Crystal structure of opsin in itsG-protein-interacting conformationPatrick Scheerer1*, Jung Hee Park1*, Peter W. Hildebrand1, Yong Ju Kim1, Norbert Krauß2, Hui-Woog Choe1,3,Klaus Peter Hofmann1,4 & Oliver P. Ernst1
Opsin, the ligand-free form of the G-protein-coupled receptor rhodopsin, at low pH adopts a conformationally distinct, activeG-protein-binding state known as Ops*. A synthetic peptide derived from the main binding site of the heterotrimeric Gprotein—the carboxy terminus of the a-subunit (GaCT)—stabilizes Ops*. Here we present the 3.2 A crystal structure of thebovine Ops*–GaCT peptide complex. GaCT binds to a site in opsin that is opened by an outward tilt of transmembrane helix(TM) 6, a pairing of TM5 and TM6, and a restructured TM7–helix 8 kink. Contacts along the inner surface of TM5 and TM6induce an a-helical conformation in GaCT with a C-terminal reverse turn. Main-chain carbonyl groups in the reverse turnconstitute the centre of a hydrogen-bonded network, which links the two receptor regions containing the conserved E(D)RYand NPxxY(x)5,6F motifs. On the basis of the Ops*–GaCT structure and known conformational changes in Ga, we discusssignal transfer from the receptor to the G protein nucleotide-binding site.
G-protein-coupled receptors (GPCRs), also known as seven-transmembrane receptors, are the largest superfamily of plasmamembrane receptors1. Binding of extracellular ligands to GPCRsmodulates their capacity to catalyse GDP–GTP exchange in hetero-trimeric G proteins (Gabc), thereby regulating the intracellular levelof secondary messengers. Rhodopsin is the photoreceptor of thevertebrate retinal rod cell and the eponym of the rhodopsin familyof GPCRs with ,670 members in the human genome2. Rhodopsinconsists of the apoprotein opsin and the chromophore 11-cis-retinal,which is bound by a protonated Schiff base to Lys 296 in the seventhtransmembrane helix to stabilize the inactive receptor state. Photonabsorption leads to cis–trans isomerization of the retinal, which trig-gers deprotonation of the retinylidene Schiff base linkage and forma-tion of the active, G-protein-binding metarhodopsin II state3,4.Metarhodopsin II decays by hydrolysis of the retinylidene Schiff baseand release of the photolysed all-trans-retinal from its binding pocketto generate ligand-free opsin. Under physiological conditions, fresh11-cis-retinal is metabolically supplied and taken up to regeneraterhodopsin3,5. Before regeneration is complete, visual sensitivity isreduced owing to a background level of GTP-bound G protein whichis maintained by a persistently active form of opsin5. In vitro, opsincan readily adopt inactive (Ops) and active (Ops*) conformations,and low pH and a synthetic peptide derived from the C terminus ofGa stabilize Ops* (ref. 6).
Besides the crystal structures of bovine and squid rhodopsin7–10
and different photointermediates11,12, the structures of two isoformsof the b-adrenergic receptor13,14 are known. All of these GPCR struc-tures contain a ligand bound in their binding site and lack a pro-minent tilt of the cytoplasmic half of TM6 out of the helical bundle,which is considered to be mandatory for G protein activation15–17. Werecently solved the crystal structure of ligand-free opsin and foundthat it was quite different from the known GPCR structures18. Themost prominent features were the activating movement of TM6 andaccompanying rearrangements in the regions of the conserved
E(D)RY and NPxxY(x)5,6F motifs3,4,19. Here we report the structureof Ops* when it actively binds to the key G protein interaction site.Major sites for receptor interaction include the C termini of the Gaand Gc subunits of transducin (Gtabc, consisting of Gat, Gb1 andGc1), the cognate G protein of rhodopsin20–22. We stabilized Ops*with an 11 amino acid synthetic peptide (GaCT, amino acid sequenceILENLKDCGLF, Gat(340–350)K341L) derived from the extreme Cterminus of the transducin Gat subunit23. The crystal structure of theOps*–GaCT complex provides insight into the structural changesinvolved in signal transfer from the receptor to the G protein.
Structure of Ops*–GaCT complex
Optimized extraction of the receptor from native rod cell disc mem-branes allowed the growth of Ops*–GaCT complex crystals. We usedtwo different approaches: co-crystallization of the GaCT peptidewith either opsin or photoactivated rhodopsin, respectively. In thelatter case, the rhodopsin and GaCT mixture was illuminated directlybefore crystallization to produce active metarhodopsin II. Loss of all-trans-retinal from the metarhodopsin II–GaCT complex occurredduring crystallization and resulted in an Ops*–GaCT complex.This was determined by the lack of electron density for retinal inthe 3.2 A crystal structure which was obtained by co-crystallizationof GaCT and photoactivated rhodopsin (Fig. 1 and SupplementaryFig. 1). When solubilized ligand-free opsin was used as the startingmaterial, Ops*–GaCT crystals were obtained which diffracted to3.8 A and showed, within the limits of resolution, the same structureand binding of GaCT (data not shown). For crystallization, datacollection, structure determination and refinement statistics seeMethods and Supplementary Table 1.
The structure of Ops*–GaCT comprises amino acids 1–326 ofopsin (lacking 22 C-terminal amino acids which are not resolved,presumably because of their known high mobility), and 11 aminoacids of the GaCT peptide with well defined electron density (Fig. 1and Supplementary Figs 1a and 2). The Ops* model includes the
*These authors contributed equally to this work.
1Institut fur Medizinische Physik und Biophysik (CC2), Charite – Universitatsmedizin Berlin, Chariteplatz 1, D-10117 Berlin, Germany. 2Queen Mary, University of London, School ofBiological and Chemical Sciences, London E1 4NS, UK. 3Department of Chemistry, College of Natural Science, Chonbuk National University, 561-756 Chonju, South Korea. 4Zentrumfur Biophysik und Bioinformatik, Humboldt-Universitat zu Berlin, Invalidenstrasse 42, D-10115 Berlin, Germany.
Vol 455 | 25 September 2008 | doi:10.1038/nature07330
seven transmembrane helices connected by extracellular (E1–E3) andcytoplasmic (C1–C3) loops, and the cytoplasmic helix 8 (H8), whichruns along the membrane. The structure of Ops*–GaCT bearsresemblance to the structure of peptide-free opsin (free Ops*)18,which was used as a search model to solve the structure by molecularreplacement. Compared to the Ops* structure18, the Ops*–GaCTstructure presented here shows subtle but distinct changes in Ops*induced by GaCT binding (Supplementary Fig. 4).
Gross structural features which are comparable in free Ops* andOps*–GaCT but are different from the 11-cis-retinal-bound dark-state rhodopsin include a short helical turn in loop C1 and rearran-gements of loops C2 and C3 (Fig. 2). Compared with dark-staterhodopsin, the cytoplasmic half of TM6 is tilted outwards of thehelical bundle by 6–7 A with Trp 265 as the pivot point, in agreementwith predictions of the active receptor state derived from electronparamagnetic resonance spectroscopy15,17. Furthermore, TM5 islonger, very straight and more inclined than in rhodopsin, with aresulting shift of the cytoplasmic end by 2–3 A towards TM6. As aconsequence, the cytoplasmic ends of TM5 and TM6 are closer andnearly parallel to each other, forming a cytoplasmic helical pair. Inthe extracellular half, the Ops*–GaCT structure also shows twoopenings between TM5 and TM6 and between TM1 and TM7,respectively (data not shown), which may act as gates for retinal18.Owing to the TM5 and TM6 rearrangement, a crevice is opened intowhich the GaCT peptide binds in an a-helical conformation with anopen reverse turn, an aL-type C-capping motif (C-cap; Fig. 2a andSupplementary Figs 2–6). Notably, the conformation of GaCTobserved in the Ops*–GaCT crystal is almost identical to the NMRsolution structure of a GaCT analogue and the parent Gat(340–350)peptide in the receptor-bound state determined by transfer nuclearOverhauser effect experiments24,25 (Supplementary Fig. 3). As seen inthe superposition of rhodopsin and GaCT (as part of Ops*–GaCT),
dipping of GaCT into the cytoplasmic part of the helical bundle is notpossible for the inactive dark state of rhodopsin with the given ori-entation of GaCT (Fig. 2b and Supplementary Fig. 5b).
The dual role of Arg 135 in the E(D)RY motif
In rhodopsin, TM3 and TM6 are tethered by a hydrogen-bondednetwork (‘ionic lock’) which includes the side chains of Arg 135and Glu 134 from the conserved E(D)RY motif in TM3, and the sidechains of Glu 247 and Thr 251 in TM6 (Fig. 3a). In the peptide-freeOps* state18 (Fig. 3b), as well as in Ops*–GaCT (Fig. 3c), the ioniclock is broken owing to the outward movement of TM6. The newlyformed cytoplasmic TM5–TM6 pair conformation is stabilized bynew interactions—that is, the side chains of Glu 247 and Thr 251 arereleased from Arg 135 and engage with the TM5 side chain of Lys 231in a hydrogen-bonded network (not shown in Fig. 3b, c; see ref. 18).Apart from its main function in the ionic lock of rhodopsin,Arg 135—one of the most conserved residues in GPCRs—has a cru-cial role in the active receptor state. In both Ops* and Ops*–GaCT,the hydroxyl group of Tyr 223 from TM5 replaces the carboxyl groupof Glu 134 by its interaction with Arg 135. The arginine side chain isthereby liberated from Glu 134 and allowed to swing into the centreof the GaCT binding crevice to form its floor. Stabilized by Tyr 223, it
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Figure 1 | Overall structure of Ops*–GaCT complex. The asymmetric unitcontains one molecule of Ops*with one molecule of GaCT peptide bound atthe intracellular side. Shown is a crystallographic dimer of symmetry relatedOps*–GaCT monomers, with the view parallel to the membrane. The Ops*molecules (orange and yellow) and the GaCT peptides (blue) are shown inribbon representation, and the GaCT side chains are shown as sticks. One ofthe GaCT molecules is also shown as transparent space-filling model. TheGaCT peptide is derived from the Gat C terminus (Gat(340–350)K341L).The K341L substitution increases the affinity for the active receptorconformation by two orders of magnitude23. Ops* consists of seventransmembrane helices (TM), connected by extracellular (E1–E3) andcytoplasmic (C1–C3) loops and the cytoplasmic helix H8. Two antiparallelb-sheets in the N terminus (strands b1 and b2) and loop E2 (strands b3 andb4) are shown as blue arrows. Oligosaccharides at Asn 2 and Asn 15 andpalmitoyl chains at Cys 322 and Cys 323 are presented as sticks.
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Figure 2 | Comparison of Ops*–GaCT and dark-state rhodopsin structures.a, View from the cytoplasm of Ops*–GaCT. Ops* is shown in ribbon(orange) and surface (grey) representation, the Gat(340–350)K341L peptide(ILENLKDCGLF) is shown as a ribbon and stick model (blue). The anglebetween the helical axis and the membrane normal (43 6 2u) is in goodagreement with predictions from residual dipolar coupling NMR data25.b, The view from the cytoplasm of dark-state rhodopsin (PDB accession1U19; green ribbon and grey surface) and superposed GaCT (blue) is shown.The scheme indicates differences in the arrangement of TM1–TM7 betweenOps*–GaCT (Ops*, orange; GaCT, blue) and dark-state rhodopsin (green).
then interacts with the backbone carbonyl group of Cys 347 at the tipof GaCT (Fig. 3c). Conversely, Glu 134 is not involved in an inter-action with GaCT. Glu 134 faces towards TM2 and TM4, and furtherelectron density is observed close to the carboxyl group of Glu 134.This electron density could be interpreted as a water molecule pro-viding a weak linkage of Glu 134 to the main-chain carbonyl ofAla 153 and peptide nitrogen of Ile 75 (Supplementary Fig. 7). In this
rather non-polar environment, Glu 134 is probably protonatedwhich explains the proton uptake measured in the course of the light-activation of rhodopsin4,26, which does not occur in the E134Qmutant27. The outwardly tilted position of TM6 and the new locationof the Arg 135 side chain in Ops* and Ops*–GaCT is stabilized byTyr 306 as part of the conserved NPxxY(x)5,6F motif connecting TM7and H8 (Fig. 3b, c). In rhodopsin, the side chains of Tyr 306 (TM7)and Phe 313 (H8) interact with each other, whereas in Ops* andOps*–GaCT, Tyr 306 is extended into the helical bundle belowArg 135 and hinders the inward tilt of TM6.
GaCT and retinal-binding pockets
The inner side of the cytoplasmic TM5 and TM6 pair in Ops* pro-vides a hydrophobic surface formed by Leu 226, Val 230 and Ala 233in TM5, and by Thr 242, Thr 243, Ala 246 and Val 250 in TM6, forhydrophobic interaction with GaCT (Fig. 4a, Supplementary Figs 8and 9 and Supplementary Table 2). Further hydrophobic contacts areprovided by Val 138 and Val 139 at the junction of TM3 and loop C2,and by Leu 72 in loop C1. Some of the elements that contribute to thispart of the binding site were predicted from earlier mappingapproaches by mutational analysis28,29 and photocrosslinking30. Thefully induced C-capped helical structure of GaCT, which is not pre-sent in aqueous solution24 or in GDP-bound Gtabc
31,32, is used toconnect the E(D)RY and NPxxY(x)5,6F regions in Ops* (Fig. 4b). Themain-chain carbonyl groups of Cys 347 and Lys 345 of GaCT are thebridge heads in an extended hydrogen-bonded network reachingfrom Cys 347 via Arg 135 (TM3) to Tyr 223 (TM5) on one side,and from Lys 345 via Gln 312 (H8) to Asn 310 (TM7) on the otherside, explaining why mutations in the TM7–H8 kink affect binding ofGaCT homologues33. For the Ops*-induced formation of the C-cap,the hydrogen bridges to Arg 135 and Gln 312 are probably the maindeterminants, together with GaCT-intramolecular hydrogen bonds.We assume that the C-cap is needed for precise recognition betweenGPCRs and G proteins. Contacts occur from fixed main chains andnot flexible side chains (a mode of interaction known from trans-membrane helices34,35), arguing for recognition of GaCT by means ofthe topology and specifics of local geometry.
Apart from local effects, GaCT binding also induces long-rangestabilization effects into the ligand binding pocket. In Ops*, no elec-tron density was observed for the side chain of Lys 296—the retinalattachment site. In Ops*–GaCT, however, electron density isobserved, which indicates that the Lys 296 side chain is stabilizedby a potential network of weak interactions between the e-aminogroup of Lys 296 and the side chains of Ser 186 and Glu 181 in loopE2 (Fig. 4c). In this network, the carboxyl group of Glu 181 is alsostabilized by the phenolic hydroxyl group of Tyr 268 (Fig. 4c, d).Notably, Glu 113—which is thought to form the counterion in inact-ive opsin36—is more than 6 A from Lys 296 within Ops*–GaCT,which is too far to interact. Instead, the side chain of Glu 113 formsa hydrogen bridge with the main-chain nitrogen of Cys 187 in loopE2. Cys 187 in turn is further tethered to Cys 110 in TM3 by a con-served disulphide bridge. Concerning Lys 296, the resolution of thestructure does not provide details of water in the changed hydrogen-bonded networks at the chromophore-binding site and the nearbyNPxxY(x)5,6F motif8,9, so that not all the contributions to fixation ofLys 296 can be derived. However, it seems that G protein and ligand-binding sites are coupled in the active receptor conformation, fittingin with the classical receptor theory, in which the active conforma-tion is stabilized by the ligand and/or G protein37.
Ops* conformation in signal transduction and regeneration
The present analyses show that the same set of structural elementsand conserved GPCR residues in the E(D)RY and NPxxY(x)5,6Fregions, which stabilize inactive rhodopsin, are used in differentinteractions in ligand-free Ops*, and are decisive in building newinteractions of Ops* with GaCT. The same residues were crucial inprevious analyses of the determinants of the metarhodopsin II
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Figure 3 | Interactions of Arg 135 of the conserved E(D)RY motif. a, Indark-state rhodopsin (PDB accession 1U19), Arg 135 of the conservedE(D)RY motif interacts with Glu 134 and links TM3 with TM6 by Glu 247and Thr 251. The TM7–H8 kink is stabilized by electrostatic interactionbetween Tyr 306 and Phe 313. b, In opsin (PDB accession 3CAP), Tyr 223(TM5) interacts with Arg 135, which is released from Glu 134. Tyr 306 isrotated to face into the helical bundle. c, In Ops*–GaCT (Ops*, orange;GaCT, blue), Arg 135 and Tyr 223 interact with the main-chain carbonyl ofGaCT residue Cys 347. In addition to the rotated Tyr 306 side chain, Phe 313shows a different rotamer conformation. Parts of the helices in front wereremoved for clarity.
state19,27,38,39, with the hallmark of TM6 motion4,15,17. The structuralinformation thus provides insights into how the Ops* state can act asthe principal element in two different functional modules of the rodcell, that is, the signal transduction and the rhodopsin regenerationmodules40. In signal transduction, the Ops* conformation is presentin the highly active metarhodopsin II photoproduct, in which thecovalently bound all-trans-retinal agonist shifts the pK for the tran-sition to the active conformation into the neutral pH range3,41. Inrhodopsin regeneration, Ops* facilitates the uptake of 11-cis-retinalinto the binding site42 and causes the visual system to behave as if it isexperiencing a phenomenon equivalent to light (‘equivalent back-ground light’; ref. 5) owing to its capability—like metarhodopsinII—to activate the G protein.
A conceptual model for signal transfer to the G protein
We next studied whether the Ops*–GaCT structure provides a clueas to how the signal propagates from the receptor–GaCT interface tothe 35–40 A distant nucleotide-binding site in the G protein forGDP–GTP exchange. The Ops*–GaCT structure shows a well-defined interaction of a short fragment of Gat with the receptor, sothat the position of the Gtabc holoprotein in relation to the receptorand to the membrane can be determined. Therefore, we first mod-elled the Gat C-terminal a5 helix as part of the Ops*–GaCT complex.For this, GaCT in Ops*–GaCT was amino-terminally elongatedusing the standard geometry of an a-helix (Supplementary Fig. 5).In a superposition of the resulting Ops*–GaCT and a5 model withthe GDP-bound Gtabc crystal structure31, Gtabc clashes with thelipid membrane (Fig. 5a). To avoid this, the Gtabc body (residues1–324) has to be tilted relative to the helical C terminus (shown in red
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Figure 4 | Stabilizing effects of GaCT on Ops*. a, Pairing of the cytoplasmicparts of TM5 and TM6 in Ops* exposes hydrophobic side chains for van derWaals interaction with GaCT, which adopts a near-ideal a-helix terminatedby an aL-type C-cap. Amino acids of GaCT are labelled in blue font. b, Thehydrogen-bonded network linking GaCT peptide with the two conservedE(D)RY (Arg 135) and NPxxY(x)5,6F (Gln 312 and Asn 310) regions of Ops*.c, Long-range effects of GaCT binding lead to stabilization of the Lys 296side chain in a potential network of weak interactions comprising Lys 296 inTM7, Ser 186 and Glu 181 in loop E2, and Tyr 268 in TM6. A direct
stabilizing effect on Lys 296 by Glu 113 is negligible owing to a large distanceof 6.2 A between the side-chain amino and carboxyl groups. The numbersdenote distances in A (b, c). d, The view of the retinal binding pocket fromthe extracellular side is shown. 11-cis-retinal was superimposed fromrhodopsin (PDB accession 1U19) to illustrate the empty retinal binding site.The side chains of Tyr 268 and Trp 265 moved into the space filled by retinalin rhodopsin. The blue mesh represents 2Fo 2 Fc electron density contouredat 1.0s (c, d). Parts of the helices in front were removed for clarity.
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Figure 5 | Conceptual model for signal transmission from the activereceptor to the G protein by the Ga C terminus. a, The Ops*–Gtabccomplex modelled by superposition of Gtabc (PDB accession 1GOT; grey)and Ops*–GaCT (orange and blue, respectively) with a completed (red)C-terminal a5 helix (see Supplementary Fig. 5 for details of a5 helixmodelling). b, To avoid a clash with the lipid bilayer, the remainder of Gtabcwas tilted by about 40u relative to the a5 helix as indicated by the arrow. Inthis conformation, GDP is assumed to be released43.
and blue in Fig. 5). A simple possibility is a change within the b6–a5loop, which is involved in guanine-ring binding and connects the b6strand (Gat residues 316–320) with the C-terminal a5 helix and itsC-cap (Gat residues 325–350). A suitable rigid-body rotation andtranslation of the a5 helix relative to the Gtabc body on binding toactivated rhodopsin was proposed from electron paramagnetic reso-nance studies on Gai1, a close homologue of Gat, and considered tobe necessary for receptor-catalysed GDP release22,43. We may thusassume that the newly formed Ops* and GaCT interaction triggersGDP release using the a5 helix as a transmission rod, with extraconstraints fixing the G protein relative to the receptor, to alter theb6–a5 loop conformation. An intact a5 helix was previously iden-tified as a necessary element in this regard43–45. The model in Fig. 5btherefore probably represents a state in which the receptor hasalready induced emptying of the Gat nucleotide-binding pocket43.
Future work is needed to clarify the role of further structural ele-ments involved in the receptor and G protein interaction. AlthoughGbc is not mandatory for catalysed nucleotide exchange46, modu-lation of the Ga–Gbc interface in the holo G protein seems tofacilitate GDP release (ref. 22 and references therein). A receptor-mimetic peptide derived from the N-terminal portion of loop C3 ofthe D2-dopamine receptor activates the Gai and Gao subunitsdirectly47. This peptide binds between the a4 helix and the b6 strandof Gai1–GDP, suggesting that the receptor loop C3 can contribute bymeans of the b6 strand to b6–a5 loop modulation22,48. It remains tobe investigated how the active receptor recognizes the unstructureddistal Ga C terminus and how an initial encounter/docking com-plex49 between receptor and GDP-bound G protein is formed. Asequential interaction model has been proposed in which the G pro-tein docks first by the farnesylated Gbc subunit to the receptor tomake the Ga C terminus available for receptor interaction23,46.
METHODS SUMMARY
Native rhodopsin was purified by selective extraction from bovine rod
outer-segment disc membranes using b-D-octylglucopyranoside. In a mixture
with the GaCT peptide, rhodopsin was light-activated immediately before co-
crystallization. Crystals grew within 10 days using hanging-drop vapour dif-
fusion in a mixture of ammonium sulphate and MES or sodium citrate buffer
at pH 6. Crystals were cryoprotected in 10% trehalose in precipitation buffer and
frozen in liquid nitrogen for X-ray analysis at the synchrotron BESSY (Berlin,
Germany). The Ops*–GaCT structure was solved by molecular replacement
using the ligand-free opsin monomer (Protein Data Bank (PDB) accession
3CAP) as a search model.
Full Methods and any associated references are available in the online version ofthe paper at www.nature.com/nature.
Received 29 May; accepted 8 August 2008.
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Supplementary Information is linked to the online version of the paper atwww.nature.com/nature.
Acknowledgements We thank J. Engelmann and C. Koch for technical assistance;P. Henklein for peptide synthesis; C. Enenkel and M. Sommer for critically readingthe manuscript; U. Muller and the scientific staff of the Protein Structure Factoryand the Freie Universitat Berlin at beamlines BL 14.1 and BL 14.2 at BESSY forcontinuous support of the project. This work was supported by the DeutscheForschungsgemeinschaft Sfb449 (to O.P.E.), Sfb740 (to O.P.E. and K.P.H.),DFG-KOSEF international cooperation ER 294/1-1 (to O.P.E.) andF01-2004-000-10054-0 (to H.-W.C.), and CBNU funds for overseas research2006–2007 (to H.-W.C.) and a fellowship of the Leibniz Graduate School ofMolecular Biophysics, Berlin (to Y.J.K.).
Author Information The atomic coordinates and structure factors have beendeposited in the Protein Data Bank under accession number 3DQB. Reprints andpermissions information is available at www.nature.com/reprints.Correspondence and requests for materials should be addressed to O.P.E.([email protected]), K.P.H. ([email protected]) or H.-W.C.([email protected]).
METHODSPeptide synthesis. Peptide GaCT (corresponding to Gat(340–350)K341L,
amino acid sequence ILENLKDCGLF) was derived from the extreme C terminus
of the Gat subunit. It was synthesized with unmodified N and C termini as
described23,50. The K341L substitution was found previously to increase the
affinity of the peptide by two orders of magnitude compared with the native
Gat(340–350) peptide23,50.
Protein preparation and crystallization. Bovine rod outer segment membranes
and opsin were prepared as described18,51. Rod outer segment membranes
(10 mg ml21 rhodopsin) and opsin (7 mg ml21) within rod outer segment mem-branes were solubilized essentially as described, using 1%b-D-octylglucopyrano-
side9,52. Synthetic GaCT peptide was added in a 4:1 molar ratio of peptide to
solubilized rhodopsin. The mixture was incubated on ice for 1 h and illuminated
for 15 s with green light (500 6 20 nm). The illuminated sample was used with-
out further purification for crystallization. Alternatively, GaCT was mixed with
solubilized opsin in a 4:1 molar ratio for crystallization. Crystallization screens
by the sparse matrix crystallization method53 were carried out using the hanging-
drop vapour diffusion method and more than 2,000 crystallization conditions.
Promising conditions were systematically screened further by altering protein
concentration, precipitation agents and pH. Ops*–GaCT crystals could be
grown at 277 K using 24-well Linbro plates. Each hanging drop was prepared
on a siliconized coverslip by mixing equal volumes (2ml each) of receptor and
GaCT mixture and reservoir solution. The reservoir solution contained 3.2 M
ammonium sulphate in 0.1 M MES or sodium citrate buffer, pH 6.0. Crystals
appeared within 5 days and continued to grow for 5 days. Fully grown crystals
had dimensions of 0.05 3 0.05 3 0.15 mm3.
Structure analysis. X-ray data collection of Ops*–GaCT crystals was performed
at 100 K using cryoprotectant consisting of 90% (v/v) reservoir solution and 10%(w/v) trehalose. Diffraction data were collected at synchrotron beamline BL 14.2
(wavelength 0.9184A) of the Protein Structure Factory and Freie Universitat
Berlin at BESSY with a MAR-165CCD detector. The crystal to detector distance
was fixed at 240 mm. The rotation increment for each frame was 0.2u with an
exposure time of 10 s. All images were indexed, integrated and scaled using
HKL2000 (ref. 54). Ops*–GaCT crystals belong to rhombohedral space group
H32 (a 5 238.02 A, b 5 238.02 A, c 5 109.13 A, a5b5 90u, c5 120u).
Supplementary Table 1 summarizes the statistics for crystallographic data col-
lection and structural refinement.
Initial phases for Ops*–GaCT were obtained by conventional molecular
replacement protocol (rotation, translation and rigid body fitting) using the
model of ligand-free opsin structure (PDB accession 3CAP) as an initial search
trial. Molecular replacement was achieved using the CCP4 program PHASER55
by first placing the seven transmembrane bundle of the opsin monomer (rota-
tion function: Z 5 8.8; translation function: Z 5 54.0, as defined by PHASER). In
subsequent steps, torsion angle molecular dynamics, simulated annealing using a
slow-cooling protocol and a maximum likelihood target function, energy mini-
mization, and B-factor refinement by the program CNS56 were carried out in the
resolution range 48.22–3.2 A. After the first round of refinement, the GaCT
peptide was clearly visible in the electron density of both sA-weighted Fo 2 Fc
maps, as well as in the simulated annealing omit density maps (as shown in
Supplementary Fig. 2). Restrained individual B-factors were refined and the
crystal structure was finalized by REFMAC5 and other programs in CCP4 (ref.
55). The final model had agreement factors Rfree and Rcryst of 24.8% and 21.3%,
respectively. Manual rebuilding of the opsin model and electron density inter-
pretation were performed after each refinement cycle using the program
COOT57. Structure validation was performed with the programsPROCHECK58 and WHAT_CHECK59. Potential hydrogen bonds and van der
Waals contacts (Supplementary Table 2) were analysed using the programs
HBPLUS60 and LIGPLOT61 (Supplementary Fig. 8). All crystal structure super-
positions of backbone alpha carbon traces were performed using CCP4 program
LSQKAB (Figs 2b and 4d and Supplementary Figs 3b, c, 4a–c and 5)55. All
molecular graphics representations were created using PyMol62.
50. Herrmann, R. et al. Rhodopsin-transducin coupling: role of the Ga C-terminus innucleotide exchange catalysis. Vision Res. 46, 4582–4593 (2006).
51. Sachs, K., Maretzki, D. & Hofmann, K. P. Assays for activation of opsin by all-trans-retinal. Methods Enzymol. 315, 238–251 (2000).
52. Murakami, M., Kitahara, R., Gotoh, T. & Kouyama, T. Crystallization and crystalproperties of squid rhodopsin. Acta Crystallogr. F 63, 475–479 (2007).
53. Jancarik, J. & Kim, S.-H. Sparse matrix sampling: a screening method forcrystallization of proteins. J. Appl. Crystallogr. 24, 409–411 (1991).
54. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected inoscillation mode. Methods Enzymol. 276, 307–326 (1997).
55. Collaborative Computational Project, Number 4. The CCP4 suite: programs forprotein crystallography. Acta Crystallogr. D 50, 760–763 (1994).
56. Brunger, A. T. et al. Crystallography & NMR system: A new software suite formacromolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998).
57. Emsley, P. & Cowtan, K. Coot: Model-Building Tools for Molecular Graphics. ActaCrystallogr. D 60, 2126–2132 (2004).
58. Laskowski, R. A., MacArthur, M. W., Moss, D. S. & Thornton, J. M. PROCHECK: aprogram to check the stereochemical quality of protein structures. J. Appl.Crystallogr. 26, 283–291 (1993).
59. Hooft, R. W., Vriend, G., Sander, C. & Abola, E. E. Errors in protein structures.Nature 381, 272 (1996).
60. McDonald, I. K. & Thornton, J. M. Satisfying hydrogen bonding potential inproteins. J. Mol. Biol. 238, 777–793 (1994).
61. Wallace, A. C., Laskowski, R. A. & Thornton, J. M. LIGPLOT: a program to generateschematic diagrams of protein-ligand interactions. Protein Eng. 8, 127–134 (1995).
62. DeLano, W. L. The PyMOL Molecular Graphics System. ,http://www.pymol.org. (2002).
