriate at the function of rhodopsin, s4 Figure 7(b) shows some of medium resolution. However, the weights of the the H-bonds from the conserved Pro170, Pro171, restraints (Table 3) were set to be 5-12 times smaller Gly174, Trp175, Tyr178 and Asp190, which fasten than those on peptide bonds or aromatic rings, so the B-hairpin to the helix bundle. The hairpin is they could not have induced a planar conformation anchored to H3 by the disulphide bond between had it not been rooted in the X-ray data. Thus our Cys110 and Cys187, and its first and last residues, structure provides a credible description of the Arg177 and Asp190, form a salt-bridge (Figure 7(b)) retinal-protein interactions in the dark state. whose removal causes release of retinal from The torsion angle about the 11-cis bond is rhodopsin in the dark without apparently determined to be -13(+2) ° (Table 3 and Figure 6). increasing the accessibility of the Schiff base to This value is compatible with the calculation, based bulk solventY This destabilisation is probably on analysis of resonance Raman intensities, that the due to perturbation of the H-bonding network Cl1=C12 torsion angle increases, within 50 fs after (Figure 6(b)) that helps to position 134 at the "floor" excitation, from -15 ° in the dark state towards of the retinal-binding pocket. the 90 ° transition state for the cis-to-trans isomerisation. 86 Both the rapidity and stereospeci- Conformation and environment of the ficity of the initial torsional dynamics are driven by chromophore the strain on this non-planar segment, s6"87 The chirality and pucker of the ionone ring are also in Table 3 lists the torsion angles along the agreement with NMR data. s8 conjugated double bonds of the 11-cis-retinal and Figure 8(a) shows the electron density map its imine linkage to Lys296. Uncertainties were around the retinal. Figure 8(b) is a schematic estimated by comparing values in the two NCS diagram of the retinal-binding pocket, showing copies after one cycle of mock refinement in the H-bonds of Lys296 and Glu113, and the mostly absence of NCS restraints on the retinal moiety. The hydrophobic °contacts with the retinal within a chromophore is confirmed as containing three distance of 4 A. Those contact distances are given in planar segments, 2° with deviations from the Table 4. The elongated 11-cis-retinal buries about planarity of the n-bonding system about the 6s-cis, 220/~2 of surface area on a total of 22 residues that and 11-cis, 12s-trans bonds because of the steric form the binding pocket. Roughly one-third of this interactions between 5-CH3 and 8-H, and between area is contributed by Trp265 and Tyr268 in H6, 13-CH3 and 10-H, respectively. The conformation of another third is from Thr118, Glu122 in H3 and the chromophore in our structure is similar to that Met207, Phe212 in H5, and the rest is made up by 16 in the 1F88 coordinates for the tetragonal crystal residues from helices H3 to H7 and the plug 14 form, 14 and disagrees with the 1HZX 18 and 1L9H 19 (Ala117, Lys296, Cys187, Phe261, Ala269, Phe208, coordinates. For example, 1L9H shows a planar Ala292, Tyr191, Gly121, His211, Gly188, Cys167, 11-cis bond but significantly non-planar C8-C9 and Ile189, Glu181, Ser186 and Ala272 in descending C14-C15 bonds. It should be pointed out that when order of contact area). The large number of we used the 1F88 coordinates for the molecular contact residues in addition to its covalent linkage 1424 Rhodopsin Structure in Trigonal Crystals

(a)

(b) C~Z ('El Phe 293 ~co~

CF2 ~ N Retinal Binding Pocket

Thr 297

~ 3..~(")" Glu122 ~ ~

Me~ (X;IcG2( 3,25/ , , , Ala117 ./ c5 , c3 ~212 / Ala 295

ceu47~ Lys 296 c,o ~ c'~6 ~Met207 o CI7 c)El~ ; ~ I.... ~lle 189 G)~113 ~-o~ x d 2.!8 WAT 16 ,..~d././..

N~ 2.8~ Cys187 Gly188

Key Phe91 Non-ligand residues involved in hydrophobic Ligand bond "##~" contact(s) Thr 25 Non-ligand residues in contact using polar Non-ligand bond ")z~¢" atoms O ..... • Hydrogen bond and its length • Corresponding ligand atoms involved in contact(s) Figure 8. The retinal-binding pocket. (a) Electron density map around the 11-cis-retinal. (b) Schematic drawing of the binding pocket showing all residues within 4 A of the retinal and Lys296. This drawing was modified from the output of LIGPLOT) 48 • ° • ° ,

0 ..o

o (~ CO U t~ C',I (~ I I

• ° e,1 t~ P~

• • ° U P1 i~ (',1

u~ 0 U

~0 r-- CO • ° U o'1 c,~

r--

U t,1

~D o f,1 i U M 0"1 ~0 r~ ~D 0 :U c,1 o"1 t~l io • ° P1

~D ol

o'1

0 ,<

o co n~ o

U~ UUU U U 0 0 U 0 L) (J Z 0 U 0 0 U Z U 0 U ~ U L) L) U L;, U rJU L) UU U ~ U 0 0 U ~U ~

o ~ .~Y~

,-'4

~ r.tl ~ [-.t

~ 0 I

0 u 1426 Rhodopsin Structure in Trigonal Crystals contribute to a favourable binding enthalpy of to the anion. The Wat16-mediated H-bonds -11 kcal mol-1. 89 In our structure, the seven delocalise the negative charge on the Glu113 residues with the lowest B-factors, Trp265, Ala295, carboxylate group and lower its pKa. Stabilisation Ala269, Thr118, Phe91, Ala117 and Tyr268, are of the anion, aided by the precise geometry of the all located around the retinal-binding pocket ion pair, in turn stabilises the proton on the Schiff (Figure 8), which attests to the stabilising inter- base, elevating its pKa in the dark state to > 16. 99 action between the chromophore and its binding Therefore spontaneous deprotonation is kept at a pocket. This stabilisation is responsible for the low rate, which is essential for dim light vision. 1°° "inverse agonist" effect of the 11-cis-retinal that Change of geometry in the Schiff base region after keeps the basal activity of rhodopsin towards Gt in the photoisomerisation can destabilise the proton the dark very low, below that of the ligand-free and pave the way for the deprotonation of Schiff opsin, which is itself 106-fold less active than the base in MII. MII state with all-trans-retinal bound. 9° The three The distance between OE2 of Glu113 and the aromatic residues showing the largest chromophore Schiff base nitrogen atom is 3.2 A (Table 4) on both contact areas, Trp265, Tyr268 and Phe212, are highly. NCS copies, and is in agreement with the 3.1 A conserved throughout rhodopsin-like GPCRs," distanceoin the P41 crystal form. 19 This is less than suggesting common elements in the mechanism the 4.3 A distance implied by a similarity of 15N for stabilising the inactive receptor conformation chemical shift of the protonated Schiff base in from the boundaries of the ligand-binding pocket. rhodopsin and that in model retinylidene with Electrostatic interactions of the binding pocket iodide as counterion. 1°1 However, delocalisation of with the chromophore affect the visible absorption the negative charge in Glu113 effectively moves maximum. 91-94 Three residues near the ionone ring the centre of the counterion further °away correspond to the major colour ttming positions in from the protonated Schiff base by about 1 A, thus red/green cone pigments identified by primate weakening their electrostatic coupling to permit genetics. 95 Among these Phe261 and Ala269 are greater delocalisation of the positive charge from direct contacts of the ionone ring (Figure 6(b)), and the Schiff base into the polyene of the retinal, 1°2 mutagenesis to Tyr and Thr, respectively, repro- which was indicated by the nitrogen chemical shift. duced the predicted red shift, 91 which is consistent Therefore, Glu113 and Wat16 form a complex with a dipolar stabilisation of the positive charge counterion in the dark state, which both stabilises redistribution in the excited state chromophore the proton on the Schiff base and determines the towards the ionone ring. 94"96 The third residue, wavelength of the visible absorption maximum. Ala164, is a second shell contact of the ionone ring Mutating Glu113 to the smaller Asp results in a red via Glu122. In rhodopsin, its substitution by Ser shift without dependence on external chloride ions produced only a minor red shift, 91 but this can be is also consistent with an increase of the ion pair explained by the dipole contribution of Ser being distance. 1°3 overshadowed by the intervening Glu122, 97 since Recently, because the Glu181 to Gln mutation was replacing Glu122 by Asp was known to cause a blue found to lower the pKa of the Schiff base under shift. 66 In the red/green cone pigments, where the MI-like conditions, the counterion was proposed to conserved residue at the position 122 is Ile, a strong switch in MI to Glu181,1°4'1°s accompanied by tuning effect can be expected. reorganisation of the E2 loop coupled to movements The structure of the binding pocket is illustrated of H3. l°s The dark state structure suggests that the in Figure 6 with the Schiff base (Figure 6(a)) or the proposed backbone movements are not required for ionone ring (Figure 6(b)) in the foreground. In the the Glu181 side-chain to get close enough to the Schiff base region, the Lys296 side-chain is fully Schiff base to measurably influence its electrostatic extended and held in a channel between Met44, environment. The Schiff base NH bond in the Leu47 and Phe91 from H1 and H2 and Phe293 from all-trans retinal may be tilted more towards Ser186 H7, as previously reported. 14 The Glu113 side-chain and Glu181 on the H6, H7 side of the polyene is in an unusual rotamer because Wat16 is backbone (out of the page in Figure 6(a)), instead of H-bonded between its peptide O and carboxyl tilting towards Glu113 on the H3 side as in the dark OE2 atoms. The OE2 atom is thus placed in-line state structure. 1°6 The NMR result, that the ionone with the NH bond of the protonated Schiff base. ring and the adjacent segment of the retinal in MI Thr94 and Gly90 from H2 make close contact with despite being more relaxed remain largely in the Glu113 and Wat16 bound to it (see below). OE1 of dark state conformation, 1°7 is consistent with the Glu113 is H-bonded to the backbone of Cys187, retinal isomerisation up to this stage involving which is in a disulphide bond with Cys110 in H3. rotations mainly on the Schiff base side of the Thus the geometry of the Lys296, Glu113 ion pair is isomerised bond. There is also evidence that the stringently defined. The structure suggests that a retinal isomerisation impinges on Tyr268. 68"78 Its salt-bridge between them can also exist in the effect on the H-bonding network (Figure 6(b)) might apoprotein, where it is required for maintaining the allow the Glu181 side-chain to rotate towards the inactive state. 98 Schiff base. Such localised rearrangements from In Figure 8(b), Wat16 forms a shorter H-bond to the known dark state structure may apply to the the negatively charged carboxyl OE2 than to the invertebrate rhodopsins, which lack an acidic neutral peptide O, showing that it is strongly bound residue at position 113 and use a conserved residue Rhodopsin Structure in Trigonal Crystals 1427 corresponding to Glu181 as the counterion, 1°8 and the three cytoplasmic loops (C1, C2, C3) and to UV-absorbing pigments, which have a neutral adjoining ends of transmembrane helices (residues Glu113 in the dark state and switch to Glu181 for Gln64-Pro71, Glu134-His152 and Gln225-Arg252), the counterion in MI. 109"110 the peripheral helix H8 and adjoining loops Steric interactions with the binding pocket allow (residues Asn310-Asn326), and a di-peptide relaxation of the photo-isomerised retinal to trigger fragment from the C-terminal tail (Figure 9). conformational changes that result in Gt activation in MII. The ionone ring is under steric restraints from residues in helices H3, H5 and H6 (Table 4; Cl loop and C-terminal fragment Figure 6(b)) up to the MI state./°7 Evidence for a The C1 loop (residues 65-69) is very similar to change in the microenvironment of adjacent that in the P41 form. 14 It is well ordered (Figure 3(a)) Trp26569 in the formation of MII may indicate a and comprised of basic and hydrophobic residues movement of the ionone ring. A ring movement will (His65, Lys66, Lys67, Leu68, Arg69), which except impinge on Trp265, which is the most ordered for His65 are more than 97% conserved among residue in the dark state structure, the residue with vertebrate opsins, more conserved than the ends of the largest contact area with the retinal, and is H1 and H2. Leu68 and Arg69 point inward, making widely conserved among GPCRs. It may also affect van der Waals and H-bonding contacts with Phe261, a conserved aromatic residue in visual residues in H1, H2 and H8; the other residues pigments which is in contact with the C3, C4 edge of point outward to form a basic patch. The 2Fo-F c the ring and located in the cytoplasmic segment of map next to the C1 loop shows a strong density H6 one helical turn above Trp265 and one below feature with the flatness of a peptide plane in Met257 (Figure 5). The residue at position 261 in the contact with the side-chains of His65 and Lys67, glycoprotein hormone receptors has been shown to and the main chain from His65 to Lys67. Its aqueous play a similar role to Met257 in rhodopsin in location and shortness make it rather unlikely to forming a stabilising helix packing interaction belong to a detergent molecule. We have interpreted with Asn302 in H7. 7¢'111 Thus retinal interactions this density as a di-peptide from the disordered with the aromatic residues following photo- C-terminal tail and tentatively assigned it to Asp330 isomerisation could precipitate a release of the and Asp331, because of the basic contact site and packing interactions between H3 and H6 (see because the distance from the last ordered residue, previous subsection on Transmembrane domain), Asn326, can be spanned by three or more residues and between H6 and H7, 7° that stabilise the inactive with reasonable main chain torsion angles. The C state. As shown in Figure 5, and in Figure 9(a), terminus of the putative di-peptide points toward when the dark state structure is placed in the H6. No other interpretable density was found for membrane frame, the ionone ring appears vertically the C-terminal tail in the P31 crystal form. The below the conserved Glu134, Arg135 charge pair at presence of a thermolysin cleavage site between the cytoplasmic border of H3, where light-induced Pro327 and Leu328 116 and an Asp-N endoprotein- protonation of Glu134 is a prerequisite for ase cleavage site between Gly329 and Asp33016 is rhodopsin to bind Gt. 112-114 The H-bonding consistent with solvent exposure of the unobserved networks and van der Waals contacts described in 327-329 segment. Site-directed spin labelling of the transmembrane domain serve as conduits for rhodopsin in dodecylmaltoside micelles showed vectorial transmission of the conformational the C-terminal tail to be in equilibrium between an changes across this distance, in details that are ordered and disordered conformation, with the still unknown. Deletion of the 19-CH3 attached to disordered component increasing dramatically C9 of the polyene chain was shown to result in a beyond Asp331.°'* The density adjacent to loop C1 looser fit of the chromophore analogue in the probably corresponds to the partially ordered bindin~ pocket, consequently reducing the entropz component. In the P41 structure, loop C1 also 115", 89 gain that drives the conversion from MI to MII. forms contact with the C-terminal tail, but the Therefore the 19-CH3 in the native structure, chain orientations (Figure 2(b)) and residue assign- wedged between Thr118 from H3, and Ile189 and ments differ. Our tentative assignment of Asp330 Tyr191 from the plug (Figure 6(a)), appears to and Asp331 pointing towards H6 is reconcilable provide a grip against the floor of the binding with the report that the Cys mutant at position 338 pocket and restrict the relaxation of the chromo- in the C-terminal tail does not form a disulphide phore so that it is directed into a productive with the Cys mutant at position 65 in the C1 loop, activation. Thus steric interactions of all parts of but it does cross-link with Cys mutants at positions the chromophore are important for the activation. 242, 245 and 246 in H6, rapidly in the dark and slowly in the light. 117 Cytoplasmic domain C2 loop and cytoplasmic ends of I-t3 and H4 Having positioned the X-ray coordinates in the membrane frame (Figure 3(a)), we regard all The C2 loop (residues 141-149) is L-shaped residues above the mean level of phosphate groups in both crystal forms, but lies more parallel with (beyond 20 A from the mid-plane of the bilayer) as the membrane surface in the present structure belonging to the cytoplasmic domain. They include (Figure 2(b)). It is as if the two conformations 1428 Rhodopsin Structure in Trigonal Crystals

(a) H4 R147 C2 Loop

F148 A153 V137 C1 Loop H2 P71 J Y136 ~k~O ~ • - ....." : T229 H1 ~/A233 t L72 E24; Y306

A234 E249 t ~ M317 T251 K248 M309 C3 Loop 1307 T243 R314 T242

(b) I.tAt

Figure 9. The cytoplasmic domain. (a) Residues implicated in interaction with the heterotrimeric G protein transducin. The main chain is coloured in the four segments that project above the lipid phosphate groups and form the cytoplasmic domain: C1 loop region (residues 64-71), C2 loop region (134-152), C3 loop region (225-252), and H8 and C-terminal tail (310-326). The side-chain colours indicate: green, mutations causing constitutive Gt activation; red, residues cross-linked to Gt~; pink, mutations inhibiting stabilisation of MII by Gtc~ 340-350; yellow, mutations reducing Gt activation; greenish-yellow, gain-of-function for Gt activation upon reverse mutation from Ala; blue, Arg135 of the conserved E(D)RY motif; blue-grey, other residues discussed in the text. Residues falling in more than one category are coloured according to the former category. Long broken chains joining pairs of C a atoms marked by backbone spheres indicate inter - h ehcal'" cross-links• that inhibit-~ -~ • activation.• . 35 ' 119 The - footprintv. = of the 11-cis-retinal in the membrane plane (light pink) is shown in the background to illustrate its vertical alignment with key residues on the cytoplasmic surface. (b) Topography of the cytoplasmic surface showing a cavity adjacent to H3, with Leu131 at its bottom, and the Glu134- Arg135 pair forming a salt-bridge in its walls. Note the close contact of Arg135 with H6. Part Co) was prepared using PYMOL (http://pymol.sourceforge.net/). Rhodopsin Structure in Trigonal Crystals 1429 were related through a hinge movement, and Va1137-Va1139 may also contribute stabilising inter- comparisons with the NCS differences showed actions for Gt binding, as Va1138 is adjacent that it is most likely caused by different packing to Glu134 and Arg135 (Figure 9(a)). Mutating contacts in the two crystal forms. The coordinate individual residues of Tyr136-Va1139 to cysteine differences are above background level for also caused partial loss of Gt activation. 27 A spin residues 136-150 (Figure 2(a)), starting at the point label attached to Lys140 showed a rise and a fall of where the highly tilted H3 becomes accessible from mobility with time constants corresponding to the the surface (Figure 9). Thus the cytoplasmic tip of formation and decay of the MII state, la2 Therefore, H3 appears to bend with C2 as the loop is the cytoplasmic end of H3 is involved in the re-oriented. The middle of C2, where B-factors formation of the active state and binding of Gt. exceed 100 A 2 in both crystal forms, is probably In the C2 loop (residues 141-149), mutating rather flexible. Lys141 to Cys reduced Gt activation. 126 Reagents Peptides corresponding to the C2 and C3 loops, attached to this residue cross-linked photoactivated and H8, have been shown to interact with Gt.n18 rhodopsin to Gt~. 38 Replacing the sequence Residues important for Gt activation (Figure 9(a)) Lys141-Arg147 caused partial loss of Gt activation are found in the C terminus of H3 and in the C2 but did not prevent binding of Gt~ 340-350. 28 After loop. Glu134, Arg135 and Tyr136 form the highly mutating Lys141-Phe148 to polyalanine abolished conserved E(D)RY motif found in H3 of all activation, restoring only two residues from each rhodopsin-like GPCRs. In the dark state, Glu134 is end, namely Cys140, Lys141, Arg147 and Phe148, negatively charged and forms a salt-bridge with restored 50% of the activity, but the middle residues Arg135. The latter faces H6 and forms van der also contribute to activation, possibly by influen- Waals contacts with Va1250, Thr251 and Va1254 in cing the conformation of the loop.3gThe flexible H6. Protonation of Glu134, in addition to deproto- middle section might become more ordered when nation of the Schiff base, is required for rhodopsin MII binds to Gt and help to increase the affinity of to activate Gt. 114 The cytoplasmic surface of Gt binding. rhodopsin shows a cavity next to H3 (Figure 9(b)). Leu131 from the hydrophobic cluster that seals the inter-helical H-bonding network (Figure 5) from C3 loop and cytoplasmic ends of H5 and 1-16 the cytoplasm is visible from the opening. But the Helices H5 (residues 200-230) and H6 (residues cavity extends like a tunnel under Pro71 and 241-276) in our structure are both longer by Arg147 to reach Phe148 and Ala153, and may one helical turn at the cytoplasmic boundary in provide a site for transducin to interact with the P41 form. 19 Therefore the C3 loop is elevated rhodopsin. The Glu134, Arg135 charge pair is above the membrane surface (Figure 2(b)). This sequestered in the wall of this cavity in the dark loop has the highest B-factors in the whole state, so that they can form a salt-bridge, but Glu134 rhodopsin molecule (Figure 3(a)). However, these also has access to the aqueous phase to take up a B-factors are authentic indicators of main chain proton. Protonation is thought to cause Glu134 to flexibilities, since the relative scale of B-factors in move to a non-polar environment and favours the C3 loop compared to those in the C2 loop is binding to Gt. 113 Loss of the salt-bridge partner similar to the relative mobilities of these surface might also cause Arg135 to transfer to the aqueous loops measured by spin-label studies in detergent phase, thus breaking its van der Waals contacts with micelles. 43 During model building, main chain the residues along H6 (Figure 9(a)). This would fragments selected from a data base of well-refined, favour a parting of the cytoplasmic ends of H3 and high-resolution structures by the program O 48 for H6, which is believed to occur in the activation of their similarity to the densities of the cytoplasmic rhodopsin as indicated by studies using spin ends of H5 and H6 and the loop were tightly labelling, 4 metal chelation, 119 and disulphide clustered and all showed helix-like sections. After cross-linking (Figure 9(a)). 35 The Gln134 mutant, refinement with the best fitting fragment inserted which mimics the protonation, favours binding of into the C3 loop density, the real space correlation 113 rhodopsin to Gt and causes constitutive activity was 0.72 in the C3 loop, whereas the average for the of opsin. 112 This mutant shows increased mobility whole structttre was 0.95. The density was poor for of spin label attached to Cys140 in H3 or Cys316 in residues Ala233-Ala235 and Gln238, which have H8 in the dark, 12° which is otherwise associated the highest B-factors. Coordinates for the C3 loop with photoactivation. 121-123 Eliminating the charge represent the most probable conformation in a on Arg135, the other half of the salt-bridge, does not population of overlapping, variable conformations. cause the same mobility change. 12° However, the extra helical turn at the ends of H5 The binding of Gt or the C-terminal peptide of and H6 compared with the P41 coordinates is its ~-subunit (Gt~ 340-350) stabilises MII. 124'125 unambiguous. Replacing the Tyr136-Va1139 sequence reduced From residue 231 to 236 the C3 loop rises from the Gt activation by ~ 80% and abolished stabilisation membrane like an extension of H5, but with the axis of MII by Gt~ 340-350. 28 Tyr136, which points tilted from H5 towards the cytoplasmic tip of H3; outwards and does not interact with the rest of from residue 236 onwards, the loop follows another the protein, is an important determinant for spiral path running parallel with the membrane the stabilisation of MII by Gt~ 340-350, but until it joins H6. The tip of H6 from residue 244 to 1430 Rhodopsin Structure in Trigonal Crystals

241 is bent towards H5 to meet with the loop. The the membrane-aqueous interface by buried side- secondary structure of loop C3 consists of chain contacts with residues in H1 and H7 under- hydrogen-bonded turns and not regular 0~-helices,33 neath, such as the contacts of Phe313 with Thr58, but the periodicity of accessibilities detected by a Va161, Thr62 and Tyr306, and of Met317 with Leu57, previous spin-label study 33 is accounted for. Va161 and Ile307. The presence of Asn310 and Therefore the P31 structure and the spin label Lys311 are required for correct folding of the study both show the C3 loop to be well elevated rhodopsin. 39 If these two residues are kept, then from the bilayer and mobile but not unstructured. replacing all other residues in H8 except Phe313 and Residues around the C3 loop that were shown by Met317 by Ala, including replacing all outward- mutagenesis to affect Gt activation map to the pointing residues, produced a mutant pigment with cytoplasmic end of H5 (Leu226, Thr229 and a potency for Gt activation equal to the wild-type Va1230) 127 and the first turn of H6 (Thr242, Thr243 rhodopsm.-- • 39 The contacts of these four residues just and Gln244), 39"127 which are ordered in the dark described suggest that they are required for the state, and to the spiral extension of H5 (Ala233, correct tertiary structure and orientation of H8. Ala234), 29"127 which is highly mobile. All three Deleterious effects of mutations in the N-terminal regions project well above the cytoplasmic surface part of H8 are all interpretable in terms of in our structure (Figures 2(b) and 3(a)). Ser240 next perturbation of the interaction between H8 and to the N terminus of H6 is the residue most elevated the transmembrane domain. 75-77 It has been shown above the membrane. Reagents attached to the that trtmcating rhodopsin following Asn315 does Ser240Cys side-chain cross-linked the light- not diminish Gt activation. 13° Therefore, although activated rhodopsin mutant to Gto~.37"38'128 The H8 participates in binding of Gt, its role in cross-linked sites were located within the Gt~ activation of Gt is secondary to the C2 and C3 peptides 19-28, 38 310-313 and 342-345,128 which loop regions. overlap respectively with the three rhodopsin- binding regions of Gt~ mapped with synthetic peptides, 8-23, 311-323 and 340-350. I25 The Expected location of G protein interaction sites in mutants Lys141Cys and Lys248Cys can also be other GPCRs cross-linked to Gt~z.38 Replacing Glu247-Thr251 in In the C2 loop, residues important to activation H6 by unrelated sequence reduced Gt activation have been mapped in the conserved E(D)RY motif tenfold and abolished Gt0t 340-350 binding. 28 or just C-terminal to this. The C3 loop varies widely Restoring the sequence of Glu247-Glu249 restored in length among GPCRs. As seen in the bovine Gt0~ 340-350 binding, but only part of the Gt rhodopsin structure, however, residues believed to activation. 28 Tyr136-Va1139 and Glu247-Glu249 participate in Gt binding and activation are clus- were proposed to form a binding pocket for Gt= tered around the cytoplasmic ends of H5 and H6, 340-350.28In the dark state structure they map to and the spiral extension immediately above H5, H3 and H6 at similar heights from the membrane while residues shown by alanine scanning muta- (Figure 3(a)). The effects of the mutations may have genesis to have no effect on Gt activation29"map to resulted from interactions with other sites, since the C-terminal section of C3, which runs across the Va1250 and Thr251 are in contact with Arg135 in H3, gap between H5 and H6. We may expect that, in the Thr251 with Leu226 in H5 (Figure 9(a)), and Glu249 C3 region of other GPCRs, the Gt interaction sites with residues in H7 and H8 (see next subsection on will be similarly clustered with respect to the H8). In chimeras of the Gt-activating bovine transmembrane helices. In the cannabinoid recep- rhodopsin and Go-activating scallop rhodopsin, tor, the C3 loop is about 30 residues long and a small replacing the C2 loop with the scallop sequence helix was detected by NMR adjacent to the reduced activation of both G proteins, but replacing c y to p lasrmc" end of H5,*131 which is analogous to the C3 loop with the scallop sequence enhanced the H-bonded spiral extension of H5 in bovine activation of Go fourfold while depressing acti- rhodopsin, even though there is no sequence vation of Gt by 60%. 129 Therefore the C3 loop homology in this region. In the muscarinic acetyl- contains sites for specific activation of G protein. choline receptors, the C3 loops are very large, but The ability of the C3 loop of bovine rhodopsin to deletions of over 100 residues to reduce it to the discriminate between different Gt~ 340-345 length of C3 in bovine rhodopsin affected only the sequences may be a mechanism to ensure specificity desensitisation process and left the G protein- of coupling. 129 dependent functions intact, such as agonist and antagonist binding and phospholipase acti- vanon... -- 132 ' 133 Therefore it is likely that these residues H8 and adjoining loops mapped in the C2 and C3 regions of bovine The structure of H8 (residues 311-321) is very rhodopsin are the common G protein-binding sites similar in both crystal forms. Asn310 is the only in all GPCRs. non-helical residue between H7 and H8. Several side-chain to main chain H-bonds stabilise the Bound lipid and detergent molecules H7/H8 corner, such as from Glu249 to Met309 and Lys311, from Asn310 to Phe313 and from The presence of a bound phospholipid molecule Arg314 to Ile307. In addition, H8 is anchored at near the cytoplasmic ends of H6 and H7 Rhodopsin Structure in Trigonal Crystals 1431

(a) (b)

9-.~: '-dJ' ~" . " : '-~:~:~..\ ,,~? (

{~: '"->4"' ~(" 1't1~2u4 \ . . I't1~212 / .

" v'.:L" ; ,.:~.~ __.~ " r,.,,_',,s ~ :s > -7' _-..4:

t>th'J',t',~i~,/!l'li% %~T,, - >..- " :;~ ' .3- s's,<:,. L ] ". :X "/

"" <, . \ ,- i~ ,\ , # I'r,,2Ul 0 ~" ).,,-

(c) , :~,J " .:-<-C~~ ~:~ j

I,,..,. , 5- -~" ' l£~Y "~"': /,4 / / ~ ~ >,i //"! .1

.~ ~" tu/isn; Ilalh9 . I ll/.¢llll ttql~tgl [ _~,, ~ +- .

Figure 10. Bound lipid and detergent molecules. (a) Two acyl chains of a phospholipid molecule bound to H6 and H7 in the cytoplasmic half of rhodopsin. The cytoplasmic segment of H6 is orientated roughly along the membrane normal. The acyl chains are inclined relative to it. Met309 is the C-terminal residue of H7. (b) An ordered LDAO molecule bound to H5, H6 and H7just to the extracellular side of the kinks in H6 and H7. Pro267 is one of the contact residues. (c) A C8E4 molecule bound in the crevice between stacked helix bundles of the P31 crystals. 1432 Rhodopsin Structure in Trigonal Crystals

(Figure 10(a)) is indicated by densities for a pair to-hydrophilic contacts between the C8E4 and two of acyl chains with a mean separation of protein molecules, one of them presenting the 4.15(+0.02)A, which is characteristic of close- cytoplasmic end of H7 and H8 and the other packed hydrocarbon tails in an ordered bilayer. presenting the extracellular end of H4 and H5 The lipid acyl chains form contacts with residues in (Figure 10(c)). The choice of C8E4 as the detergent H6, Ile256, Ala260 and I1e263 in molecule A and and its interactions in this crevice most likely Ile256, Ala260 in molecule B, and in H7, Thr297, precipitated the P31 crystal form. Tyr301, Va1304, Ile305, Met309 in molecule A and Thr297, Ty301, Ile305 and Met309 in molecule B. The identity of this phospholipid is unknown, Methods because no density was observed for the head group, which is located in the solvent channel and is apparently disordered. Data collection and processing The addition of 0.05% LDAO to CsE4-solubilised rhodopsin was the key to obtaining isotropically Crystallisation and preparation of heavy atom derivatives are described in the ~companying paper) 6 diffracting crystals of this form. 16 The structure Complete data sets were obtained:~6~ combining wedges shows a single ordered LDAO molecule in addition of 20-30° collected from different positions on one or more to several partially ordered C8E4 molecules bound crystals. Data were processed using programs in the to each rhodopsin molecule. The LDAO is bound CCP4 suite. 136 Intensities were ii~tegrated to anisotropic just to the extracellular side of the H6 kink resolution limits using MOSFLM, and merged using (Figure 10(b)) and is itself kinked at the C5 atom, SCALA. A consistent indexing regime was maintained, with the amine oxide arm pointing roughly along by re-indexing each wedge under the four indexing H6 towards the extracellular face and the hydro- regimes of point group 3, and selecting the one leading to carbon arm running from H6 towards H5. The the lowest R-factor on amplitudes (Rderiv) , calculated using SCALEIT,relative to a previously merged reference contacts are with H6 mainly (Leu266, Pro267, data set or partial data set. Using the same procedure, Gly270, Va1271, Phe273, Tyr274), but also with data sets from non-isomorphous crystals were placed H5 (Phe208 and Phe212) and H7 (Phe287). In under a common indexing regime prior to cross-crystal wrapping around these helices, the LDAO may be averaging. The intensities were scaled in two rounds. The re-enforcing the more ordered molecular structure first round was done in the "batch" mode, with the batch originating from the extracellular leaflet (see showing the least negative B-factor serving as reference Figure 3(b)), therefore stabilising a particular for B-factor normalisation; batches found with relative protein conformation resulting in better ordered B-factors above 13, indicating significant radiation crystals. Pro267 in the binding site of LDAO has damage, were rejected from further scaling. The second been proposed as a possible hinge in H643"79 for the round used smooth scale and B-factors, in which secondary beam corrections in the form of spherical outward tilt of its cytoplasmic segment upon harmonic functions were determined for each crystal. photoactivation.4'3sm9 The presence of a hinge A "tails" correction for contributions from diffuse midway along H6 is supported by disulphide scattering was applied during both batch and smooth linking experiments showing a lack of relative scaling rounds. Twinning was detected by inspecting the movement between the extracellular ends of H6 cumulative intensity distribution given by TRUNCATE. and H5 during activation.TM Note that in the dark When it was present, the twin fraction was estimated and state structure H6 is kinked in a plane tangential intensities were de-twinned using the program to the helix bundle. In the activated form the DETWIN. TM cytoplasmic segment of H6 is kinked about the same place but in a radial plane instead. For this Molecular replacement from the 1 F88 coordinates and change to occur, the structure of the hinge region refinement may have to undergo a rotation first. Recently, cryo-EM of rhodopsin 2D crystals highly enriched Molecular replacement was done using the program for the MI state showed a local density change next AMoRe137 and the° rhodopsin coordinates in the P41 crystal form at 2.8 A resolution (PDB code 1F8814"17) as to the dark state position of Trp265, 135"_which would search model. The two rhodopsin molecules in the P41 be consistent with mobilisation of the hinge prior to asymmetric unit were superimposed to form one search the larger conformational changes required to enter model, but only the more ordered and more complete of the active MII state. Thus the LDAO molecule the two molecules (chain A) was used to calculate initial wrapped around the kink in H6 appears to stabilise phases. Non-protein atoms were omitted from the search the inactive conformation around a potential hinge. model and initial phase calculation. In the calculated The most completely modelled CsE4 molecule maps, the density for the retinal moiety, which was shows the octyl chain and three ordered oxyethy- omitted from the model, was used to assess the quality of lene groups out of the total four. The elongated the phasing. detergent molecule is intercalated in a tapered Molecular replacement was done in parallel against each data set in Table 1A, and each solution was subjected crevice between rhodopsin helix bundles, as they to rigid body refinement, using first one molecule, then are stacked along the 31-screw axis. Together with one helix, per rigid group. Cross-crystal averaging was the palmitoylate chains linked to Cys322 and carried out among Native-2, EMTS-1 and EMTS-2, Cys323 it fills this crevice (Figure 10(c)). There together with solvent flattening, 2-fold NCS averaging are hydrophobic-to-hydrophobic and hydrophilic- and histogram matching, in DMMULTI.138 The atomic Rhodopsin Structure in Trigonal Crystals 1433

model was rebuilt using 0 48 into the averaged map of cycle of solvent-flattening using the program dm 139 and a Native-2. solvent content of 75%, which was estimated from the The model was refined using CNS 22 with simulated volume per molecule in the P31 crystal 16 and a unit cell annealing and group B-factors. NCS restraints were thickness of 200 A in the 2D crystal. applied between the two molecules in the asymmetric The solvent-flattened EM map was re-calculated to 6 unit, but the restraints were relaxed in the loop regions. resolution for skeletonisafion, r4° The bones within the This was because in simulated annealing trials, the loop boundary of a single rhodopsin molecule were edited in residues showed systematically larger inter-molecular O to follow the high-density ridges along the helix axes, coordinate differences than residues in the helices, and they were annotated in O for the helices represented, regardless of whether the NCS restraints have been in accordance with the model of helix arrangement imposed over the loops, whereas excluding the loop proposed by Baldwin et al. 12 Using the bone_mask option regions from NCS restraints was found to reduce the in O and a 6 A radius for the bones atoms, a molecular free-R-factor and improve overall stereochemistry. mask was formed. The mask was edited using MAMATM Native-3 was not averaged with the other data sets, to remove internal cavities, and converted to the CCP4 but was subjected to 20 cycles of solvent flattening format on a 1 A sampling grid using MAMA2ccp4.136 (46% solvent) and 2-fold NCS averaging, during which The solvent-flattened EM map was interpolated onto the averaging radius was reduced and the resolution the 1A grid inside the molecular mask using extended from 2.9 A to 2.65 A. The resulting map showed MAPROT, 1~2 to yield the masked density of a single clear densities for the retinal and carbohydrate residues, rhodopsin molecule. The centre of gravity for the masked the palmitoyl modifications to Cys322 and Cys323, as well region was shifted to the origin of a P1 unit cell that has as a few tightly bound water and detergent molecules. 150 A cell edges and 90° cell angles, by editing the map The 1F88 model used for molecular replacement was header using the program 1MEDIT in the MRC image extensively rebuilt, especially the side-chain confor- processing suite.14°The asymmetric unit was filled with mations. Coordinates for the retinal and carbohydrate zeros outside the mask, using the program moieties and cytoplasmic loops were taken from the MAPMASK. TM This density of a single rhodopsin partially refined Native-2 model and rebuilt. Refinement molecule, cut out from the EM map, and placed in a against Native-3 data was done using simulated large P1 cell was the "EM model" for molecular annealing and individual B-factors, under NCS restraints replacement. imposed over the helical segments. When the free- Structure factors for the EM model were calculated R-factor decreased to 24.5%, ordered water molecules using SFALL.TM Cross-rotation function was calculated were located in the difference map using the water pick using GLRF,TM in which the best signal-to-noise ratio was task file in CNS. After another round of refinement with obtained by using a 28 A radius of Patterson integration individual B-factors, NCS copies of the bound water and a resolution limit of 9-5 A. Using MAPROT14Zagain, molecules were located in the aA-weighted 2Fo- Fc map the EM model was rotated about its centre of gravity to and confirmed in the solvent-flattened EDEN23 map the cross-rotation angles, and re-interpolated into the (kindly calculated by Dr Luca Jovine). Detergent and actual P31 unit cell at 1 A sampling intervals. Translation phospholipid molecules were added during refinement. searches were conducted using TFFC 145 in both P31 and Geometric parameters for the 11-cis-retinal were taken P32. The molecular orientation found 16 was subsequently from the small molecule structure, 2° except the 12-s-cis confirmed by molecular replacement from the 1F88 bond was made trans, and parameters for the protonated coordinates, once these became available. The molecular Schiff base were taken from the retinylidene structure. 21 orientations found using the EM model agreed with those In early rounds of the refinement, dihedral angles in the found subsequently from the atomic model to within 2 °. polyene section of retinal were tightly constrained to the However, the map of translation search from the EM plane using energy constants for the tryptophan ring model showed only streaks rather than discrete peaks. (500 kcal/mol), but for dihedrals about the C6-C7, Cll-C12 and C12-C13 bonds the energy constants were Docking X-ray coordinates to the EM map reduced tenfold. After addition of water molecules, the tight dihedral restraints along the polyene were relaxed to The C a coordinates of the 1F88 model and of the refined 100 kcal/mol. structure from this work were first manually aligned with Initial models for the detergent molecules LDAO and the "bones" for the EM map, which trace out the helix C8E4, and the hydrocarbon chains of phospholipids were axes. Then using the RSR_rigid option in O, in the taken from structures in the Protein Data Bank as convolution mode, the fit of all atoms of the protein provided by the HIC-UP web site% Topology parameters molecule to the EM density was optimised. for these groups and for the thioester linkage of palmitoylated cysteine were derived from structures in the Cambridge Small Molecule Database. Protein Data Bank accession numbers

Coordinates and structure factors have been released Molecular replacement from an EM model through the RCSB Protein Data Bank (code 1GZM, 1GZM-SF). From 2D crystals of bovine rhodopsin imaged by electron cryomicroscopy, a three-dimensional map has been calculated to 5 A resolution in the plane and 13.5 A normal to it. 1° The seven transmembrane helices appear as density rods of unknown azimuthal orientations. To Acknowledgements reduce the distortion caused by the missing cone in data collected from 2D crystals, this map was subjected to one We thank the ESRF, SPring-8, APS and Daresbury Laboratory for providing synchrotron radiation t http://alpha2.bmc.uu.se/hicup/ facilities and staff support during data collection, 1434 Rhodopsin Structure in Trigonal Crystals and in particular the ESRF for a long-term award to 17. Okada, T., Le Trong, I., Fox, B. A., Behnke, C. A., G.F.X. for use of the microfocus beamline ID13. We Stenkamp, R. E. & Palczewski, K. (2000). X-ray are grateful to Drs Richard Henderson, Paul diffraction analysis of three-dimensional crystals of Hargrave, Joyce Baldwin, Harry Powell and Alexei bovine rhodopsin obtained from mixed micelles. Murzin for helpful discussions; Luca Jovine for J. Struct. Biol. 130, 73--80. calculating the EDEN map from partially refined 18. Teller, D. C., Okada, T., Behnke, C. A., Palczewski, K. coordinates; Richard Henderson, Phil Evans, & Stenkamp, R. E. (2001). Advances in determination of a high-resolution three-dimensional structure of Andrew Leslie and Dan Oprian for comments on rhodopsin, a model of G-protein-coupled receptors the manuscript; colleagues at the MRC Laboratory (GPCRs). Biochemistry, 40, 7761-7772. of Molecular Biology for encouragement. We 19. Okada, T., Fujiyoshi, Y., Silow, M., Navarro, J., acknowledge the use of the GPCRDB data base. Landau, E. M. & Shichida, Y. (2002). Functional role of internal water molecules in rhodopsin revealed by X-ray crystallography. Proc. Natl Acad. References Sci. USA, 99, 5982-5987. 20. Gilardi, R. D., Karle, I. L. & Karle, J. (1972). The 1. Baldwin, J. M. (1993). The probable arrangement of crystal and molecular structure of 11-cis-retinal. Acta the helices in G protein-coupled receptors. EMBO J. Crystallog. sect. B, 28, 2605-2612. 12, 1693-1703. 21. Santarsiero, B. D. & James, M. N. G. (1990). Crystal 2. Hubbard, R. & Kropf, A. (1958). The action of light structure of N-methyl-N-phenylretinal iminium per- on rhodopsin. Proc. Natl Acad. Sci. USA, 44, 130-139. chlorate: a structural model for the bacteriorhodop- 3. Wald, G. (1968). Molecular basis of visual excitation. sin chromophore. J. Am. Chem. Soc. 112, 9416-9418. Science, 162, 230-239. 22. Briinger, A., Adams, P., Clore, G., DeLano, W., Gros, 4. Farrens, D. L., Altenbach, C., Yang, K., Hubbell, W. L. P., Grosse-Kunstleve, R. et al. (1998). Crystallography & Khorana, H. G. (1996). Requirement of rigid-body & NMR system: a new software suite for macro- motion of transmembrane helices for light activation molecular structure determination. Acta Crystallog. of rhodopsin. Science, 274, 768--770. sect. D, 54, 905-921. 5. Sakmar, T. E (1998). Rhodopsin: a prototypical G 23. Sz6ke, A. (1993). Holographic methods in X-ray protein-coupled receptor. Prog. Nucl. Acid Res. Mol. crystallography. II. Detailed theory and connections Biol. 59, 1-34. 6. Baylor, D. A., Matthews, G. & Yau, K. W. (1980). Two to other methods of crystallography. Acta Crystallog. components of electrical dark noise in toad retinal sect. A, 49, 853-866. rod outer segments. J. Physiol. 309, 591-621. 24. Read, R. (1986). Improved Fourier coefficients for 7. Scherfler, G. F., Villa, C. & Henderson, R. (1993). maps using phases from partial structures with Projection structure of rhodopsin. Nature, 362, errors. Acta Crystallog. sect. A, 42, 140-149. 770-772. 25. K6nig, B., Welte, W. & Hofmann, K. P. (1989). 8. Scherfler, G. E & Hargrave, P. A. (1995). Projection Photoactivation of rhodopsin and interaction with structure of frog rhodopsin in two crystal forms. transducin in detergent micelles-effect of doping Proc. Natl Acad. Sci. USA, 92, 11578-11582. with steroid molecules. FEBS Letters, 257, 163-166. 9. Unger, V. M. & Schertler, G. F. (1995). Low resolution 26. Franke, R. R., Konig, B., Sakmar, T. P., Khorana, H. G. structure of bovine rhodopsin determined by elec- & Hofmann, K. P. (1990). Rhodopsin mutants that tron cryo-microscopy. Biophys. J. 68, 1776-1786. bind but fail to activate transducin. Science, 250, 10. Krebs, A., Edwards, P. C., Villa, C., Li, J. & Schertler, 123-125. G. F. (2003). The three-dimensional structure 27. Ridge, K. D., Zhang, C. & Khorana, H. G. (1995). of bovine rhodopsin determined by electron Mapping of the amino acids in the cytoplasmic loop cryomicroscopy. J. Biol. Chem. 278, 50217-50225. connecting helices C and D in rhodopsin. Chemical 11. Davies, A., Gowen, B. E., Krebs, A. M., Schertler, G. E reactivity in the dark state following single cysteine & Saibil, H. R. (2001). Three-dimensional structure of replacements. Biochemistry, 34, 8804-8811. an invertebrate rhodopsin and basis for ordered 28. Acharya, S., Saad, Y. & Karnik, S. S. (1997). alignment in the photoreceptor membrane. J. Mol. Transducin-alpha C-terminal peptide binding site Biol. 314, 455-463. consists of C-D and E-F loops of rhodopsin. J. Biol. 12. Baldwin, J. M., Schertler, G. F. & Unger, V. M. (1997). Chem. 272, 6519-6524. An alpha-carbon template for the transmembrane 29. Shi, W., Osawa, S., Dickerson, C. D. & Weiss, E. R. helices in the rhodopsin family of G-protein-coupled (1995). Rhodopsin mutants discriminate sites receptors. J. Mol. Biol. 272, 144-164. important for the activation of rhodopsin kinase 13. Unger, V. M., Hargrave, P. A., Baldwin, J. M. & Schertler, G. F. (1997). Arrangement of rhodopsin and Gt. J. Biol. Chem. 270, 2112-2119. transmembrane alpha-helices. Nature, 389, 203-206. 30. Thurmond, R. L., Creuzenet, C., Reeves, P. J. & 14. Palczewski, K., Kumasaka, T., Hori, T., Behnke, C. A., Khorana, H. G. (1997). Structure and function in Motoshima, H., Fox, B. A. et al. (2000). Crystal rhodopsin: peptide sequences in the cytoplasmic structure of rhodopsin: a G protein-coupled receptor. loops of rhodopsin are intimately involved in Science, 289, 739-745. interaction with rhodopsin kinase. Proc. Natl Acad. 15. Bourne, H. R. & Meng, E. C. (2000). Structure. Sci. USA, 94, 1715-1720. Rhodopsin sees the light. Science, 289, 733-734. 31. Kisselev, O. G., Downs, M. A., McDowell, J. H. & 16. Edwards, P. C., Li, J., Burghammer, M., McDowell, J. H., Hargrave, P. A. (2004). Conformational changes in Villa, C., Hargrave, P. A. & Schertler, G. F. X. (2004). the phosphorylated C-terminal domain of rhodopsin Crystals of native and modified bovine rhodopsins during rhodopsin arrestin interactions. J. Biol. Chem. and their heavy metal derivatives. J. Mol. Biol. In the press. jmb56619 accompanying. 32. McDowell, J. H., Ubel, A., Brown, R. A. & Hargrave, Rhodopsin Structure in Trigonal Crystals 1435

P. A. (1986). Transglutaminase modification of 48. Jones, T. A., Zou, J. Y., Cowan, S. W. & Kjeldgaard, M. rhodopsin in retinal rod outer segment disk (1991). Improved methods for building protein membranes. Arch. Biochem. Biophys. 249, 506-514. models in electron density maps and the location 33. Altenbach, C., Yang, K., Farrens, D. L., Farahbakhsh, of errors in these models. Acta Crystallog. sect. A, 47, Z. T., Khorana, H. G. & Hubbell, W. L. (1996). 110-119. Structural features and light-dependent changes in 49. Horn, F., Bettler, E., Oliveira, L., Campagne, F., the cytoplasmic interhelical E-F loop region of Cohen, F. E. & Vriend, G. (2003). GPCRDB infor- rhodopsin: a site-directed spin-labeling study. mation system for G protein-coupled receptors. Biochemistry, 35, 12470-12478. Nucl. Acids Res. 31, 294-297. 34. Langen, R., Cai, K., Altenbach, C., Khorana, H. G. & 50. Sakmar, T. P., Franke, R. R. & Khorana, H. G. (1989). Hubbell, W. L. (1999). Structural features of the Glutamic acid-113 serves as the retinylidene Schiff C-terminal domain of bovine rhodopsin: a base counterion in bovine rhodopsin. Proc. Natl Acad. site-directed spin-labeling study. Biochemistry, 38, Sci. USA, 86, 8309-8313. 7918-7924. 51. Zhukovsky, E. A. & Oprian, D. D. (1989). Effect of 35. Cai, K., Klein-Seetharaman, J., Hwa, J., Hubbell, W. L. carboxylic acid side chains on the absorption & Khorana, H. G. (1999). Structure and function in maximum of visual pigments. Science, 246, 928--930. rhodopsin: effects of disulfide cross-links in the 52. Nathans, J. (1990). Determinants of visual pigment cytoplasmic face of rhodopsin on transducin acti- absorbance: identification of the retinylidene Schiff's vation and phosphorylation by rhodopsin kinase. base counterion in bovine rhodopsin.1. Biochemistry, Biochemistry, 38, 12893--12898. 16, 9746--9752. 36. McDowell, J. H., Nawrocki, J. P. & Hargrave, P. A. 53. Probst, W. C., Snyder, L. A., Schuster, D. I., Brosius, J. (1993). Phosphorylation sites in bovine rhodopsin. & Sealfon, S. C. (1992). Sequence alignment of the Biochemistry, 32, 4968-4974. G-protein coupled receptor superfamily. DNA Cell 37. Resek, J. F., Farrens, D. & Khorana, H. G. (1994). Biol. 11, 1-20. Structure and function in rhodopsin: covalent 54. Ballesteros, J. A. & Weinstein, H. (1995). Integrated crosslinking of the rhodopsin (metarhodopsin II)- methods for the construction of three dimensional transducin complex-the rhodopsin cytoplasmic face models and computational probing of structure- links to the transducin alpha subunit. Proc. Natl Acad. function relations in G-protein coupled receptors. Sci. USA, 91, 7643-7647. Methods Neurosci. 25, 366-428. 38. Itoh, Y., Cai, K. & Khorana, H. G. (2001). Mapping of 55. Perlman, J. H., Colson, A. O., Wang, W., Bence, K., contact sites in complex formation between light- Osman, R. & Gershengorn, M. C. (1997). Interactions activated rhodopsin and transducin by covalent between conserved residues in transmembrane crosslinking: use of a chemically preactivated helices 1, 2, and 7 of the thyrotropin-releasing reagent. Proc. Natl Acad. Sci. USA, 24, 4883-4887. hormone receptor. J. Biol. Chem. 272, 11937-11942. 39. Natochin, M., Gasimov, K. G., Moussaif, M. & 56. Flanagan, C. A., Zhou, W., Chi, L., Yuen, T., Rodic, V., Artemyev, N. O. (2003). Rhodopsin determinants for transducin activation: a gain-of-function Robertson, D. et al. (1999). The functional micro- domain in transmembrane helices 2 and 7 regulates approach. J. Biol. Chem. 278, 37574-37581. 40. Blaurock, A. E. & Wilkins, M. H. F. (1972). Structure expression, activation, and coupling pathways of the of retinal photoreceptor membranes. Nature, 236, gonadotropin-releasing hormone receptor. J. Biol. 313-314. Chem. 274, 28880-28886. 41. Saibil, H., Chabre, M. & Worcester, D. (1976). 57. Zhou, W., Flanagan, C., Ballesteros, J. A., Konvicka, Neutron diffraction studies of retinal rod outer K., Davidson, J. S., Weinstein, H. et al. (1994). segment membranes. Nature, 262, 266-270. A reciprocal mutation supports helix 2 and helix 7 42. Lewis, B. A. & Engelman, D. M. (1983). Lipid bilayer proximity in the gonadotropin-releasing hormone thickness varies linearly with acyl chain length in receptor. Mol. Pharmacol. 45, 165-170. fluid phosphatidylcholine vesicles. J. Mol. Biol. 166, 58. Sealfon, S. C., Chi, L., Ebersole, B. J., Rodic, V., 211-217. Zhang, D., Ballesteros, J. A. & Weinstein, H. (1995). 43. Hubbell, W. L., Altenbach, C., Hubbell, C. M. & Related contribution of specific helix 2 and 7 Khorana, H. G. (2003). Rhodopsin structure, residues to conformational activation of the dynamics, and activation: a perspective from crystal- serotonin 5-HT2A receptor. J. Biol. Chem. 270, lography, site-directed spin labeling, sulfhydryl 16683-16688. reactivity, and disulfide cross-linking. Advan. Protein 59. Donnelly, D., Maudsley, S., Gent, J. P., Moser, R. N., Chem. 63, 243-290. Hurrell, C. R. & Findlay, J. B. (1999). Conserved polar 44. Fukuda, M. N., Papermaster, D. S. & Hargrave, P. A. residues in the transmembrane domain of the (1979). Rhodopsin carbohydrate. Structure of small human tachykinin NK2 receptor: functional roles oligosaccharides attached at two sites near the NH2 and structural implications. Biochem. J. 339, 55-61. terminus. J. Biol. Chem. 254, 8201-8207. 60. Fahmy, K., Jager, F., Beck, M., Zvyaga, T. A., Sakmar, 45. Doi, T., Molday, R. S. & Khorana, H. G. (1990). Role of T. P. & Siebert, F. (1993). Protonation states of the intradiscal domain in rhodopsin assembly and membrane-embedded carboxylic acid groups in function. Proc. Natl Acad. Sci. USA, 87, 4991-4995. rhodopsin and metarhodopsin II: a Fourier- 46. Illing, M. E., Rajan, R. S., Bence, N. F. & Kopito, R. R. transform infrared spectroscopy study of (2002). A rhodopsin mutant linked to autosomal site-directed mutants. Proc. Natl Acad. Sci. USA, 90, dominant retinitis pigmentosa is prone to aggregate 10206--10210. and interacts with the ubiquitin proteasome system. 61. Nagata, T., Terakita, A., Kandori, H., Kojima, D., J. Biol. Chem. 277, 34150-34160. Shichida, Y. & Maeda, A. (1997). Water and peptide 47. Chothia, C. & Lesk, A. M. (1986). The relation backbone structure in the active center of bovine between the divergence of sequence and structure rhodopsin. Biochemistry, 36, 6164-6170. in proteins. EMBO J. 5, 823-826. 62. Wang, Q., Schoenlein, R. W., Peteanu, L. A., Mathies, 1436 Rhodopsin Structure in Trigonal Crystals

R. A. & Shank, C. V. (1994). Vibrationally coherent of the fourth cytoplasmic loop of rhodopsin modu- photochemistry in the femtosecond primary event of lates rhodopsin-transducin interaction. ]. Biol. Chem. vision. Science, 266, 422--424. 275, 1930-1936. 63. Kandori, H., Shichida, Y. & Yoshizawa, T. (2001). 76. Ernst, O. P., Meyer, C. K., Marin, E. P., Henklein, P., Photoisomerization in rhodopsin. Biochemistry Fu, W. Y., Sakmar, T. P. & Hofmann, K. P. (2000). (Mosc), 66, 1197-1209. Mutation of the fourth cytoplasmic loop of rhodop- 64. Nagata, T., Terakita, A., Kandori, H., Shichida, Y. & sin affects binding of transducin and peptides Maeda, A. (1998). The hydrogen-bonding network of derived from the carboxyl-terminal sequences of water molecules and the peptide backbone in the transducin alpha and gamma subunits. J. Biol. Chem. region connecting Asp83, Gly120, and Glu113 in 275, 1937-1943. bovine rhodopsin. Biochemistry, 37, 17216-17222. 77. Cai, K., Klein-Seetharaman, J., Farrens, D., Zhang, C., 65. Rath, P., DeCaluwe, L. L., Bovee-Geurts, P. H., Altenbach, C., Hubbell, W. L. & Khorana, H. G. DeGrip, W. J. & Rothschild, K. J. (1993). Fourier (1999). Single-cysteine substitution mutants at amino transform infrared difference spectroscopy of acid positions 306-321 in rhodopsin, the sequence rhodopsin mutants: light activation of rhodopsin between the cytoplasmic end of helix VII and the causes hydrogen-bonding change in residue aspartic palmitoylation sites: sulfhydryl reactivity and acid-83 during meta II formation. Biochemistry, 32, transducin activation reveal a tertiary structure. 10277-10282. Biochemistry, 38, 7925-7930. 66. Nakayama, T. A. & Khorana, H. G. (1991). Mapping 78. DeGrip, W. J., DeLange, F., Klaassen, C. H., of the amino acids in membrane-embedded helices Verdegem, P. J., Wallace-Williams, S., Creemers, that interact with the retinal chromophore in bovine A. F. et al. (1999). Photoactivation of rhodopsin: rhodopsin. J. Biol. Chem. 266, 4269-4275. interplay between protein and chromophore. In 67. Nakayama, T. A. & Khorana, H. G. (1990). Novartis Found Symposium (Yoshizawa, T., ed.), Orientation of retinal in bovine rhodopsin deter- vol. 224, pp. 102-123, Wiley, Chichester. mined by cross-linking using a photoactivatable 79. DeLange, F., Klaassen, C. H., Wallace-Williams, S. E., analog of 11-cis-retinal. J. Biol. Chem. 265, Bovee-Geurts, P. H., Liu, X. M., DeGrip, W. J. & 15762-15769. Rothschild, K. J. (1998). Tyrosine structural changes 68. Kim, J. E., Pan, D. & Mathies, R. A. (2003). detected during the photoactivation of rhodopsin. Picosecond dynamics of G-protein coupled receptor J. Biol. Chem. 273, 23735-23739. activation in rhodopsin from time-resolved UV 80. Beck, M., Sakmar, T. P. & Siebert, E (1998). Spectro- resonance Raman spectroscopy. Biochemistry, 42, scopic evidence for interaction between trans- 5169-5175. membrane helices 3 and 5 in rhodopsin. 69. Lin, S. W. & Sakmar, T. P. (1996). Specific tryptophan Biochemistry, 37, 7630-7639. UV-absorbance changes are probes of the transition 81. Furutani, Y., Kandori, H. & Shichida, Y. (2003). of rhodopsin to its active state. Biochemistry, 35, Structural changes in lumirhodopsin and meta- 11149-11159. rhodopsin I studied by their photoreactions at 70. Han, M., Smith, S. O. & Sakrnar, T. P. (1998). 77 K. Biochemistry, 42, 8494-8500. Constitutive activation of opsin by mutation of 82. Subramaniam, S. & Henderson, R. (2000). Molecular methionine 257 on transmembrane helix 6. mechanism of vectorial proton translocation by Biochemistry, 37, 8253--8261. bacteriorhodopsin. Nature, 406, 653-657. 71. Altenbach, C., Cai, K., Khorana, H. G. & Hubbell, 83. Levitt, M. & Perutz, M. E (1988). Aromatic rings W. L. (1999). Structural features and light-dependent act as hydrogen bond acceptors. J. Mol. Biol. 201, changes in the sequence 306-322 extending from 751-754. helix VII to the palmitoylation sites in rhodopsin: a 84. Horn, F., Bettler, E., Oliveira, L., Campagne, F., site-directed spin-labeling study. Biochemistry, 38, Cohen, E E. & Vriend, G. (2003). GPCRDB infor- 7931-7937. mation system for G protein-coupled receptors. 72. Altenbach, C., Cai, K., Klein-Seetharaman, J., Nucl. Acids Res. 31, 294-297. Khorana, H. G. & Hubbell, W. L. (2001). Structure 85. Janz, J. M., Fay, J. E & Farrens, D. L. (2003). Stability and function in rhodopsin: mapping light- of dark-state rhodopsin is mediated by a conserved dependent changes in distance between residue 65 ion-pair in intradiscal loop E-2. J. Biol. Chem. 278, in helix TM1 and residues in the sequence 306-319 at 16982-16991. the cytoplasmic end of helix TM7 and in helix H8. 86. Mathies, R. A. (1999). Photons, femtoseconds and Biochemistry, 40, 15483-15492. dipolar interactions: a molecular picture of the 73. Altenbach, C., Klein-Seetharaman, J., Cai, K., primary events in vision. In Rhodopsins and Photo- Khorana, H. G. & Hubbell, W. L. (2001). Structure transduction (Yoshizawa, T., ed.), vol. 224, pp. 70-89, and function in rhodopsin: mapping light- Wiley, Chichester. dependent changes in distance between residue 87. Kochendoerfer, G. G., Verdegem, P. J., van der Hoef, 316 in helix 8 and residues in the sequence 60-75, I., Lugtenburg, J. & Mathies, R. A. (1996). Retinal covering the cytoplasmic end of helices TM1 and analog study of the role of steric interactions in the TM2 and their connection loop CL1. Biochemistry, 40, excited state isomerization dynamics of rhodopsin. 15493-15500. Biochemistry, 35, 16230--16240. 74. Fritze, O., Filipek, S., Kuksa, V., Palczewski, K., 88. Creemers, A. E, Kiihne, S., Bovee-Geurts, P. H., Hofmann, K. P. & Ernst, O. P. (2003). Role of the DeGrip, W. J., Lugtenburg, J. & de Groot, H. J. (2002). conserved NPxxY(x)5,6F motif in the rhodopsin (1)H and (13)C MAS NMR evidence for pronounced ground state and during activation. Proc. Natl Acad. ligand-protein interactions involving the ionone Sci. USA, 100, 2290-2295. ring of the retinylidene chromophore in rhodopsin. 75. Marin, E. P., Krishna, A. G., Zvyaga, T. A., Isele, J., Proc. Natl Acad. Sci. USA, 99, 9101-9106. Siebert, F. & Sakmar, T. P. (2000). The amino terminus 89. Cooper, A. & Converse, C. A. (1976). Energetics of Rhodopsin Structure in Trigonal Crystals 1437

primary processes in visual excitation: photo- 106. Pan, D., Ganim, Z., Kim, J. E., Verhoeven, M. A., calorimetry of rhodopsin in rod outer segment Lugtenburg, J. & Mathies, R. A. (2002). Time- membranes. Biochemistry, 15, 2078-2970. resolved resonance Raman analysis of chromophore 90. Melia, T. J., Cowan, C., Angleson, J. & Wensel, T. structural changes in the formation and decay of (1997). A comparison of the efficiency of G protein rhodopsin's BSI intermediate. J. Am. Chem. Soc. 124, activation by ligand-free and light-activated forms of 4857-4864. rhodopsin. Biophys. J. 93, 3182-3191. 107. Spooner, P. J., Sharpies, J. M., Goodall, S. C., Seedorf, 91. Chan, T., Lee, M. & Sakmar, T. P. (1992). Introduction H., Verhoeven, M. A., Lugtenburg, J. et al. (2003). of hydroxyl-bearing amino acids causes bathochro- Conformational similarities in the beta-ionone ring mic spectral shifts in rhodopsin. Amino acid region of the rhodopsin chromophore in its ground substitutions responsible for red-green color pig- state and after photoactivation to the metarhodop- ment spectral tuning. J. Biol. Chem. 267, 9478-9480. sin-I intermediate. Biochemistry, 42, 13371-13378. 92. Lin, S. W., Kochendoerfer, G. G., Carroll, K. S., 108. Terakita, A., Yamashita, T. & Shichida, Y. (2000). Wang, D., Mathies, R. A. & Sakmar, T. P. (1998). Highly conserved glutamic acid in the extracellular Mechanisms of spectral tuning in blue cone IV-V loop in rhodopsins acts as the counterion in visual pigments. Visible and Raman spectroscopy retinochrome, a member of the rhodopsin family. of blue-shifted rhodopsin mutants. J. Biol. Chem. Proc. Natl Acad. Sci. USA, 97, 14263-14267. 273, 24583-24591. 109. Birge, R. R. & Knox, B. E. (2003). Perspectives on the 93. Kochendoerfer, G. G., Lin, S. W., Sakmar, T. P. & counterion switch-induced photoactivation of the G Mathies, R. A. (1999). How color visual pigments are protein-coupled receptor rhodopsin. Proc. Natl Acad. tuned. Trends Biochem. Sci. 24, 300-305. Sci. USA, 100, 9105-9107. 94. Fasick, J. I., Lee, N. & Oprian, D. D. (1999). Spectral 110. Kusnetzow, A. K., Dukkipati, A., Babu, K. R., Ramos, tuning in the human blue cone pigment. L., Knox, B. E. & Birge, R. R. (2004). Vertebrate Biochemistry, 38, 11593-11596. ultraviolet visual pigments: protonation of the 95. Neitz, M., Neitz, J. & Jacobs, G. H. (1991). Spectral retinylidene Schiff base and a counterion switch tuning of pigments underlying red-green color during photoactivation. Proc. Natl Acad. Sci. USA, vision. Science, 252, 971-974. 101, 941-946. 96. Birge, R. R. & Hubbard, L. M. (1980). Molecular 111. Govaerts, C., Lefort, A., Costagliola, S., Wodak, S. J., dynamics of cis-trans isomerization in rhodopsin. Ballesteros, J. A., Van Sande, J. et al. (2001). J. Am. Chem. Soc. 102, 2195-2205. A conserved Asn in transmembrane helix 7 is an 97. Imai, H., Kojima, D., Oura, T., Tachibanaki, S., on/off switch in the activation of the thyrotropin Terakita, A. & Shichida, Y. (1997). Single amino receptor. J. Biol. Chem. 276, 22991-22999. acid residue as a h~nctional determinant of rod and 112. Cohen, G. B., Yang, T., Robinson, P. R. & Oprian, D. D. cone visual pigments. Proc. Natl Acad. Sci. USA, 94, (1993). Constitutive activation of opsin: influence of 2322-2326. charge at position 134 and size at position 296. 98. Rao, V. R. & Oprian, D. D. (1996). Activating Biochemistry, 32, 6111-6115. mutations of rhodopsin and other G protein-coupled 113. Fahmy, K. & Sakmar, T. P. (1993). Regulation of receptors. Annu. Rev. Biophys. Biomol. Struct. 25, the rhodopsin-transducin interaction by a highly 287-314. conserved carboxylic acid group. Biochemistry, 32, 99. Steinberg, G., Ottolenghi, M. & Sheves, M. (1993). 7229-7236. pKa of the protonated Schiff base of bovine 114. Arnis, S. & Hofmann, K. P. (1993). Two different rhodopsin. A study with artificial pigments. Biophys. forms of metarhodopsin II: Schiff base deprotonation J. 64, 1499-1502. precedes proton uptake and signaling state. Proc. 100. Hecht, S., Schlaer, S. & Pirenne, M. H. (1942). Energy, Natl Acad. Sci. USA, 90, 7849-7853. quanta, and vision. J. Gen. Physiol. 25, 819~840. 115. Vogel, R., Fan, G. B., Sheves, M. & Siebert, F. (2000). 101. Creemers, A. F. L., Klaassen, C. H. W., Bovee-Geurts, The molecular origin of the inhibition of transducin P. H. M., Kelle, R., Kragl, U., Raap, J. et al. (1999). activation in rhodopsin lacking the 9-methyl group Solid state 15N NMR evidence for a complex Schiff of the retinal chromophore: a UV-vis and FTIR base counterion in the visual G-protein-coupled spectroscopic study. Biochemistry, 39, 8895-8908. receptor rhodopsin. Biochemistry, 38, 7195-7199. 116. Bayramashvili, D. I., Drachev, A. L., Drachev, L A., 102. Irving, C. S., Byers, G. W. & Leermakers, P. A. (1970). Kaulen, A. D., Kudelin, A. B., Martynov, V. I. & Spectroscopic model for the visual pigments. Skulachev, V. P. (1984). Proteinase-treated photo- Influence of microenvironmental polarizability. receptor discs. Photoelectric activity of the partially- Biochemistry, 9, 858-864. digested rhodopsin and membrane orientation. 103. Sakmar, T. P., Franke, R. R. & Khorana, H. G. (1991). Eur. J. Biochem. 142, 583-590. The role of the retinylidene Schiff base counterion in 117. Cai, K., Langen, R., Hubbell, W. L. & Khorana, H. G. rhodopsin in determining wavelength absorbance (1997). Structure and function in rhodopsin: and Schiff base pKa. Proc. Natl Acad. Sci. USA, 88, topology of the C-terminal polypeptide chain in 3079-3083. relation to the cytoplasmic loops. Proc. Natl Acad. Sci. 104. Yan, E. C., Kazmi, M. A., De, S., Chang, B. S., Seibert, USA, 94, 14267-14272. C., Marin, E. P. et al. (2002). Function of extracellular 118. K6nig, B., Arendt, A., McDowell, J. H., Kahlert, M., loop 2 in rhodopsin: glutamic acid 181 modulates Hargrave, P. A. & Hofmann, K. P. (1989). Three stability and absorption wavelength of metarhodop- cytoplasmic loops of rhodopsin interact with sin II. Biochemistry, 41, 3620-3627. transducin. Proc. Natl Acad. Sci. USA, 86, 6878-6882. 105. Yan, E. C., Kazmi, M. A., Ganim, Z., Hou, J. M., Pan, 119. Sheikh, S. P., Zvyaga, T. A., Lichtarge, O., Sakmar, D., Chang, B. S. et al. (2003). Retinal counterion T. P. & Bourne, H. R. (1996). Rhodopsin activation switch in the photoactivation of the G protein- blocked by metal-ion-binding sites linking trans- coupled receptor rhodopsin. Proc. Natl Acad. Sci. membrane helices C and F. Nature, 383, 347-350. USA, 100, 9262-9267. 120. Kim, J. M., Altenbach, C., Thurmond, R. L., Khorana, 1438 Rhodopsin Structure in Trigonal Crystals

H. G. & Hubbell, W. L. (1997). Structure and function threonine-rich domain regulates internalization of in rhodopsin: rhodopsin mutants with a neutral muscarinic cholinergic receptors. J. Biol. Chem. 268, amino acid at E134 have a partially activated 6862-6865. conformation in the dark state. Proc. Natl Acad. Sci. 133. Hulme, E. C. & Curtis, C. A. (1998). Purification of USA, 94, 14273-14278. recombinant M1 muscarinic acetylcholine receptor. 121. Resek, J. F., Farahbakhsh, Z. T., Hubbell, W. L. & Biochem. Soc. Trans. 26, $361. Khorana, H. G. (1993). Formation of the meta II 134. Struthers, M., Yu, H. & Oprian, D. D. (2000). photointermediate is accompanied by conforma- G protein-coupled receptor activation: analysis of a tional changes in the cytoplasmic surface of highly constrained "straitjacketed" rhodopsin. rhodopsin. Biochemistry, 32, 12025-12032. Biochemistry, 39, 7938-7942. 122. Farahbakhsh, Z. T., Hideg, K. & Hubbell, W. L. 135. Ruprecht, J. J., Mielke, T., Vogel, R., Villa, C. & (1993). Photoactivated conformational changes in Schertler, G. F. X. (2004). Electron crystallography rhodopsin: a time-resolved spin label study. Science, reveals the structure of metarhodopsin I. EMBO J. 23, 262, 1416-1419. 3609-3620. 123. Farahbakhsh, Z. T., Ridge, K. D., Khorana, H. G. & 136. The Collaborative Computational Project, Number 4. Hubbell, W. L. (1995). Mapping light-dependent (1994). The CCP4 suite: programs for protein crystal- structural changes in the cytoplasmic loop connect- lography. Acta Crystallog. sect. D, 50, 760-763. ing helices C and D in rhodopsin: a site-directed spin 137. Navaza, J. (2001). Implementation of molecular labeling study. Biochemistry, 34, 8812-8819. replacement in AMoRe. Acta Crystallog. sect. D, 57, 124. Emeis, D., Kuhn, H., Reichert, J. & Hofmann, K. P. 1367-1372. (1982). Complex formation between metarhodopsin 138. Cowtan, K. (1994). "dm": an automated procedure II and GTP-binding protein in bovine photoreceptor for phase improvement by density modification. membranes leads to a shift of the photoproduct Joint CCP4 ESRF-EABM Newsletter Protein Crystallog. equilibrium. FEBS Letters, 143, 29-34. 31, 34-38. 125. Hamm, H. E., Deretic, D., Arendt, A., Hargrave, P. A., 139. Cowtan, K. & Main, P. (1998). Miscellaneous Koenig, B. & Hofmann, K. P. (1988). Site of G protein algorithms for density modification. Acta Crystallog. binding to rhodopsin mapped with synthetic peptides sect. D, 54, 487-493. from the alpha subunit. Science, 241, 832-835. 140. Greer, J., Kleywegt, G. J. & Jones, T. A. (1996). 126. Ridge, K. D., Lu, Z., Liu, X. & Khorana, H. G. (1995). xdlMAPMAN and xdlDATAMAN-programs for Structure and function in rhodopsin. Separation and reformatting, analysis and manipulation of bioma- characterization of the correctly folded and mis- cromolecular eletron-density maps and reflection folded opsins produced on expression of an opsin data sets. Acta Crystallog. sect. D, 52, 826-828. mutant gene containing only the native intradiscal 141. Kleywegt, G. J. & Jones, T. A. (1999). Software for cysteine codons. Biochemistry, 34, 3261-3267. handling macromolecular envelopes. Acta Crystallog. 127. Yang, K., Farrens, D. L., Hubbell, W. L. & Khorana, sect. D, 55, 911-914. H. G. (1996). Structure and function in rhodopsin. 142. Stein, P. E., Boodhoo, A., Tyrrell, G. J., Brunton, J. L. Single cysteine substitution mutants in the cyto- & Read, R. J. (1992). Crystal structure of the cell- plasmic interhetical E-F loop region show position- binding B oligomer of verotoxin-1 from E. coli. specific effects in transducin activation. Biochemistry, Nature, 355, 748-750. 35, 12464-12469. 143. Crowther, R. A., Henderson, R. & Smith, J. M. (1996). 128. Cai, K., Itoh, Y. & Khorana, H. G. (2001). Mapping of MRC image processing programs. J. Struct. Biol. 116, contact sites in complex formation between trans- 9-16. ducin and light-activated rhodopsin by covalent 144. Tong, L. A. & Rossmann, M. G. (1990). The locked crosslinking: use of a photoactivatable reagent. Proc. rotation function. Acta Crystallog. sect. A, 46, 783-792. Natl Acad. Sci. USA, 98, 4877-4882. 145. Tickle, I. J. (1992). Fast Fourier translation functions. 129. Terakita, A., Yamashita, T., Nimbari, N., Kojima, D. In Molecular Replacement (Dodson, E. J., Gover, S. & & Shichida, Y. (2002). Functional interaction between Wolf, W., ed.s), vol. DL/SCI/R33, pp. 20-32, SERC, bovine rhodopsin and G protein transducin. J. Biol. Daresbury Laboratory, Warrington, UK. Chem. 277, 40-46. 146. Kraulis, P. J. (1991). MOLSCRIPT: a program to 130. Weiss, E. R., Osawa, S., Shi, W. & Dickerson, C. D. produce both detailed and schematic plots of protein (1994). Effects of carboxyl-terminal truncation on the structures. J. Appl. Crystallog. 24, 946-950. stability and G protein-coupling activity of bovine 147. Merritt, E. A. & Bacon, D. J. (1997). Raster3D: rhodopsin. Biochemistry, 33, 7587-7593. photorealistic molecular graphics. Methods Enzymol. 131. Ulfers, A. L., McMurry, J. L., Kendall, D. A. & 277, 505-524. Mierke, D. F. (2002). Structure of the third intra- 148. Wallace, A. C., Laskowski, R. A. & Thornton, J. M. cellular loop of the human cannabinoid 1 receptor. (1995). LIGPLOT: a program to generate schematic Biochemistry, 41, 11344-11350. diagrams of protein-ligand interactions. Protein Eng. 132. Moro, O., Lameh, J. & Sadee, W. (1993). Serine- and 8, 127-134.

Edited by R. Huber

(Received 18 June 2004; received in revised form 26 August 2004; accepted 27 August 2004)