Crystal structure of opsin in its G-protein-interacting conformation

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1 Vol Septemer 2008 doi:0.038/nture0330 Crystl structure of opsin in its G-protein-intercting conformtion ARTICLES Ptrick Scheerer *, Jung Hee Prk *, Peter W. Hildernd, Yong Ju Kim, Norert Kruß 2, Hui-Woog Choe,3, Klus Peter Hofmnn,4 & Oliver P. Ernst Opsin, the lignd-free form of the G-protein-coupled receptor rhodopsin, t low ph dopts conformtionlly distinct, ctive G-protein-inding stte known s Ops*. A synthetic peptide derived from the min inding site of the heterotrimeric G protein the croxy terminus of the -suunit (GCT) stilizes Ops*. Here we present the 3.2 Å crystl structure of the ovine Ops* GCT peptide complex. GCT inds to site in opsin tht is opened y n outwrd tilt of trnsmemrne helix (TM), piring of nd, nd restructured helix 8 kink. Contcts long the inner surfce of nd induce n -helicl conformtion in GCT with C-terminl reverse turn. Min-chin cronyl groups in the reverse turn constitute the centre of hydrogen-onded network, which links the two receptor regions contining the conserved E(D)RY nd NPxxY(x) 5, F motifs. On the sis of the Ops* GCT structure nd known conformtionl chnges in G, we discuss signl trnsfer from the receptor to the G protein nucleotide-inding site. G-protein-coupled receptors (GPCRs), lso known s seventrnsmemrne receptors, re the lrgest superfmily of plsm memrne receptors. Binding of extrcellulr lignds to GPCRs modultes their cpcity to ctlyse GDP GTP exchnge in heterotrimeric G proteins (Gc), therey regulting the intrcellulr level of secondry messengers. Rhodopsin is the photoreceptor of the verterte retinl rod cell nd the eponym of the rhodopsin fmily of GPCRs with,0 memers in the humn genome 2. Rhodopsin consists of the poprotein opsin nd the chromophore -cis-retinl, which is ound y protonted Schiff se to Lys 29 in the seventh trnsmemrne helix to stilize the inctive receptor stte. Photon sorption leds to cis trns isomeriztion of the retinl, which triggers deprotontion of the retinylidene Schiff se linkge nd formtion of the ctive, G-protein-inding metrhodopsin II stte 3,4. Metrhodopsin II decys y hydrolysis of the retinylidene Schiff se nd relese of the photolysed ll-trns-retinl from its inding pocket to generte lignd-free opsin. Under physiologicl conditions, fresh -cis-retinl is metoliclly supplied nd tken up to regenerte rhodopsin 3,5. Before regenertion is complete, visul sensitivity is reduced owing to ckground level of GTP-ound G protein which is mintined y persistently ctive form of opsin 5. In vitro, opsin cn redily dopt inctive (Ops) nd ctive (Ops*) conformtions, nd low ph nd synthetic peptide derived from the C terminus of G stilize Ops* (ref. ). Besides the crystl structures of ovine nd squid rhodopsin 0 nd different photointermedites,2, the structures of two isoforms of the -drenergic receptor 3,4 re known. All of these GPCR structures contin lignd ound in their inding site nd lck prominent tilt of the cytoplsmic hlf of out of the helicl undle, which is considered to e mndtory for G protein ctivtion 5.We recently solved the crystl structure of lignd-free opsin nd found tht it ws quite different from the known GPCR structures 8. The most prominent fetures were the ctivting movement of nd ccompnying rerrngements in the regions of the conserved E(D)RY nd NPxxY(x) 5, F motifs 3,4,9. Here we report the structure of Ops* when it ctively inds to the key G protein interction site. Mjor sites for receptor interction include the C termini of the G nd Gc suunits of trnsducin (G t c, consisting of G t,g nd Gc ), the cognte G protein of rhodopsin We stilized Ops* with n mino cid synthetic peptide (GCT, mino cid sequence ILENLKDCGLF, G t ( )K34L) derived from the extreme C terminus of the trnsducin G t suunit 23. The crystl structure of the Ops* GCT complex provides insight into the structurl chnges involved in signl trnsfer from the receptor to the G protein. Structure of Ops* GCT complex Optimized extrction of the receptor from ntive rod cell disc memrnes llowed the growth of Ops* GCT complex crystls. We used two different pproches: co-crystlliztion of the GCT peptide with either opsin or photoctivted rhodopsin, respectively. In the ltter cse, the rhodopsin nd GCT mixture ws illuminted directly efore crystlliztion to produce ctive metrhodopsin II. Loss of lltrns-retinl from the metrhodopsin II GCT complex occurred during crystlliztion nd resulted in n Ops* GCT complex. This ws determined y the lck of electron density for retinl in the 3.2 Å crystl structure which ws otined y co-crystlliztion of GCT nd photoctivted rhodopsin (Fig. nd Supplementry Fig. ). When soluilized lignd-free opsin ws used s the strting mteril, Ops* GCT crystls were otined which diffrcted to 3.8 Å nd showed, within the limits of resolution, the sme structure nd inding of GCT (dt not shown). For crystlliztion, dt collection, structure determintion nd refinement sttistics see Methods nd Supplementry Tle. The structure of Ops* GCT comprises mino cids 32 of opsin (lcking 22 C-terminl mino cids which re not resolved, presumly ecuse of their known high moility), nd mino cids of the GCT peptide with well defined electron density (Fig. nd Supplementry Figs nd 2). The Ops* model includes the Institut für Medizinische Physik und Biophysik (CC2), Chrité Universitätsmedizin Berlin, Chritépltz, D-0 Berlin, Germny. 2 Queen Mry, University of London, School of Biologicl nd Chemicl Sciences, London E 4NS, UK. 3 Deprtment of Chemistry, College of Nturl Science, Chonuk Ntionl University, 5-5 Chonju, South Kore. 4 Zentrum für Biophysik und Bioinformtik, Humoldt-Universität zu Berlin, Invlidenstrsse 42, D-05 Berlin, Germny. *These uthors contriuted eqully to this work. 49

2 ARTICLES NATURE Vol Septemer 2008 seven trnsmemrne helices connected y extrcellulr (E E3) nd cytoplsmic (C ) loops, nd the cytoplsmic helix 8 (), which runs long the memrne. The structure of Ops* GCT ers resemlnce to the structure of peptide-free opsin (free Ops*) 8, which ws used s serch model to solve the structure y moleculr replcement. Compred to the Ops* structure 8, the Ops* GCT structure presented here shows sutle ut distinct chnges in Ops* induced y GCT inding (Supplementry Fig. 4). Gross structurl fetures which re comprle in free Ops* nd Ops* GCT ut re different from the -cis-retinl-ound drkstte rhodopsin include short helicl turn in loop C nd rerrngements of loops C2 nd (Fig. 2). Compred with drk-stte rhodopsin, the cytoplsmic hlf of is tilted outwrds of the helicl undle y Å with Trp 25 s the pivot point, in greement with predictions of the ctive receptor stte derived from electron prmgnetic resonnce spectroscopy 5,. Furthermore, is longer, very stright nd more inclined thn in rhodopsin, with resulting shift of the cytoplsmic end y 2 3 Å towrds. As consequence, the cytoplsmic ends of nd re closer nd nerly prllel to ech other, forming cytoplsmic helicl pir. In the extrcellulr hlf, the Ops* GCT structure lso shows two openings etween nd nd etween TM nd, respectively (dt not shown), which my ct s gtes for retinl 8. Owing to the nd rerrngement, crevice is opened into which the GCT peptide inds in n -helicl conformtion with n open reverse turn, n L -type C-cpping motif (C-cp; Fig. 2 nd Supplementry Figs 2 ). Notly, the conformtion of GCT oserved in the Ops* GCT crystl is lmost identicl to the NMR solution structure of GCT nlogue nd the prent G t ( ) peptide in the receptor-ound stte determined y trnsfer nucler Overhuser effect experiments 24,25 (Supplementry Fig. 3). As seen in the superposition of rhodopsin nd GCT (s prt of Ops* GCT), Intrcellulr TM Extrcellulr C2 E2 E3 N C E Figure Overll structure of Ops* GCT complex. The symmetric unit contins one molecule of Ops* with one molecule of GCT peptide ound t the intrcellulr side. Shown is crystllogrphic dimer of symmetry relted Ops* GCT monomers, with the view prllel to the memrne. The Ops* molecules (ornge nd yellow) nd the GCT peptides (lue) re shown in rion representtion, nd the GCT side chins re shown s sticks. One of the GCT molecules is lso shown s trnsprent spce-filling model. The GCT peptide is derived from the G t C terminus (G t ( )K34L). The K34L sustitution increses the ffinity for the ctive receptor conformtion y two orders of mgnitude 23. Ops* consists of seven trnsmemrne helices (TM), connected y extrcellulr (E E3) nd cytoplsmic (C ) loops nd the cytoplsmic helix. Two ntiprllel -sheets in the N terminus (strnds nd 2) nd loop E2 (strnds 3 nd 4) re shown s lue rrows. Oligoscchrides t Asn 2 nd Asn 5 nd plmitoyl chins t Cys 322 nd Cys 323 re presented s sticks. 498 dipping of GCT into the cytoplsmic prt of the helicl undle is not possile for the inctive drk stte of rhodopsin with the given orienttion of GCT (Fig. 2 nd Supplementry Fig. 5). The dul role of in the E(D)RY motif In rhodopsin, nd re tethered y hydrogen-onded network ( ionic lock ) which includes the side chins of nd Glu 34 from the conserved E(D)RY motif in, nd the side chins of Glu 24 nd Thr 25 in (Fig. 3). In the peptide-free Ops* stte 8 (Fig. 3), s well s in Ops* GCT (Fig. 3c), the ionic lock is roken owing to the outwrd movement of. The newly formed cytoplsmic pir conformtion is stilized y new interctions tht is, the side chins of Glu 24 nd Thr 25 re relesed from nd engge with the side chin of Lys 23 in hydrogen-onded network (not shown in Fig. 3, c; see ref. 8). Aprt from its min function in the ionic lock of rhodopsin, one of the most conserved residues in GPCRs hs crucil role in the ctive receptor stte. In oth Ops* nd Ops* GCT, the hydroxyl group of from replces the croxyl group of Glu 34 y its interction with. The rginine side chin is therey lierted from Glu 34 nd llowed to swing into the centre of the GCT inding crevice to form its floor. Stilized y, it 5 5 C2 3 3 C C 5 C 5 3 Figure 2 Comprison of Ops* GCT nd drk-stte rhodopsin structures., View from the cytoplsm of Ops* GCT. Ops* is shown in rion (ornge) nd surfce (grey) representtion, the G t ( )K34L peptide (ILENLKDCGLF) is shown s rion nd stick model (lue). The ngle etween the helicl xis nd the memrne norml (43 2u) is in good greement with predictions from residul dipolr coupling NMR dt 25., The view from the cytoplsm of drk-stte rhodopsin (PDB ccession U9; green rion nd grey surfce) nd superposed GCT (lue) is shown. The scheme indictes differences in the rrngement of TM etween Ops* GCT (Ops*, ornge; GCT, lue) nd drk-stte rhodopsin (green). 4 2

3 NATURE Vol Septemer 2008 ARTICLES then intercts with the ckone cronyl group of Cys 34 t the tip of GCT (Fig. 3c). Conversely, Glu 34 is not involved in n interction with GCT. Glu 34 fces towrds TM2 nd TM4, nd further electron density is oserved close to the croxyl group of Glu 34. This electron density could e interpreted s wter molecule providing wek linkge of Glu 34 to the min-chin cronyl of Al 53 nd peptide nitrogen of Ile 5 (Supplementry Fig. ). In this rther non-polr environment, Glu 34 is proly protonted which explins the proton uptke mesured in the course of the lightctivtion of rhodopsin 4,2, which does not occur in the E34Q mutnt 2. The outwrdly tilted position of nd the new loction of the side chin in Ops* nd Ops* GCT is stilized y Tyr 30 s prt of the conserved NPxxY(x) 5, F motif connecting nd (Fig. 3, c). In rhodopsin, the side chins of Tyr 30 () nd () interct with ech other, wheres in Ops* nd Ops* GCT, Tyr 30 is extended into the helicl undle elow nd hinders the inwrd tilt of. c Glu 34 Glu 34 Glu 34 Glu 24 Thr 25 Tyr 30 Tyr 30 Tyr 30 Figure 3 Interctions of of the conserved E(D)RY motif., In drk-stte rhodopsin (PDB ccession U9), of the conserved E(D)RY motif intercts with Glu 34 nd links with y Glu 24 nd Thr 25. The kink is stilized y electrosttic interction etween Tyr 30 nd., In opsin (PDB ccession 3CAP), () intercts with, which is relesed from Glu 34. Tyr 30 is rotted to fce into the helicl undle. c, In Ops* GCT (Ops*, ornge; GCT, lue), nd interct with the min-chin cronyl of GCT residue Cys 34. In ddition to the rotted Tyr 30 side chin, shows different rotmer conformtion. Prts of the helices in front were removed for clrity. GCT nd retinl-inding pockets The inner side of the cytoplsmic nd pir in Ops* provides hydrophoic surfce formed y Leu 22, Vl 230 nd Al 233 in, nd y Thr 242, Thr 243, Al 24 nd Vl 250 in, for hydrophoic interction with GCT (Fig. 4, Supplementry Figs 8 nd 9 nd Supplementry Tle 2). Further hydrophoic contcts re provided y Vl 38 nd Vl 39 t the junction of nd loop C2, nd y Leu 2 in loop C. Some of the elements tht contriute to this prt of the inding site were predicted from erlier mpping pproches y muttionl nlysis 28,29 nd photocrosslinking 30. The fully induced C-cpped helicl structure of GCT, which is not present in queous solution 24 or in GDP-ound G t c 3,32, is used to connect the E(D)RY nd NPxxY(x) 5, F regions in Ops* (Fig. 4). The min-chin cronyl groups of Cys 34 nd Lys 345 of GCT re the ridge heds in n extended hydrogen-onded network reching from Cys 34 vi () to () on one side, nd from Lys 345 vi Gln 32 () to Asn 30 () on the other side, explining why muttions in the kink ffect inding of GCT homologues 33. For the Ops*-induced formtion of the C-cp, the hydrogen ridges to nd Gln 32 re proly the min determinnts, together with GCT-intrmoleculr hydrogen onds. We ssume tht the C-cp is needed for precise recognition etween GPCRs nd G proteins. Contcts occur from fixed min chins nd not flexile side chins ( mode of interction known from trnsmemrne helices 34,35 ), rguing for recognition of GCT y mens of the topology nd specifics of locl geometry. Aprt from locl effects, GCT inding lso induces long-rnge stiliztion effects into the lignd inding pocket. In Ops*, no electron density ws oserved for the side chin of Lys 29 the retinl ttchment site. In Ops* GCT, however, electron density is oserved, which indictes tht the Lys 29 side chin is stilized y potentil network of wek interctions etween the e-mino group of Lys 29 nd the side chins of Ser 8 nd Glu 8 in loop E2 (Fig. 4c). In this network, the croxyl group of Glu 8 is lso stilized y the phenolic hydroxyl group of Tyr 28 (Fig. 4c, d). Notly, Glu 3 which is thought to form the counterion in inctive opsin 3 is more thn Å from Lys 29 within Ops* GCT, which is too fr to interct. Insted, the side chin of Glu 3 forms hydrogen ridge with the min-chin nitrogen of Cys 8 in loop E2. Cys 8 in turn is further tethered to Cys 0 in y conserved disulphide ridge. Concerning Lys 29, the resolution of the structure does not provide detils of wter in the chnged hydrogenonded networks t the chromophore-inding site nd the nery NPxxY(x) 5, F motif 8,9, so tht not ll the contriutions to fixtion of Lys 29 cn e derived. However, it seems tht G protein nd ligndinding sites re coupled in the ctive receptor conformtion, fitting in with the clssicl receptor theory, in which the ctive conformtion is stilized y the lignd nd/or G protein 3. Ops* conformtion in signl trnsduction nd regenertion The present nlyses show tht the sme set of structurl elements nd conserved GPCR residues in the E(D)RY nd NPxxY(x) 5, F regions, which stilize inctive rhodopsin, re used in different interctions in lignd-free Ops*, nd re decisive in uilding new interctions of Ops* with GCT. The sme residues were crucil in previous nlyses of the determinnts of the metrhodopsin II 499

4 ARTICLES NATURE Vol Septemer 2008 Thr 242 Phe 350 Thr 243 Leu 34 Glu 342 Al 233 Al 24 Lys 345 Asn 343 Leu 344 Vl 230 Ile 340 Asp 34 Vl 250 Gly 348 Leu 22 Leu 349 Cys 34 Ile 340 Cys Lys Gln 32 Asn Tyr 30 TM c Lys 29 Tyr 28 d Lys 29 Trp 25 Tyr Ser 8 Glu Ser 8 Glu 8 Met 20 Tyr 9 Glu 8 E2 Figure 4 Stilizing effects of GCT on Ops*., Piring of the cytoplsmic prts of nd in Ops* exposes hydrophoic side chins for vn der Wls interction with GCT, which dopts ner-idel -helix terminted y n L -type C-cp. Amino cids of GCT re lelled in lue font., The hydrogen-onded network linking GCT peptide with the two conserved E(D)RY () nd NPxxY(x) 5, F (Gln 32 nd Asn 30) regions of Ops*. c, Long-rnge effects of GCT inding led to stiliztion of the Lys 29 side chin in potentil network of wek interctions comprising Lys 29 in, Ser 8 nd Glu 8 in loop E2, nd Tyr 28 in. A direct stilizing effect on Lys 29 y Glu 3 is negligile owing to lrge distnce of.2 Å etween the side-chin mino nd croxyl groups. The numers denote distnces in Å (, c). d, The view of the retinl inding pocket from the extrcellulr side is shown. -cis-retinl ws superimposed from rhodopsin (PDB ccession U9) to illustrte the empty retinl inding site. The side chins of Tyr 28 nd Trp 25 moved into the spce filled y retinl in rhodopsin. The lue mesh represents 2F o 2 F c electron density contoured t.0s (c, d). Prts of the helices in front were removed for clrity. stte 9,2,38,39, with the hllmrk of motion 4,5,. The structurl informtion thus provides insights into how the Ops* stte cn ct s the principl element in two different functionl modules of the rod cell, tht is, the signl trnsduction nd the rhodopsin regenertion modules 40. In signl trnsduction, the Ops* conformtion is present in the highly ctive metrhodopsin II photoproduct, in which the covlently ound ll-trns-retinl gonist shifts the pk for the trnsition to the ctive conformtion into the neutrl ph rnge 3,4.In rhodopsin regenertion, Ops* fcilittes the uptke of -cis-retinl into the inding site 42 nd cuses the visul system to ehve s if it is experiencing phenomenon equivlent to light ( equivlent ckground light ; ref. 5) owing to its cpility like metrhodopsin II to ctivte the G protein. A conceptul model for signl trnsfer to the G protein We next studied whether the Ops* GCT structure provides clue s to how the signl propgtes from the receptor GCT interfce to the Å distnt nucleotide-inding site in the G protein for GDP GTP exchnge. The Ops* GCT structure shows welldefined interction of short frgment of G t with the receptor, so tht the position of the G t c holoprotein in reltion to the receptor nd to the memrne cn e determined. Therefore, we first modelled the G t C-terminl 5 helix s prt of the Ops* GCT complex. For this, GCT in Ops* GCT ws mino-terminlly elongted using the stndrd geometry of n -helix (Supplementry Fig. 5). In superposition of the resulting Ops* GCT nd 5 model with the GDP-ound G t c crystl structure 3,G t c clshes with the lipid memrne (Fig. 5). To void this, the G t c ody (residues 324) hs to e tilted reltive to the helicl C terminus (shown in red 500 βγ α GDP Figure 5 Conceptul model for signl trnsmission from the ctive receptor to the G protein y the G C terminus., The Ops* G t c complex modelled y superposition of G t c (PDB ccession GOT; grey) nd Ops* GCT (ornge nd lue, respectively) with completed (red) C-terminl 5 helix (see Supplementry Fig. 5 for detils of 5 helix modelling)., To void clsh with the lipid ilyer, the reminder of G t c ws tilted y out 40u reltive to the 5 helix s indicted y the rrow. In this conformtion, GDP is ssumed to e relesed 43.

5 NATURE Vol Septemer 2008 ARTICLES nd lue in Fig. 5). A simple possiility is chnge within the 5 loop, which is involved in gunine-ring inding nd connects the strnd (G t residues 3 320) with the C-terminl 5 helix nd its C-cp (G t residues ). A suitle rigid-ody rottion nd trnsltion of the 5 helix reltive to the G t c ody on inding to ctivted rhodopsin ws proposed from electron prmgnetic resonnce studies on G i, close homologue of G t, nd considered to e necessry for receptor-ctlysed GDP relese 22,43. We my thus ssume tht the newly formed Ops* nd GCT interction triggers GDP relese using the 5 helix s trnsmission rod, with extr constrints fixing the G protein reltive to the receptor, to lter the 5 loop conformtion. An intct 5 helix ws previously identified s necessry element in this regrd The model in Fig. 5 therefore proly represents stte in which the receptor hs lredy induced emptying of the G t nucleotide-inding pocket 43. Future work is needed to clrify the role of further structurl elements involved in the receptor nd G protein interction. Although Gc is not mndtory for ctlysed nucleotide exchnge 4, modultion of the G Gc interfce in the holo G protein seems to fcilitte GDP relese (ref. 22 nd references therein). A receptormimetic peptide derived from the N-terminl portion of loop of the D 2 -dopmine receptor ctivtes the G i nd G o suunits directly 4. This peptide inds etween the 4 helix nd the strnd of G i GDP, suggesting tht the receptor loop cn contriute y mens of the strnd to 5 loop modultion 22,48. It remins to e investigted how the ctive receptor recognizes the unstructured distl G C terminus nd how n initil encounter/docking complex 49 etween receptor nd GDP-ound G protein is formed. A sequentil interction model hs een proposed in which the G protein docks first y the frnesylted Gc suunit to the receptor to mke the G C terminus ville for receptor interction 23,4. METHODS SUMMARY Ntive rhodopsin ws purified y selective extrction from ovine rod outer-segment disc memrnes using -D-octylglucopyrnoside. In mixture with the GCT peptide, rhodopsin ws light-ctivted immeditely efore cocrystlliztion. Crystls grew within 0 dys using hnging-drop vpour diffusion in mixture of mmonium sulphte nd MES or sodium citrte uffer t ph. Crystls were cryoprotected in 0% trehlose in precipittion uffer nd frozen in liquid nitrogen for X-ry nlysis t the synchrotron BESSY (Berlin, Germny). The Ops* GCT structure ws solved y moleculr replcement using the lignd-free opsin monomer (Protein Dt Bnk (PDB) ccession 3CAP) s serch model. Full Methods nd ny ssocited references re ville in the online version of the pper t Received 29 My; ccepted 8 August Pierce, K. L., Premont, R. T. & Lefkowitz, R. J. Seven-trnsmemrne receptors. Nture Rev. Mol. Cell Biol. 3, (2002). 2. Lgerstrom, M. C. & Schioth, H. B. Structurl diversity of G protein-coupled receptors nd significnce for drug discovery. Nture Rev. Drug Discov., (2008). 3. Okd, T., Ernst, O. P., Plczewski, K. & Hofmnn, K. P. Activtion of rhodopsin: new insights from structurl nd iochemicl studies. Trends Biochem. Sci. 2, (200). 4. Knierim, B., Hofmnn, K. P., Ernst, O. P. & Huell, W. L. Sequence of lte moleculr events in the ctivtion of rhodopsin. Proc. Ntl Acd. Sci. USA 04, (200). 5. Lm, T. D. & Pugh, E. N. Jr. Drk dpttion nd the retinoid cycle of vision. Prog. Retin. Eye Res. 23, (2004).. Vogel, R. & Sieert, F. Conformtions of the ctive nd inctive sttes of opsin. J. Biol. Chem. 2, (200).. Plczewski, K. et l. Crystl structure of rhodopsin: A G protein-coupled receptor. Science 289, (2000). 8. Li, J., Edwrds, P. C., Burghmmer, M., Vill, C. & Schertler, G. F. Structure of ovine rhodopsin in trigonl crystl form. J. Mol. Biol. 343, (2004). 9. Okd, T. et l. The retinl conformtion nd its environment in rhodopsin in light of new 2.2 Å crystl structure. J. Mol. Biol. 342, (2004). 0. Murkmi, M. & Kouym, T. Crystl structure of squid rhodopsin. Nture 453, 33 3 (2008).. Nkmichi, H. & Okd, T. Locl peptide movement in the photorection intermedite of rhodopsin. Proc. Ntl Acd. Sci. USA 03, (200). 2. Slom, D. et l. Crystl structure of photoctivted deprotonted intermedite of rhodopsin. Proc. Ntl Acd. Sci. USA 03, (200). 3. Wrne, T. et l. Structure of -drenergic G-protein-coupled receptor. Nture 454, (2008). 4. Cherezov, V. et l. High-resolution crystl structure of n engineered humn 2 - drenergic G protein-coupled receptor. Science 38, (200). 5. Frrens, D. L., Altench, C., Yng, K., Huell, W. L. & Khorn, H. G. Requirement of rigid-ody motion of trnsmemrne helices for light ctivtion of rhodopsin. Science 24, 8 0 (99).. Sheikh, S. P., Zvyg, T. A., Lichtrge, O., Skmr, T. P. & Bourne, H. R. Rhodopsin ctivtion locked y metl-ion-inding sites linking trnsmemrne helices C nd F. Nture 383, (99).. Altench, C., Kusnetzow, A. K., Ernst, O. P., Hofmnn, K. P. & Huell, W. L. Highresolution distnce mpping in rhodopsin revels the pttern of helix movement due to ctivtion. Proc. Ntl Acd. Sci. USA 05, (2008). 8. Prk, J. H., Scheerer, P., Hofmnn, K. P., Choe, H.-W. & Ernst, O. P. Crystl structure of the lignd-free G-protein-coupled receptor opsin. Nture 454, 83 8 (2008). 9. Fritze, O. et l. Role of the conserved NPxxY(x) 5, F motif in the rhodopsin ground stte nd during ctivtion. Proc. Ntl Acd. Sci. USA 00, (2003). 20. Hmm, H. E. et l. Site of G protein inding to rhodopsin mpped with synthetic peptides from the suunit. Science 24, (988). 2. Kisselev, O. G., Ermolev, M. V. & Gutm, N. A frnesylted domin in the G protein c suunit is specific determinnt of receptor coupling. J. Biol. Chem. 29, (994). 22. Oldhm, W. M. & Hmm, H. E. Heterotrimeric G protein ctivtion y G-proteincoupled receptors. Nture Rev. Mol. Cell Biol. 9, 0 (2008). 23. Herrmnn, R. et l. Sequence of interctions in receptor-g protein coupling. J. Biol. Chem. 29, (2004). 24. Kisselev, O. G. et l. Light-ctivted rhodopsin induces structurl inding motif in G protein suunit. Proc. Ntl Acd. Sci. USA 95, (998). 25. Koenig, B. W. et l. Structure nd orienttion of G protein frgment in the receptor ound stte from residul dipolr couplings. J. Mol. Biol. 322, 44 4 (2002). 2. Arnis, S. & Hofmnn, K. P. Two different forms of metrhodopsin II: Schiff se deprotontion precedes proton uptke nd signling stte. Proc. Ntl Acd. Sci. USA 90, (993). 2. Arnis, S., Fhmy, K., Hofmnn, K. P. & Skmr, T. P. A conserved croxylic cid group medites light-dependent proton uptke nd signling y rhodopsin. J. Biol. Chem. 29, (994). 28. Achry, S., Sd, Y. & Krnik, S. S. Trnsducin- C-terminl peptide inding site consists of C-D nd E-F loops of rhodopsin. J. Biol. Chem. 22, (99). 29. Jnz, J. M. & Frrens, D. L. Rhodopsin ctivtion exposes key hydrophoic inding site for the trnsducin -suunit C terminus. J. Biol. Chem. 29, (2004). 30. Ci, K., Itoh, Y. & Khorn, H. G. Mpping of contct sites in complex formtion etween trnsducin nd light-ctivted rhodopsin y covlent crosslinking: use of photoctivtle regent. Proc. Ntl Acd. Sci. USA 98, (200). 3. Lmright, D. G. et l. The 2.0 Å crystl structure of heterotrimeric G protein. Nture 39, 3 39 (99). 32. Ridge, K. D. et l. Conformtionl chnges ssocited with receptor stimulted gunine nucleotide exchnge in heterotrimeric G-protein -suunit: NMR nlysis of GTPc S-ound sttes. J. Biol. Chem. 28, (200). 33. Ernst, O. P. et l. Muttion of the fourth cytoplsmic loop of rhodopsin ffects inding of trnsducin nd peptides derived from the croxyl-terminl sequences of trnsducin nd c suunits. J. Biol. Chem. 25, (2000). 34. Edwrds, M. D. et l. Pivotl role of the glycine-rich helix in gting the MscS mechnosensitive chnnel. Nture Struct. Mol. Biol. 2, 3 9 (2005). 35. Hildernd, P. W. et l. Hydrogen-onding nd pcking fetures of memrne proteins: functionl implictions. Biophys. J. 94, (2008). 3. Cohen, G. B., Oprin, D. D. & Roinson, P. R. Mechnism of ctivtion nd inctivtion of opsin: role of Glu 3 nd Lys 29. Biochemistry 3, (992). 3. De Len, A., Stdel, J. M. & Lefkowitz, R. J. A ternry complex model explins the gonist-specific inding properties of the denylte cyclse-coupled -drenergic receptor. J. Biol. Chem. 255, 08 (980). 38. Fhmy, K. & Skmr, T. P. Regultion of the rhodopsin-trnsducin interction y highly conserved croxylic cid group. Biochemistry 32, (993). 39. Frnke, R. R., König, B., Skmr, T. P., Khorn, H. G. & Hofmnn, K. P. Rhodopsin mutnts tht ind ut fil to ctivte trnsducin. Science 250, (990). 40. Hofmnn, K. P., Sphn, C. M., Heinrich, R. & Heinemnn, U. Building functionl modules from moleculr interctions. Trends Biochem. Sci. 3, (200). 4. Meyer, C. K. et l. Signling sttes of rhodopsin. Retinl provides scffold for ctivting proton trnsfer switches. J. Biol. Chem. 25, (2000). 42. Keflov, V. J., Crouch, R. K. & Cornwll, M. C. Role of noncovlent inding of -cisretinl to opsin in drk dpttion of rod nd cone photoreceptors. Neuron 29, (200). 43. Oldhm, W. M., Vn Eps, N., Preininger, A. M., Huell, W. L. & Hmm, H. E. Mechnism of the receptor-ctlyzed ctivtion of heterotrimeric G proteins. Nture Struct. Mol. Biol. 3, 2 (200). 44. Ntochin, M., Moussif, M. & Artemyev, N. O. Proing the mechnism of rhodopsin-ctlyzed trnsducin ctivtion. J. Neurochem., (200). 50

6 ARTICLES NATURE Vol Septemer Mrin, E. P., Krishn, A. G. & Skmr, T. P. Disruption of the 5 helix of trnsducin impirs rhodopsin-ctlyzed nucleotide exchnge. Biochemistry 4, (2002). 4. Herrmnn, R., Heck, M., Henklein, P., Hofmnn, K. P. & Ernst, O. P. Signl trnsfer from GPCRs to G proteins: Role of the G N-terminl region in rhodopsintrnsducin coupling. J. Biol. Chem. 28, (200). 4. Nnoff, C. et l. The croxyl terminus of the G-suunit is the ltch for triggered ctivtion of heterotrimeric G proteins. Mol. Phrmcol. 9, (200). 48. Johnston, C. A. & Siderovski, D. P. Structurl sis for nucleotide exchnge on G i suunits nd receptor coupling specificity. Proc. Ntl Acd. Sci. USA 04, (200). 49. Heck, M. & Hofmnn, K. P. Mximl rte nd nucleotide dependence of rhodopsin-ctlyzed trnsducin ctivtion: initil rte nlysis sed on doule displcement mechnism. J. Biol. Chem. 2, (200). Supplementry Informtion is linked to the online version of the pper t Acknowledgements We thnk J. Engelmnn nd C. Koch for technicl ssistnce; P. Henklein for peptide synthesis; C. Enenkel nd M. Sommer for criticlly reding the mnuscript; U. Müller nd the scientific stff of the Protein Structure Fctory nd the Freie Universität Berlin t emlines BL 4. nd BL 4.2 t BESSY for continuous support of the project. This work ws supported y the Deutsche Forschungsgemeinschft Sf449 (to O.P.E.), Sf40 (to O.P.E. nd K.P.H.), DFG-KOSEF interntionl coopertion ER 294/- (to O.P.E.) nd F (to H.-W.C.), nd CBNU funds for overses reserch (to H.-W.C.) nd fellowship of the Leiniz Grdute School of Moleculr Biophysics, Berlin (to Y.J.K.). Author Informtion The tomic coordintes nd structure fctors hve een deposited in the Protein Dt Bnk under ccession numer 3DQB. Reprints nd permissions informtion is ville t Correspondence nd requests for mterils should e ddressed to O.P.E. (oliver.ernst@chrite.de), K.P.H. (klus_peter.hofmnn@chrite.de) or H.-W.C. (hwchoe@chonuk.c.kr). 502

7 doi:0.038/nture0330 METHODS Peptide synthesis. Peptide GCT (corresponding to G t ( )K34L, mino cid sequence ILENLKDCGLF) ws derived from the extreme C terminus of the G t suunit. It ws synthesized with unmodified N nd C termini s descried 23,50. The K34L sustitution ws found previously to increse the ffinity of the peptide y two orders of mgnitude compred with the ntive G t ( ) peptide 23,50. Protein preprtion nd crystlliztion. Bovine rod outer segment memrnes nd opsin were prepred s descried 8,5. Rod outer segment memrnes (0 mg ml 2 rhodopsin) nd opsin ( mg ml 2 ) within rod outer segment memrnes were soluilized essentilly s descried, using % -D-octylglucopyrnoside 9,52. Synthetic GCT peptide ws dded in 4: molr rtio of peptide to soluilized rhodopsin. The mixture ws incuted on ice for h nd illuminted for 5 s with green light ( nm). The illuminted smple ws used without further purifiction for crystlliztion. Alterntively, GCT ws mixed with soluilized opsin in 4: molr rtio for crystlliztion. Crystlliztion screens y the sprse mtrix crystlliztion method 53 were crried out using the hngingdrop vpour diffusion method nd more thn 2,000 crystlliztion conditions. Promising conditions were systemticlly screened further y ltering protein concentrtion, precipittion gents nd ph. Ops* GCT crystls could e grown t 2 K using 24-well Linro pltes. Ech hnging drop ws prepred on siliconized coverslip y mixing equl volumes (2 ml ech) of receptor nd GCT mixture nd reservoir solution. The reservoir solution contined 3.2 M mmonium sulphte in 0. M MES or sodium citrte uffer, ph.0. Crystls ppered within 5 dys nd continued to grow for 5 dys. Fully grown crystls hd dimensions of mm 3. Structure nlysis. X-ry dt collection of Ops* GCT crystls ws performed t 00 K using cryoprotectnt consisting of 90% (v/v) reservoir solution nd 0% (w/v) trehlose. Diffrction dt were collected t synchrotron emline BL 4.2 (wvelength 0.984Å) of the Protein Structure Fctory nd Freie Universität Berlin t BESSY with MAR-5CCD detector. The crystl to detector distnce ws fixed t 240 mm. The rottion increment for ech frme ws 0.2u with n exposure time of 0 s. All imges were indexed, integrted nd scled using HKL2000 (ref. 54). Ops* GCT crystls elong to rhomohedrl spce group H32 ( Å, Å, c Å, u, c 5 20u). Supplementry Tle summrizes the sttistics for crystllogrphic dt collection nd structurl refinement. Initil phses for Ops* GCT were otined y conventionl moleculr replcement protocol (rottion, trnsltion nd rigid ody fitting) using the model of lignd-free opsin structure (PDB ccession 3CAP) s n initil serch tril. Moleculr replcement ws chieved using the CCP4 progrm PHASER 55 y first plcing the seven trnsmemrne undle of the opsin monomer (rottion function: Z 5 8.8; trnsltion function: Z , s defined y PHASER). In susequent steps, torsion ngle moleculr dynmics, simulted nneling using slow-cooling protocol nd mximum likelihood trget function, energy minimiztion, nd B-fctor refinement y the progrm CNS 5 were crried out in the resolution rnge Å. After the first round of refinement, the GCT peptide ws clerly visile in the electron density of oth s A -weighted F o 2 F c mps, s well s in the simulted nneling omit density mps (s shown in Supplementry Fig. 2). Restrined individul B-fctors were refined nd the crystl structure ws finlized y REFMAC5 nd other progrms in CCP4 (ref. 55). The finl model hd greement fctors R free nd R cryst of 24.8% nd 2.3%, respectively. Mnul reuilding of the opsin model nd electron density interprettion were performed fter ech refinement cycle using the progrm COOT 5. Structure vlidtion ws performed with the progrms PROCHECK 58 nd WHAT_CHECK 59. Potentil hydrogen onds nd vn der Wls contcts (Supplementry Tle 2) were nlysed using the progrms HBPLUS 0 nd LIGPLOT (Supplementry Fig. 8). All crystl structure superpositions of ckone lph cron trces were performed using CCP4 progrm LSQKAB (Figs 2 nd 4d nd Supplementry Figs 3, c, 4 c nd 5) 55. All moleculr grphics representtions were creted using PyMol Herrmnn, R. et l. Rhodopsin-trnsducin coupling: role of the G C-terminus in nucleotide exchnge ctlysis. Vision Res. 4, (200). 5. Schs, K., Mretzki, D. & Hofmnn, K. P. Assys for ctivtion of opsin y ll-trnsretinl. Methods Enzymol. 35, (2000). 52. Murkmi, M., Kithr, R., Gotoh, T. & Kouym, T. Crystlliztion nd crystl properties of squid rhodopsin. Act Crystllogr. F 3, (200). 53. Jncrik, J. & Kim, S.-H. Sprse mtrix smpling: screening method for crystlliztion of proteins. J. Appl. Crystllogr. 24, (99). 54. Otwinowski, Z. & Minor, W. Processing of X-ry diffrction dt collected in oscilltion mode. Methods Enzymol. 2, (99). 55. Collortive Computtionl Project, Numer 4. The CCP4 suite: progrms for protein crystllogrphy. Act Crystllogr. D 50, 0 3 (994). 5. Brunger, A. T. et l. Crystllogrphy & NMR system: A new softwre suite for mcromoleculr structure determintion. Act Crystllogr. D 54, (998). 5. Emsley, P. & Cowtn, K. Coot: Model-Building Tools for Moleculr Grphics. Act Crystllogr. D 0, (2004). 58. Lskowski, R. A., McArthur, M. W., Moss, D. S. & Thornton, J. M. PROCHECK: progrm to check the stereochemicl qulity of protein structures. J. Appl. Crystllogr. 2, (993). 59. Hooft, R. W., Vriend, G., Snder, C. & Aol, E. E. Errors in protein structures. Nture 38, 22 (99). 0. McDonld, I. K. & Thornton, J. M. Stisfying hydrogen onding potentil in proteins. J. Mol. Biol. 238, 93 (994).. Wllce, A. C., Lskowski, R. A. & Thornton, J. M. LIGPLOT: progrm to generte schemtic digrms of protein-lignd interctions. Protein Eng. 8, 2 34 (995). 2. DeLno, W. L. The PyMOL Moleculr Grphics System., (2002).