Current Topics Advances in Determination of a High ...pharmacology.case.edu/department/faculty/primary/pages/Palczewski/... · heterogeneous preparations of these proteins isolated

Current Topics

Advances in Determination of a High-Resolution Three-Dimensional Structure ofRhodopsin, a Model of G-Protein-Coupled Receptors (GPCRs)†,‡

David C. Teller,*,§,| Tetsuji Okada,⊥,# Craig A. Behnke,§,|,$ Krzysztof Palczewski,⊥,@,% and Ronald E. Stenkamp|,&

Departments of Ophthalmology, Pharmacology, Chemistry, Biochemistry, and Biological Structure andBiomolecular Structure Center, UniVersity of Washington, Seattle, Washington 98195

ReceiVed February 5, 2001; ReVised Manuscript ReceiVed March 7, 2001

Membrane proteins, encoded by∼20% of genes in almostall organisms, including humans, are critical for cellularcommunication, electrical and ion balances, structural in-tegrity of the cells and their adhesions, and other functions.Atomic-resolution structures of these proteins furnish im-portant information for understanding their molecular orga-nization and constitute major breakthroughs in our under-standing of how they participate in physiological processes.However, obtaining structural information about these pro-teins has progressed slowly (1, 2), mostly because oftechnical difficulties in the purification and handling ofintegral membrane proteins. Instability of the proteins in

environments lacking phospholipids, the tendency for themto aggregate and precipitate, and/or difficulties with highlyheterogeneous preparations of these proteins isolated fromheterologous expression systems have hindered applicationof standard structure determination techniques to thesemolecules.

Among membrane proteins, G-protein-coupled receptors(GPCRs)1 are of special importance because they form oneof the largest and most diverse groups of receptor proteins.More than 400 nonsensory receptors identified in the humangenome are involved in the regulation of virtually allphysiological processes. Drug addiction, mood control, andmemory (via 5-HT6 or neuropeptide receptors) are just ashort list of processes in which GPCRs are criticallyimplicated. Another even larger group of GPCRs consist ofsensory receptors involved in the fundamental process oftranslation of light energy (rhodopsin and cone pigments),the detection of chemoattractant molecules, or the detectionof compounds stimulating the taste buds (3, 4). The activityof GPCRs comes about when binding of diffusable extra-cellular ligands causes them to switch from quiescent formsto an active conformation capable of interaction withhundreds of G-proteins. Their roles as extracellular ligand-binding proteins make them attractive targets for drug design.GPCRs account for∼40% of all therapeutic intervention,

† This research was supported by NIH Grant EY-09339, awards fromResearch to Prevent Blindness, Inc. (RPB), to the Department ofOphthalmology at the University of Washington, the FoundationFighting Blindness, the Ruth and Milton Steinbach Fund, and the E.K. Bishop Foundation. K.P. is a RPB Senior Investigator.

‡ The coordinates have been deposited in the Protein Data Bank asentry 1HZX.

* To whom correspondence should be addressed: Department ofBiochemistry, University of Washington, Box 357350, Seattle, WA98195-7350. Phone: (206) 543-1756. Fax: (206) 685-1792. E-mail:[email protected].

§ Department of Biochemistry.| Biomolecular Structure Center.⊥ Department of Ophthalmology.# Present address: Department of Biophysics, Graduate School of

Science, Kyoto University, Kyoto 606-8502, Japan.$ Present address: Emerald Biostructures, Bainbridge Island, WA

98110.@ Department of Pharmacology.% Department of Chemistry.& Department of Biological Structure.

1 Abbreviations: GPCR, G-protein-coupled receptor; MAN,â-D-mannose; NAG, 2-N-acetyl-â-D-glucose; BNG,â-nonylglucoside;|Fo|,observed structure factor;|Fc|, calculated structure factor.R ) ∑||Fo|- |Fc||/∑|Fo|. Rcryst is the value for the working set of|Fo|, while Rfree

is that calculated for the 5% of reflections set aside for cross validation.

© Copyright 2001 by the American Chemical Society Volume 40, Number 26 July 3, 2001

10.1021/bi0155091 CCC: $20.00 © 2001 American Chemical SocietyPublished on Web 04/14/2001

and major GPCR research projects are found throughout thepharmaceutical industry (5, 6).

A paucity of structural data is available for GPCRs. Thecrystal structure of a member of the largest subgroup (I) ofGPCRs, rhodopsin (7), and a ligand-binding domain of themetabotropic glutamate receptor with and without the ligand(8) have been determined recently. The data allow models,firmly based on the atomic-resolution structural information,to be further tested as to the conformational changes thatthese receptors undergo in going from the quiescent to thesignaling state. In this article, we describe the furtherrefinement of rhodopsin (7) and provide some clues abouthow the receptor could be activated by light.

RHODOPSIN

Rhodopsin is involved in the molecular transformation oflight energy into a neuronal signal transmitted to thesecondary neurons of the retina and ultimately to the brain.The role of rhodopsin in this physiologically interesting andimportant process is that of a classic G-protein-coupledreceptor. These receptors interact with environmental signalsand initiate, via G-proteins, intracellular responses to thosesignals. Rhodopsin is also a member of the largest subfamilyof the membrane receptors, constituting∼90% of all GPCRs.The family includes cone pigments and adrenergic and otherligand receptors (9-15). Most GPCRs respond to bindingof a ligand. In the case of rhodopsin, the signal is made upof two components: the bound chromophore, which under-goes cisf trans photoisomerization, and a photon. In fact,the binding site for retinal is viewed as representative of thebinding mode of an inverse agonist in other GPCRs. Indeed,a rhodopsin mutant lacking Lys296, the residue to which 11-cis-retinal binds via a protonated Schiff base linkage (16,17), can bind and is inactivated by a noncovalently bound11-cis-retinal analogue (18).

Rhodopsin is an integral membrane protein located in theouter segments of rod cells. It provides an environment inwhich its 11-cis-retinal chromophore can undergo a cisftrans conformational switch in response to absorption of aphoton with a very high quantum yield of 0.67 (19). Thisprocess is completed in<200 fs (20). Due to this fastphotochemical process, one of the fastest chemical reactionsknown, it is believed that a large fraction of the energy of aphoton, 32 kcal/mol, is first stored in the chromophore-rhodopsin complex (21, 22). Further structural changes inphotoactivated rhodopsin lead to a well-defined set ofphotostates and are well-characterized spectroscopically bylow-temperature trapping experiments (for example, see refs23 and 24). Interestingly, other GPCRs may also undergomultiple conformational changes upon agonist binding (25).In milliseconds, the formation of the active species ofrhodopsin, so-called metarhodopsin II, allows photoactivatedrhodopsin to interact with a G-protein, transducin (Gt), andactivate it (26, 27). The resulting signal transduction cascadeinvolves activation of cGMP phosphodiesterase, reductionin levels of cGMP, closure of cGMP cation channels in thecellular membrane, hyperpolarization of the cells, andsynaptic signaling (28-33).

The crystal structure of rhodopsin (7) provided the firstdetailed three-dimensional structural model for a GPCR.

Earlier low-resolution work on rhodopsin had revealed theorganization of this receptor at 7.5 Å resolution in the planeof the membrane and 16.5 Å resolution perpendicular to themembrane (34-39). This low-resolution three-dimensionalstudy of rhodopsin predicted the location of the seven rodsof density that correspond to transmembrane helices and wasour first view of rhodopsin. The electron density map alsoallowed an estimation of the tilt angles for these seven helicesand showed clear differences between rhodopsin, bacterior-hodopsin, and other retinylidene-binding proteins (39, 40;for theoretical models, see refs41-44). Further refinementof the structural model reported at 2.8 Å resolution (7) hasresulted in an improved model for ground-state bovinerhodopsin with many structural details that were not seenvia cryoelectron microscopy.

REFINEMENT OF THE RHODOPSIN STRUCTURE

The crystals of rhodopsin are twinned. Two refinementsof rhodopsin were carried out to account for the twinning.In one refinement protocol, the twin law relating the indicesof the overlapping reflections was used in conjunction witha twinning parameter. Alternatively, once a twinning pa-rameter was available, the intensities of the overlappingreflections could be calculated and used in a standardrefinement. Since this latter procedure involves fitting ofderived data (de-twinned) rather than the observationsthemselves, we consider it a less desirable procedure.

The data set used for the refinements was part of the sameset published previously (APS 19-ID) (7). The starting modelwas PDB entry 1F88. The entire refinement was carried outusing CNS (45), with weak noncrystallographic symmetryrestraints applied to the two molecules in the asymmetricunit (molecules A and B). For the refinement, reflectionswith F < 2σ(F) were omitted. This was done because thereflections in the last shell are rather weak with an⟨I/σ(I)⟩of <2.

Palmitoyl groups, heptanetriol, andâ-nonylglucosidetopology and parameter files were taken from the HIC-Upserver (46) and modified as necessary. The bond angles andbond lengths of retinal, which were used in this refinement,differ slightly from those previously used (7). They weretaken from Cambridge Structural Database entry cretal10(47). XtalView (48) was used for the model building stages.For the twinned data, the twin fraction was repeatedlydetermined on the basis of the model and this fraction usedfor the next steps in refinement. The twin fraction initiallywas close to 0.29 and decreased throughout the refinementto a final value of 0.277. Additionally, the statistical methodof Yeates as implemented in CNS was used to de-twin thedata; the value determined using the average value ofH was0.252 (49). This value appeared to give cleaner maps thanthe value of 0.242 determined from⟨H2⟩.

TheR-values and statistics for these two refinements areshown in Table 1. TheR-values for refinement using thede-twinned data differ from those from the refinement againstthe twinned data by the expected ratio of 1/x2 (50). Thetwo resulting refined models differ slightly, but likely notsignificantly. Accordingly, the model obtained using twinneddata will be the focus of the following discussion. This modelhas been deposited in the Protein Data Bank (1HZX).

7762 Biochemistry, Vol. 40, No. 26, 2001 Current Topics

DOMAINS OF RHODOPSIN

The overall shape of the various parts of the rhodopsinmolecule indicates the dimensions of the cytoplasmic,transmembrane, and extracellular regions and their footprintof the molecule in the plane of the membrane. These regions,however, are not domains in the protein folding sense.

Examination of the figures in the “standard view” (Figure1) shows an ellipsoidal shape. The dimensions of the ellipsoidare∼75 Å perpendicular to the membrane,∼48 Å wide inthe standard view, and∼35 Å thick in this view.

The transmembrane domain was determined by bisectinghelix VIII as the cytoplasmic limit. The extracellular limitof the transmembrane region was determined by locating aplane parallel to the membrane where the external residuesof rhodopsin shift from hydrophobic to polar. The shape ofthis transmembrane domain is that of an elliptic cylinder∼41Å in height. The length and width of the elliptic footprinton the plane at the middle of the membrane are roughly 45and 37 Å, respectively.

The cytoplasmic domain was taken simply as the residuesabove the transmembrane domain, and it is reasonably wellfitted by an ellipsoid of axes 44 Å× 30 Å × 18 Å alongx,y, andz, respectively. Similarly, the extracellular domain wasdetermined as an ellipsoid of axes 38 Å× 32 Å × 27 Å.These two ellipsoids encompass about 95% of the atoms ofthe respective domains.

It is of some interest to project the molecule into themembrane plane and determine the area. This area isconnected to the density of the molecules in the membraneand their closest approaches. The area is close to 1500 Å2.The molecule has a strong dipole perpendicular to themembrane, and one would anticipate that these paralleldipoles would repel one another due to the electrostaticrepulsion at the two ends.

Due to the elongation of the cytoplasmic region byinclusion of helix VIII, the area of the cytoplasmic domainis sufficient to dock a single transducin Gt trimer (R-, â-,and γ-subunits) on the surface. However, the C-terminalresidues must move aside to allow all the residues implicatedin binding to be on the surface. A reasonable but speculativemodel of this complex has been built taking into accountthe known interactions of Gt and rhodopsin (data not shown).

In such a complex, Gt would form a 1:1 complex withphotoactivated rhodopsin as proposed previously (51).

MODEL OF RHODOPSIN

Bovine rhodopsin contains 348 amino acids, has a mo-lecular mass of∼40 kDa, and folds into seven transmem-brane helices varying in length from 19 to 34 residues andone cytoplasmic helix (Figure 1, blue rods encapsulatingR-helices; Figure 2). These helices differ in their length andare irregular, and they tilt at various angles with respect tothe expected membrane surface (Figure 1). They contain a

Table 1: Refinement Statistics

twinneda

refinement“de-twinned”b

refinement

resolution range (Å) 30-2.8 30-2.8no. of reflections in working set 42036 39588no. of reflections in test set 2026 1913Rcryst 0.175c 0.248d

Rfree 0.212c 0.277d

no. of atoms 5552 5552rms deviation in bond lengths (Å) 0.010 0.011rms deviation in bond angles (deg) 1.47 1.65rms deviation in dihedral angles (deg) 20.1 19.8rms deviation in improper angles (deg) 0.85 1.27averageB-value (Å2) 45.3 39.0

a The twinned data set is part of the APS 19-ID set as reported inref 7. The data were collected atλ ) 1.03320 Å. Friedel mates wereretained and used in refinement.b The de-twinned data set was obtainedby applying a twin fraction of 0.252 and the twinning law within CNS(45). c When this model is tested against the de-twinned data,Rcryst )0.236 andRfree ) 0.266.d When this model is tested against the twinneddata,Rcryst ) 0.197 andRfree ) 0.227 using a twin fraction of 0.277.

FIGURE 1: Three-dimensional crystal structure of rhodopsin withbound detergent and amphiphile molecules. Helical portions of theprotein, including the seven transmembrane helices, are shown asblue rods, andâ-strands are shown as blue arrows. The polypeptideconnecting the helices appears as blue coils. A transparent envelopearound the protein represents the molecular surface. The dark blueball-and-stick groups at the bottom of the figure denote carbohydrategroups attached to the protein. Two palmitoyl groups covalentlyattached to the protein are shown in green. Nonylglucoside andheptanetriol molecules located near the hydrophobic surface of theprotein are shown in yellow. The figure was drawn using Molscript(116) and Raster3d (117).

Scheme 1: Connections between Helices via HydrogenBondinga

a The helix-helix hydrogen bond interactions parallel the loss ofaccessible surface.

Current Topics Biochemistry, Vol. 40, No. 26, 20017763

mix of R- and 310-helices, and they possess a large numberof kinks, twists, and bends (52). Further refinement has notreduced the number of these conformational anomalies. Manyof the bends and twists were also seen in the two-dimensionalcryoelectron microscopy study of frog rhodopsin (36), andthe helical axes from that study can be readily superposedwith the helices in our refined model. This indicates thatthe irregular helices are not artifacts of data set resolutionor the methods used for crystallographic refinement.

To appraise the tilt, the membrane plane was assumed tobe perpendicular to the long axis of rhodopsin molecule A.The helix tilt angles are the angles between the helical axesand the long axis of the molecule. Thus, a helix at 0° isperpendicular to the membrane, while a helix (such as helixVIII) with a tilt angle of 90° lies parallel to the membraneplane. The second method was to measure the bending ofthe helices from an ideal straight helix. Helix I (Figure 2) is44 Å long and tilted from the plane of the membrane by

FIGURE 2: Two-dimensional model of bovine rhodopsin adopted after that of Hargrave (9). About1/4 of the polypeptide chain of opsins hascytoplasm exposure;1/4 is sequestered in the intradiscal space, and1/2 forms a core of transmembrane helices. Green cylinders representhelices that in large part are imbedded in the disk membranes (19-34 residues each). The violet cylinder represents the amphiphilic helix(H-VIII) that runs parallel to the membranes. The color of rhodopsin results from the protonated Schiff base linkage of the 11-cis-retinalchromophore (100). Lys296 forms this protonated Schiff base with retinal and is shown as a black filled circle. Glu113 is a counterion of theSchiff base and is shown as a red filled circle. The disulfide bridge conserved among GPCRs is shown as purple filled circles (60). Twocarbohydrate moieties at Asn2 and Asp15 and two palmitoyl groups at Cys322 and Cys323 (15, 64-71) are shown as light yellow filled circles(32, 72, 73). Light red filled circles represent acidic residues, and blue filled circles represent basic residues. Photoisomerization of the11-cis-retinal chromophore of rhodopsin toall-trans-retinal leads to a conformational change in the protein, including the cytoplasmicsurface (9-14, 32, 118, 119) leading to activation of Gt. Loops CII and CIII and helix VIII are involved in the recognition of Gt and aremarked with filled yellow circles at the center, changing to red filled circles in the periphery. These residues are also important in bindingof two regulatory proteins: rhodopsin kinase and arrestin. The functional phosphorylation occurs in the C-terminal region (represented bylight brown filled circles) at phosphorylation sites denoted by dark brown filled circles (86-91, 96). The last five C-terminal residues werepostulated to be involved in the vectorial transport within highly differentiated rod photoreceptors (92, 93, 120). The carbohydrate chainsand the disulfide linkage (in pink filled circles) are oriented toward the lumen (extracellular) face of rhodopsin, and the C-terminal domainis cytoplasmic. To date, more than 100 mutations in the human rhodopsin gene have been associated with recessive and dominant retinitispigmentosa (RP), as well as congenital stationary night blindness (CSNB).


25° and contains a 12° bend within it (see Table 2 in theSupporting Information), mostly due to the presence of Pro53.This helix forms multiple hydrogen bond interactions withhelix II and helix VII, but it appears that it does not interactby hydrogen bonding with helices III-VI (Scheme 1).Furthermore, the interactions of helices I and VII arestrengthened by hydrophobic interactions between Leu40

(helix I) and Phe293 (helix VII).Helix II is tilted from the plane of the membrane about

the same as helix I, 25°, and deviates 30° from an ideal helix(Table 2 in the Supporting Information), in the region ofGly89 and Gly90. Because of this bending, Gly90 is in thevicinity of Glu113, the counterion for the protonated Schiffbase of the chromophore. This helix forms multiple hydrogenbond interactions with helices I, III, IV, and VII (Scheme1). Helix III is the longest helix, most tilted from the planeof the membranes, and has two small internal bends. Thehelix begins at Pro107, near Cys110 which is engaged in adisulfide bridge with Cys187 and bends first at residues Gly120

and Gly121. The second bend occurs at Ser127, but this bendhas no apparent correlation with the sequence composition.It seems to be related to the packing of the helix among theothers. The tilt of this 48 Å helix is 33°, and the two bendsare 12° and 11°. The 33° tilt results in running the helixacross the helical bundle and making contacts with fourhelices (II, IV, V, and VII) (Scheme 1). The C-terminal endof helix III is particularly important, because it contains theE(D)RY motif implicated in the regulation of the receptor’sinteraction with its G-protein. This motif is in a veryhydrophobic environment formed among residues fromhelices II and IV-VI, and there is a salt bridge betweenGlu134 and Arg135. Reversing these residues through mu-tagenesis leads to formation of metarhodopsin II, andabolishes binding and activation of transducin (53). Duringphotoactivation, it is possible that this Glu residue becomesprotonated (12, 54, 55). This salt bridge disruption wouldbe one of the constraints abolished as rhodopsin assumesthe metarhodopsin II conformation. In the current model,Glu247 faces the solvent, but it is in a position such that itcould hydrogen bond to Arg135 by rotation aboutø1 andpossibly trigger disruption of the salt bridge. Helix IV is theshortest one, and runs almost perpendicular to the mem-branes, but it is significantly bent from an ideal helix due toPro170 and Pro171 (Table 2 in the Supporting Information).We hypothesize that it is involved in the stabilization of thedark-state rhodopsin through additional hydrophobic interac-tions with helices II, III, and V.

Helix V is 35 Å long and tilted from the membrane normalby 26°. It has two internal kinks of 25° and 15° (Table 2 ofthe Supporting Information). The bends occur at residuesPhe203 and His211 with no apparent correlation with thesequence, apart from a striking number of aromatic residuesin the region of the bend. The helix forms multiple hydrogenbond interactions with helix III and helix IV (Scheme 1).Helix VI requires special considerations, as movement ofthis helix could be a clue to receptor activation (56, 121). Itis the second longest and most bent helix (36°) because ofthe presence of Pro267, one of the most conserved residuesamong GPCRs. Overall, it is almost perpendicular to themembrane plane. The presence of specific hydrogen bondinteractions with only helix VII would allow movement ofVI relative to the rest of the helices (Scheme 1). In contrast

to the rest of the helices, helix VI interacts with helices II,III, and V only through van der Waals interactions. HelixVII shows a considerable distortion and elongation in theregion around the retinal attachment site Lys296 (shown inred in Figure 1) and contains Pro291 and Pro303, a part of thehighly conserved NPXXY motif. The NPXXY motif mightbe involved in the formation of a structural domain thatwould allow interaction with helix VI (e.g., Met253 isinvolved). These interactions could be one of the criticalconstraints holding rhodopsin in the inactive state. Helix VII(containing the chromophore) is nicely situated in the bundleof helices and interacts with all of them, except helices IVand V.

In addition to these transmembrane helices, another shorthelix in the cytoplasmic surface, termed helix VIII, is locatedat the extension of helix VII. This helical region, in additionto loops CII and CIII (reviewed in ref26) of rhodopsin, isa part of the binding sites for the Gt R-subunit and plays arole in the regulation of Gtγ binding (57). Helix VIII isstraight and amphipathic. The helix starts at Lys311 andcontains Arg314, whose side chains hydrogen bond tosurrounding residues. Cys322 is also on the cytoplasmic faceof the helix, but the attached palmitoyl moiety makes aU-turn to lie adjacent to the transmembrane portion of thestructure (Figure 1). Cys323 is the C-terminal residue of thehelix, and its palmitoyl portion associates with molecule Bin the crystals of rhodopsin. This particular arrangement maydistort the C-terminus of helix VIII.

The cytoplasmic loops are poorly determined in thestructure presented here. This is the region of the proteinwith the highestB-factors, and these loops are probablymobile in solution. Residues missing from the current modelare residues 236-240 in cytoplasmic loop II and residues331-333 in the C-terminal region. C-Terminal residues334-348 possess electron density, but it is of poor quality.Diffuse density between molecule A and a crystallographi-cally symmetric molecule is sufficiently good to build thestructure, but its reliability is probably poor. In molecule B,more residues are missing, including the C-terminus beyondresidue Asn326 (Figure 2). Almost certainly, the C-terminalresidues of rhodopsin are flexible and mobile in the physi-ological milieu, lacking a definite single conformation (58,59).

The extracellular region of rhodopsin consists of theglobular N-terminus from residue 1 to 33, the short loop ofresidues 101-105, “plug” residues 173-198 between helicesIV and V, and residues 277-285 between helices VI andVII. The current model does not differ significantly fromthe previous report (7) in this region. Residues 1-33 forma compact, glycosylated unit which overlays the rest of theloops. Prior to the determination of the rhodopsin structure,we had thought of this region as unstructured because it wasnot part of the helices, so its observation as a globular unitcame somewhat as a surprise. The extended structure ofresidues 173-198 was another aspect that had not beenanticipated. This sequence forms a twistedâ-hairpin fromresidue 177 to 190 and contains the Cys187 portion of thedisulfide bridge. As previously noted, this structure forms aplug upon which the retinal lies. The carbonyl oxygen ofCys187 approaches retinal C12 at only 3.00 Å (see Table 2 ofthe Supporting Information), so the retinal appears to lie onthe plug. Mutation of Cys187 gives rise to proteins that are


abnormally glycosylated and cannot bind retinal (60). Theplug structure also serves to bring Glu181 into proximity (4.4Å) of retinal at the C12 position. This residue is highlyconserved in rhodopsins and short- and middle-wavelengthvisual pigments (15), and a corresponding Glu residue inthis position serves as a counterion in retinochrome, amember of the rhodopsin family (61). In long-wavelengthvisual pigments, such as red visual pigments, a His residueoccupies this position. The His residue and a Lys residuepresent three residues toward the C-terminus, and form achloride ion-binding site (62). Chloride ion, when bound,further causes a bathochromic shift by∼40 nm for thesecone visual pigments (63).

MOLECULES BOUND TO RHODOPSIN

In addition to the chromophore, discussed below, rhodop-sin is modified by two palmitoyl groups at Cys322 and Cys323

in the cytoplasmic region (Figure 2) (15, 64-71). Improvedelectron density for three of the four palmitoyl groupscovalently attached to the two protein molecules in theasymmetric unit has allowed their identification duringrefinement (Figure 1, in green). Two palmitoyl side chainsare attached in molecule A of the model. The palmitoyl groupattached to Cys322 is roughly aligned with the transmembranehelices in an orientation where it could interact with thehydrophobic region of the membrane bilayer (Figure 1). Thepalmitoyl bound to Cys322 in molecule B also takes on thisorientation. The second palmitoyl bound to molecule A atCys323 interacts with a neighboring protein molecule in thiscrystal form, is extended away from the protein, and is likelynot mimicking the hydrophobic parts of the membrane. Nodensity is observed for the palmitoyl bound to Cys323 inmolecule B, presumably due to the mobility of the hydro-carbon tail.

Rhodopsin is also modified by two carbohydrate moietiesat Asn2 and Asn15 that are oriented toward the lumen(extracellular) face (Figure 1, blue) (32, 72, 73). Two majorspecies identified as Man3GlcNAc3 are attached to Asn2 andAsn15 in bovine rhodopsin (74). Additional mannose groupslead to formation of a minor component. This earlier analysishas been confirmed using mass spectrometry (75). Onlypartial three-dimensional structural models have been as-sembled for these groups. In the latest model of rhodopsin,we have been able to build three additional carbohydrateresidues, extending the original model (7).

All the carbohydrate chains are in contact with proteinresidues of crystallographically related molecules; carbohy-drates of molecule A contact crystallographically relatedmolecules of B and vice versa. The intermolecular contactsappear to be mediated by water molecules or Zn2+. Super-position of the carbohydrate chains shows significant devia-tions, however. It is not possible to say at present if this isreal or due to disorder; theB-factors of the most deviantparts of the chains are quite large.

Noncrystallographic symmetry was imposed on the proteinresidues where the carbohydrate is attached, but was notimposed for the carbohydrate. Superposition of the NAG-NAG-MAN residues attached to Asn15 gave a root-mean-square deviation of 2.18 Å. The main discrepancy betweenthe two chains is that the mannose rings are flipped. For the

carbohydrate chains attached to residue 2, the two NAGresidues of molecule A fit to those of molecule B with aroot-mean-square deviation of 1.24 Å. The major deviationin this case is between theN-acetyl moieties of the terminalNAG residues.

At position Asn15, we have built a mannose residue as anaddition to the NAG-NAG residues previously reported onmolecule A (7). Two mannose residues have also been addedto the NAG-NAG chain of molecule B. Asn15 is close to alocal 2-fold axis between molecule A and symmetry-relatedmolecule B. This particular NAG-NAG-MAN chain isaligned adjacent to the Lys16 and Thr17 of molecule B in aneighboring cell. The interaction of the two moleculesappears to be mediated by a water molecule or Zn2+ ion withlow occupancy. Protein linkages to the carbohydrate are inthe â-configuration. All linkages areâ-1,4 for this NAG-NAG-MAN chain. For the carbohydrate attached to residueAsn2, there is also a water- or Zn2+-mediated associationwith a B molecule in the crystal. In this case, only tworesidues of the carbohydrate can be observed and theseconnect through a probable Zn2+ (Zn2+ 956, occupancy)0.5) to His195. Glu197 makes a long contact (3.4 Å) with NAG;this may be a hydrogen bond. This carbohydrate chain alsocontacts one of the BNG (â-nonylglucoside) ligands with along contact of 4.2 Å between NAG O7 and O3 and O4 ofBNG (BNG 1503). For molecule B and the carbohydratechain attached to Asn15, one additional mannose residue wasincluded in the model. This residue has anR-1,3 linkage tothe remainder of the chain according to ref74, and we haveused that configuration in the model. The interactions of thiscarbohydrate with molecule A are with residues Lys16 andThr17 due to the 2-fold axis. Like the carbohydrate moleculesthat interact with Asn2 of molecule A, those attached toresidue 2 of molecule B interact through a water or Zn2+

with a crystallographically related molecule. A water mol-ecule forms a hydrogen bond between the first NAG andHis195. Glu197 makes a long (4.0 Å) contact with the secondNAG. No density was observed for carbohydrate beyondthese two residues.

Interactions of rhodopsin with phospholipid could be veryimportant during the photoactivation of rhodopsin andmovement of the helices (76, 77). When rhodopsin isreconstituted into saturated phospholipids, the formation ofmetarhodopsin II is inhibited or does not occur (78-82).There is also lipid restructuring during the photoactivationprocess (83). Lipids are not removed in the purification andcrystallization steps used here (84, 85), but no electrondensity due to phosphorus atoms could be found. However,six heptanetriol molecules and sevenâ-nonylglucosides arenow part of the structural model (Figure 1, yellow). Oneheptanetriol and one nonylglucoside are located on thecytoplasmic face of molecule A and are probably artifactsof crystallization. The other additive and detergent moleculesare found near the hydrophobic surfaces of the transmem-brane helices (Figure 1). The hydrophobic surface ofmolecules A and B are not completely masked by theseamphipathic molecules. About half of the surface is notcovered by ordered detergent or additives. Crystal packinginteractions among the rhodopsin molecules in the unit celldo not entirely account for the unmasked hydrophobicsurfaces.


Other significant additives in the crystals of rhodopsin arethe metal ions. Zn2+ ions are an integral component of thepurification and crystallization protocols (84), and Hg2+

compounds were bound to the protein to provide theanomalous scatterers used in obtaining phases for thestructure determination. Seven Zn2+ ions (occupancy of 0.4-1.0) and six Hg2+ ions (occupancy of 0.5-1.0) have beenincluded in the structural model. Identification of electrondensity peaks as Zn2+ or Hg2+ ions has been based on thenature of the protein ligands. The occupancies andB-factorswere adjusted using theB-factors of liganding atoms and|Fo| - |Fc| difference electron density maps. All of the Hg2+

ions are associated with cysteine residues at positions 222,264, and 316. Cys140 resides on the cytoplasmic surface butdoes not appear to bind ions. Zn2+ ions are found near Cys167,but the Zn2+ does not appear to bind to the sulfur atom.

Small improvements in the protein portion of the modelare distributed across the entire polypeptide chain. Whileamino acid side chains have been added for the residues atthe C-terminus of molecule A, the refinement has not im-proved the electron density for the C-terminal tail of moleculeB or for the missing residues of the cytoplasmic loops.

The cytoplasmic C-terminal region has two importantfunctions (Figure 2). In photoactivated rhodopsin, the C-terminal is a substrate for rhodopsin kinase at the C-terminalSer and Thr residues (86-91). The last five amino acids arepart of the vectorial transport machinery involved in export-ing rhodopsin to outer segments in highly differentiated rodcells of the retina (92, 93). The structural work reported hereadds another interesting twist to this physiologically impor-tant region. The C-terminal structure of rhodopsin does nothave any structural elements that would stabilize this regionin a rigid conformation. As described previously (7), theC-terminal region from Gly324 to Asp330 folds back over helixVIII so that Asp330 is located close to Lys311 at the beginningof helix VIII. Residues 331-333 have no discernible electrondensity. At Thr335, the backbone forms hydrogen bonds toGln312 of helix VIII, but diverges until Ser338. At Ser338, thebackbone forms hydrogen bonds to the carbonyl O of His65

at the end of helix I. The chain makes a loop from Ser338 toGlu341 and then associates with another molecule A in thecrystal at the C-terminal Ala348. In molecule B, the electrondensity cannot be fitted beyond residue Asn326.

Our rhodopsin structure represents the inactive form ofthe receptor, but in conjunction with other biochemical datafrom previous studies, it provides us with a better under-standing of allowed changes upon activation. The sites oflight-dependent multiple phosphorylations are six to sevenSer/Thr residues at the C-terminal end. These sites werediscovered by specific cleavage of rhodopsin by Asp-Nendoproteinase (94). The resultant 19-amino acid C-terminalpeptide contained all of the phosphorylation sites. Hetero-geneity and multiple phosphorylation of rhodopsin in vitrohas been well documented (87-89, 91, 95, 96). In vivo thereare three sites, which were found to be phosphorylated bydirect and quantitative methods after 20-40% bleachingof the protein (97, 98). Recently, Mendez and colleaguesused a combination of transgenic mice lacking selectivephosphorylation sites and electrophysiological recordings totest the effect of mutation on the physiological responses.They concluded that multiple phosphorylation events areneeded to shut off photoactivated rhodopsin (90). An

abundance of Ser/Thr residues at this region of rhodopsincould speed phosphorylation, and removal of these sitescould slow it. It is also known that arrestin binding de-pends on the presence of hydroxyl groups for high affinity(99).

RHODOPSIN CHROMOPHORE

Rhodopsin is a red-colored protein due to a prostheticgroup chromophore, 11-cis-retinal (100, 101) (Figure 1,chromophore in red). The chromophore is bound in thehydrophobic core of the molecule, causing its absorptionmaximum at approximately 380 nm to be shifted bathochro-mically to that characteristic of intact rhodopsin at 500 nm(102). The chromophore is covalently linked to Lys296 onhelix VII in bovine rhodopsin (Figure 2) (16, 17) through aprotonated Schiff base. When extracted into detergent,rhodopsin maintains its chromophore (103, 104). The coun-terion for the protonated Schiff base is provided by Glu113,which is highly conserved among all known vertebrate visualpigments (105, 106). The counterion has two importantfunctions. (1) It stabilizes the protonated Schiff base byincreasing theKa for this group by up to 107 (e.g., ref107)and preventing its spontaneous hydrolysis. (2). It also causesa bathochromic shift in the maximum absorption for visualpigments to make them more sensitive to longer wavelengthsas UV light is filtered by the front of the eye in most animals.

The 11-cis-retinylidene moieties in the molecular modelhave been refined independently. Aligning these structuresgives an estimate of the uncertainty in positions, bond angles,and bond lengths. The chromophore, albeit a conjugatedsystem, is not planar. It could be speculated that the visualcone opsins might make use of this lack of planarity inmodulating the absorption maxima of their chromophore.Table 2 (Supporting Information) contains the torsion anglesfound for the chromophore. It should be noted that the torsionangle restraints for the chromophore were removed from ourrefinement except for the cis restraint for the C11-C12 bond.This relieves the torsion angles of the chromophore fromany computational artifact due to the refinement method orrestraints.

Another interesting conformational issue concerns theorientation of theâ-ionone ring with respect to the rest ofthe chromophore. The diffraction data at 2.8 Å resolutionare sufficient to identify and orient the ring, although thereis a possibility of partial occupancy of rings rotated by 180°about the C6-C7 bond. Solid-state NMR measurements haveshown the orientation of various C-C vectors in the ringwith respect to the membrane normal (108). The NMR resultsare consistent with a ring rotated 180° from the ring foundin the crystal structure; see Figure 3B. However, other studiesindicate that the retinal chromophore is in the twisted 6-s-cis conformation in rhodopsin, in contrast to the planar 6-s-trans conformation found in bacteriorhodopsin (109). Theinconsistency between the X-ray crystallographic and solid-state NMR studies requires further study, but it should bepointed out that the binding site for theâ-ionone ring wasdemonstrated to accommodate a variety of substituents (110,111). Thus, it can be oriented as either 6s-cis or -trans inthe ground state (K. Palczewski, unpublished).


CAVITIES WITHIN THE RHODOPSINMOLECULE

An accessible surface calculation for rhodopsin revealsseveral cavities within the molecule (Figure 4). This mightbe expected for proteins containing helices since the diver-gence of the helices will provide spaces between them thatmight not be fully occupied by amino acid side chains. Thecavities could play a role in the conformational changeleading to activation of rhodopsin. In particular, severalcavities are near the cytoplasmic end of helix III, andwholesale motion of the helix to occupy them seems possible.Likewise, residues from helix III contribute to the bindingsite for the ionone ring and conjugated system of thechromophore. What is not clear is how, or whether, the cis-trans conformation change of the chromophore would becoupled to movement of helix III.

A large connected cavity is found lying parallel to the twoâ-strands of the plug. On the other side of the cavity, thepolypeptide chain runs roughly parallel to theâ-strands.Clearly, some packing interaction keeps the twoâ-strandsfrom adding another to make a three-stranded sheet. Onequestion arising from the structure of rhodopsin concernshow the chromophore makes its way to the binding pocketwhen the protein is reconstituted. This question is of greatimportance when considering the binding of ligands to otherGPCRs. If ligands pass directly to the binding site from theextracellular space and do not pass into the site from thehydrophobic center of the bilayer, large portions of the

structure (including the plug) must move. This cavity mightbe part of a “loosened” structure that can easily move forligand binding.

Two of the cavities are located near the nitrogen atom ofthe Schiff base. Water molecules have been implicated inthe hydrolysis of the Schiff base, but no electron densityappropriate for bound water molecules near 296 NZ has beenseen in our crystallographic studies. The current molecularmodel contains very few bound water molecules, but this isappropriate for a 2.8 Å resolution study. Series terminationeffects and experimental and computational noise complicatethe identification of water at this resolution. At this point,we have chosen to add water molecules to the model onlywhen electron density is found for both the twinned and de-twinned refined models. In this case, the only water moleculenear the retinal chromophore is located near C13 andhydrogen bonded to Glu181 OE2 and Ser186 OG. It is foundin only one molecule in the asymmetric unit, molecule A.This water molecule is 4.4 Å from the carboxylate of Glu113

and also 4.4 Å from the Schiff base nitrogen atom. The lackof electron density in molecule B argues for caution inascribing biochemical significance to this water molecule.A small cavity is found at this site in molecule B, consistentwith the possibility of water binding at this site.

On the other hand, the two cavities near the Schiff baseare more likely candidates for water molecules involved inthe chemistry of the chromophore since the cavities exist inall views of the molecular structure available to us at this

FIGURE 3: Conformation of the retinylidine chromophore. (A) Stereoview of the chromophores from the refined rhodopsin models showingthe small variation in the conformation and orientation of the group in the four refined subunits. The rms deviation among the four moleculesis 0.26 Å for all atoms. (B) Stereoview showing the chromophore from molecule A of the crystallographic model refined against twinneddata (yellow) and the chromophore structure obtained by Watts and colleagues (108) (green).


time. One of the cavities is close to Glu113, and the other islocated on the other side of the Schiff base nitrogen atomaway from Glu113. Both cavities are buried in the protein

with no access to the surface of the protein, but dynamicalmotions of the structure should be sufficient to allow waterto diffuse into these sites.

RHODOPSIN VERSUS BACTERIORHODOPSIN

It is interesting to compare the rhodopsin helices with thosein bacteriorhodopsin (PDB entry 1C3W) (112). Panels A andB of Figure 5 show a superposition of rhodopsin moleculeA with bacteriorhodopsin. The models were superimposedusing structural alignments only. No amino acid sequenceinformation was used in the superposition. After an initialalignment, residues from each of the molecules were usedfor superposition if they were located within 3.8 Å of eachother. Iteration of the superposition procedure eventually ledto a superposition using 79 CR atoms and resulted in a root-mean-square difference between the molecules of 2.13 Åbased on these 79 CR atoms alone.

Rhodopsin and bacteriorhodopsin have the same overalltopology of their polypeptide fold as seen in Figure 5A. Thehelices in rhodopsin are slightly longer than those inbacteriorhodopsin. We have compared the individual helixpositions by maintaining the same superposition as shownin Figure 5A but isolating the individual helices. In thiscomparison, helices I-III superimpose reasonably well.Pairwise comparison of the remaining helices (Figure 5C,left) shows that helices IV and V do not superimpose.Additionally, the twists and kinks in the helices createsubstantial differences between the two molecules. Thesedifferences are clearly large enough to affect homologymodeling efforts for other GPCRs which are based on thebacteriorhodopsin structure.

The major function of rhodopsin is to couple the confor-mational change of a retinal chromophore (caused byabsorption of a photon) with a structural change in the proteingenerating a signal on the cytoplasmic face of the molecule.Accordingly, there is great interest in the retinal environmentand conformation within the protein. The comparisonbetween the rhodopsin model and the high-resolution struc-ture of bacteriorhodopsin (113-115) demonstrates howuniquely nature adapted each protein to amplify the lightsignal and to pump protons across plasma membranes,respectively. However, when the activation process is dis-sected into fine details at atomic resolution, it is possiblethat both mechanisms for rhodopsin and bacteriorhodopsinmay share many similarities, such as movement of protonswithin the hydrophobic core of both proteins. In Figure 5C,on the right is shown a comparison of helix VII ofbacteriorhodopsin and rhodopsin. The Schiff base attachmentis in a different position relative to the membrane plane, butsurprisingly, the ionone rings are rather close. The signifi-cance of this, if any, is unknown.

CONCLUSIONS

Due to the importance of GPCRs in vast numbers ofphysiological processes, understanding how rhodopsin isactivated, as well as other GPCRs, is one of the mostfundamental problems currently unsolved in neuroscience.Our refined model, in conjunction with many biochemicalstudies, including our most recent work using rhodopsinregenerated with ring-constrained 11-cis-retinal analogues,suggests that cis-trans isomerization is merely a mechanism

FIGURE 4: Cavities within the rhodopsin molecule. (A) CR tracing(blue) with cavities shown by molecular surface dots around thetest probes (1.4 Å radius). Glu113 is depicted in cyan and is about3.6 Å from the Lys296 NZ. The cavities are of sufficient size tohold one or more water molecules. (B) Closeup of two cavitiesadjacent to the protonated Schiff base portion of the chromophore.


for repositioning theâ-ionone ring that ultimately determinesreceptor activation (K. Palczewski, unpublished). However,more molecular information is needed to understand howrhodopsin and other GPCRs are activated. Now with the firstfundamental step taken by determination of the rhodopsinstructure, further investigations will fill our gaps in under-

standing how this and other GPCRs switch into the signalingstate.

ACKNOWLEDGMENT

We thank Drs. Klaus Peter Hofmann and Paul A. Hargravefor their comments on the manuscript.

FIGURE 5: Superposition of bacteriorhodopsin (pink transparent cylinders and connecting coil) on molecule A of rhodopsin (colored helicalribbons and connecting coils). (A) At the left are molecules in the same orientation as in Figure 1. At the right is shown a similar view, withthe molecules rotated 180° about the vertical axis. (B) At the left is a view of the top surface of the molecules. This is the cytoplasmicsurface of rhodopsin. Note the substantial differences between helices IV and V in the two molecules. At the right is a bottom view of themolecules. (C) At the left, helices IV and V in the two molecules do not overlap significantly. At the right is helix VII. Note the irregularand kinked helix in rhodopsin as well as the differences in the location of the Schiff base attachment and orientation of the retinal chromophore.


SUPPORTING INFORMATION AVAILABLE

Tabular listing of the following topics: tilt of the helices,bends within helices, possible hydrogen bonds betweenhelices, closest atoms to the 11-cis-retinal, retinal atomsclosest to these protein atoms, and torsion angles for thechromophore. This material is available free of charge viathe Internet at http://pubs.acs.org.

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