Two distinct conformations of helix 6 observed in antagonist-bound structures of a β1- receptor

Rouslan Moukhametzianov, Tony Warne, Patricia C. Edwards, Maria J. Serrano-Vega, Andrew G. W. Leslie, Christopher G. Tate1, and Gebhard F. X. Schertler,1,2

Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, United Kingdom

Edited by Martin J. Lohse, Universitat Wurzburg Germany, Wurzburg, Germany, and accepted by the Editorial Board March 25, 2011 (received for review January 5, 2011)

β β The 1- ( 1AR) is a G-protein-coupled receptor homologues are unique amongst GPCRs because they contain whose inactive state structure was determined using a thermosta- a covalently bound inverse agonist, 11-cis retinal, which is con- β – bilized mutant ( 1AR M23). However, it was not thought to be in a verted by a photon of light to all-trans retinal, which is a full ago- fully inactivated state because there was no salt bridge between nist for the receptor. Thus rhodopsin functions as a binary switch, Arg139 and Glu285 linking the cytoplasmic ends of transmembrane the photon of light converting an inactive form of receptor to one helices 3 and 6 (the R3.50 − D∕E6.30 “ionic lock”). Here we compare that is fully competent to activate G proteins (reviewed in ref. 22). β – eight new structures of 1AR M23, determined from crystallogra- It is essential that rhodopsin shows no G-protein-coupling activity phically independent molecules in four different crystals with three in the absence of light and, therefore, it has evolved to have ab- different antagonists bound. These structures are all in the inactive solutely no basal activity within the lifetime of rhodopsin in the R state and show clear electron density for cytoplasmic loop 3 cell. Consequently, it is accepted that the structure of dark-state linking transmembrane helices 5 and 6 that had not been seen pre- rhodopsin represents the fully inactive state of a GPCR. Upon viously. Despite significantly different crystal packing interactions, activation by light, numerous conformational changes around there are only two distinct conformations of the cytoplasmic end the retinal binding pocket result in the cytoplasmic end of trans- of helix 6, bent and straight. In the bent conformation, the Arg139- membrane helix 6 (H6) straightening and moving 6 Å away from Glu285 salt bridge is present, as in the crystal structure of dark- the center of the molecule, accompanied by a smaller movement state rhodopsin. The straight conformation, observed in previously and rotation of transmembrane helix 5 (H5). The movements of solved structures of β-receptors, results in the ends of helices 3 and H5 and H6 create a cleft in the cytoplasmic face of the opsin that 6 being too far apart for the ionic lock to form. In the bent confor- allows the binding of the C-terminal helix of Gα, thus activating 3.50 − 6.30 mation, the R E distance is significantly longer than in rho- the G protein. dopsin, suggesting that the interaction is also weaker, which could A key feature of rhodopsin activation is the outward move- β explain the high basal activity in 1AR compared to rhodopsin. ment of H6 away from H3 (6, 7, 23). In the rhodopsin structure, Many mutations that increase the constitutive activity of G-pro- there is a salt-bridge R3.50 − E6.30 (Ballesteros-Weinstein number- tein-coupled receptors are found in the bent region at the cytoplas- ing system is in superscripts; ref. 24), which links the cytoplasmic mic end of helix 6, supporting the idea that this region plays an ends of H6 and H3. Both R3.50 and D∕E6.30 are highly conserved important role in receptor activation. throughout the GPCR superfamily and, prior to the structure determination of rhodopsin, had already been suggested to pro- ∣ ∣ membrane protein seven-transmembrane receptor conformational vide a constraint that stabilized the inactive state of GPCRs, lead- thermostabilization ing to this interaction being termed the ionic lock (25). In the case of rhodopsin, the R3.50 − E6.30 salt bridge was proposed to be one he G-protein-coupled receptor (GPCR) superfamily is re- of the major determinants for the stability of the inactive state. Tsponsible for the transduction of a wide variety of extracellu- Hormone receptors have a far more complex pharmacology lar signals, and they are found in almost all eukaryotes, with over than does rhodopsin. Many hormone-binding GPCRs can par- 800 different GPCRs in humans (1, 2). Their common seven- tially activate their respective G proteins in the absence of agonist transmembrane-domain architecture has been preserved and ligand (26). The extent of this activity varies among GPCRs and evolved to sense agonists with remarkable structural diversity and their subtypes, reflecting different needs for a basal level of sig- to couple to a variety of specific signaling pathways. The struc- naling in a physiological context. Receptors with no basal activity, tures of rhodopsin (3–7), the β1- and β2-adrenergic receptors (β1AR and β2AR) (8–11). the adenosine A2A receptor (A2AR) (12), the D3 receptor (13), and the CXCR4 chemokine Author contributions: R.M., T.W., C.G.T., and G.F.X.S. designed research; R.M., T.W., P.C.E., receptor (14) show that there is a similar overall architecture and M.J.S.-V. performed research; R.M. and A.G.W.L. analyzed data; and R.M., A.G.W.L., for all family A GPCRs (reviewed in refs. 15–17), and there is C.G.T., and G.F.X.S. wrote the paper. probably a corresponding underlying conservation of the activa- The authors declare no conflict of interest. tion process (18, 19). With this wealth of structural information, This article is a PNAS Direct Submission. M.J.L. is a guest editor invited by the Editorial the challenge is now to correlate the pharmacological properties Board. of the hormone-binding receptors with their structure. One func- Freely available online through the PNAS open access option. tional aspect that still requires a structural explanation is the Data deposition: The atomic coordinates and structure factors have been deposited in the — Protein Data Bank, www.pdb.org [PDB ID codes 2YCW (t1118), 2YCX (t148), 2YCY (t468), phenomenon of basal activity i.e., most GPCRs show some abil- 2YCZ (t756)].

ity to activate G proteins even in the absence of agonists (20, 21) 1 — To whom correspondence may be addressed. E-mail: [email protected] and cgt@ and this will be the focus of this report. mrc-lmb.cam.ac.uk. Our current understanding of the structural basis for receptor 2Present address: Paul Scherrer Institut, Laboratory of Biomolecular Research, OFLC 109, activation is based largely on the structures determined for the CH-5232 Villigen PSI, Switzerland. – dark-state of rhodopsin (R state) (3 5) and its light-activated This article contains supporting information online at www.pnas.org/lookup/suppl/ conformation, opsin (R* state) (6, 7). Rhodopsin and its close doi:10.1073/pnas.1100185108/-/DCSupplemental.

8228–8232 ∣ PNAS ∣ May 17, 2011 ∣ vol. 108 ∣ no. 20 www.pnas.org/cgi/doi/10.1073/pnas.1100185108 Downloaded by guest on September 30, 2021 such as rhodopsin, seem appropriate for mediating sensory inputs that control how well we can see in the dark, where any basal activity would add undesirable noise. In contrast, increased levels of basal activity may be more useful for the controlled modulation of an activity such as heart rate that varies over a range of only two- to fivefold. It is thought that basal activity is caused by a subset of receptor conformations, which resemble the fully acti- vated agonist-bound state despite the absence of bound agonist, and that these can bind and activate their respective G proteins. Recent structures of β1AR and β2AR have greatly increased our understanding of how ligands are recognized and the overall architecture of the receptors (8–11, 27). However, there are also many questions that remain unresolved. One feature of all of the β-receptor structures is that they appear to represent inactive states (i.e., they are most similar to the structure of dark-state rhodopsin), yet the ionic lock between helices 3 and 6 is invariably broken. This observation raises the question of whether the β-re- ceptors can adopt a conformation analogous to dark-state rho- dopsin and, if so, how this affects their basal activity. Existing β-receptor crystal structures cannot address this issue, because either the cytoplasmic end of H6 is disordered and unresolved or it is potentially perturbed by the interaction with an antibody fragment or a T4 lysozyme fusion. Here we present eight struc- tures of the β1AR, which show that the receptor can indeed form an inactive state with an ionic lock analogous to the dark-state of rhodopsin, in addition to an inactive state where the ionic lock is broken. Results Crystallization and Structure Determination. The turkey β1AR con- struct that was crystallized contains amino acid deletions in the flexible regions at the N and C termini and in cytoplasmic loop 3 (CL3), and is called β36 (28). Six thermostabilizing mutations were introduced to make the receptor more tolerant to short- chain detergents (29), resulting in the β36–m23 construct whose

structure was determined to 2.7-Å resolution with BIOCHEMISTRY bound (11). During the extensive screening required to obtain these crystals (29), lower resolution datasets were collected from a variety of crystals containing either bound cyanopindolol (crys- tals t148, t468, resolution limits 3.25 and 3.15 Å) or iodocyano- (crystal t756, resolution limit 3.65 Å). Cocrystallization β – Fig. 1. Crystal contacts involving CL3. Lattice contacts between different of 36 m23 with the high-affinity antagonist also led monomers of the antiparallel crystallographic dimer are compared for differ- to crystals suitable for structure determination (crystal t1118, ent crystal forms. The thickness of the strand representing the Cα backbone resolution limit 3.00 Å). All structures were solved by molecular reflects the temperature factor (increasing thickness reflects higher B values). replacement using the structure of β36–m23 (PDB ID code Residues making crystal contacts are shown explicitly. In the case of t148, 2VT4) as a model. There are two monomers (A and B) in the t468, t756 C2 monoclinic crystals, CL3 is in contact with CL3 of the symmetry crystallographic asymmetric unit of each crystal, resulting in a related molecule. total of eight structures for β36–m23. Data collection and refine- ment statistics are shown in Table S1. are not the result of different crystal growth conditions. In addi- tion, the two conformations show no correlation to the nature of Crystal Packing Interactions. Crystals of β36–m23 containing bound antagonist ligand. The importance of the CL3 region in forming carazolol grew in the monoclinic space group P21, whereas crys- lattice contacts probably explains the success of the deletions tals of β36–m23 with cyanopindolol or bound made to enhance crystallization (residues 244–271 and 277–278). grew in the monoclinic space group C2 and displayed significant The full length CL3 is likely to be conformationally mobile, hin- variations in lattice parameters (Table S1) as a result of different dering formation of a stable crystal lattice. cryoprotection protocols (Methods; ref. 28). The packing interac- tions between receptors in the four crystals are quite different, Overall Comparison of the β1AR Structures although in each case the crystals appear to be built from similar The eight receptor structures, regardless of the type of antagonist nonphysiological antiparallel dimers with the dimer interface bound, are very similar to the previously reported cyanopindolol- comprising H4 and H5 (Fig. S1). Analysis of the crystal packing bound structure with respect to the hydrophobic transmembrane interactions (Table S2) shows that the third CL3 region linking regions (residues 54–67, 78–101, 114–143, 158–179, 208–234, α transmembrane helices 5 and 6 is always involved in intermole- 289–306, 323–345) with rmsds for C atoms ranging from 0.03 to cular contacts (although to different extents) and suggests that 0.53 Å (Table S3). There are also only minor differences in the one of the two distinct conformations of this region (see following previously defined extracellular and intracellular loops (rmsds in section) is selected by the ability to provide stabilizing crystal con- Cα atoms 0.6–0.7 Å). However, in the case of CL3, there were tacts (Fig. 1). Both CL3 conformations are observed (in different substantial differences between some of the monomers. CL3 monomers) in the carazolol-bound and in the iodocyanopindolol- comprises the cytoplasmic extensions to transmembrane helices bound structures, demonstrating that the different conformations H5 and H6 and a relatively short loop in an extended conforma-

Moukhametzianov et al. PNAS ∣ May 17, 2011 ∣ vol. 108 ∣ no. 20 ∣ 8229 Downloaded by guest on September 30, 2021 tion between them. In the previous structure of β1AR (PDB ID code 2VT4), 15 residues in the CL3 region (239–283, but note that residues 244–271 and 277–278 are not in the construct) were disordered and therefore not modeled (11). Amongst the eight structures reported here, two distinct conformations of the CL3 region were observed (Fig. 2). Both conformations are clearly in the inactive R state as neither show the characteristic conforma- tion changes observed in opsin. Structural Differences in CL3 and H6 Transmembrane helix H6 in the published structure of cyanopin- 6.30 dolol-bound β1AR–M23 (11) extends from residue Glu285 to Phe3156.60, with a single kink formed by Pro3056.50. The cytoplas- mic membrane boundary is estimated to be in the region of Ala288 to Leu289, so the cytoplasmic helical extension of H6 is relatively short (5–6 residues). In one of the two distinct con- formations of CL3 observed in the new structures, the conforma- tion of H6 is unchanged but the cytoplasmic extension is longer by approximately 10 Å, starting at residue Lys276. In the second conformation, there is a marked kink in the cytoplasmic extension of H6 at residue Ala2886.33 (t1118, t148) or Lys2876.32 (t756). Residues 274–286 are still α-helical and the helix is just as long, but the helix axis is bent by about 35° toward CL2 and rotated about 10° around the helix axis clockwise if viewed from the cytoplasmic side. The two conformations are therefore referred to as “straight” or “bent” accordingly (Fig. 3). The folding of the

Fig. 3. Interhelical polar contacts at the cytoplasmic face of β1AR–M23. (A) The structure of the bent conformation is depicted, with the intact ionic lock between Arg139 and Glu285. (B) The structure of the straight conforma- tion is shown, with the ionic lock broken. Structures are shown as cartoons in rainbow coloration (N terminus in blue, C terminus in red), except for CL2 and the cytoplasmic extension of H6 which are in gray and residues predicted to form interhelical hydrogen bonds shown as sticks.

CL3 region is highly conserved within these two conformations (rmsds in Cα atoms of 0.28–0.39 Å for four structures in the straight conformation and 0.97–1.21 Å for four structures in the bent conformation; Fig. 2, Tables S3 and S4). In structures with H6 in the bent conformation, H5 is extended by one helical turn relative to H6 in the straight conformation. The remaining residues Lys240–Ser273 (bent conformation), Gln237–Arg275 (straight conformation) form an extended loop. The omit densities for the CL3 region in the carazolol structure are shown in Fig. 2. The electron density for the bent conforma- tion is systematically better defined in all structures. The straight conformation has breaks in the density that correspond to the residues with very high B factors in this region. The bent conformation of H6 is partially stabilized by van der Fig. 2. Two conformations of CL3. (A) Superposition of the straight and bent Waals interactions primarily involving Met281 and Leu282 with β – H6 conformations of the carazolol-bound structures of 1AR M23 (crystal side chains on H5. The salt bridge between the highly conserved t1118) colored according to the B factor (increasing values from blue to 1393.50 2856.30 red). (B) Close up of the CL3 region from panel A.(C and D) Omit densities Arg of the E(D)RY motif on H3 and Glu on H6 of the CL3 for the bent and straight conformations, respectively (t1118 (the ionic lock) will provide additional stabilization. However, crystal). Densities are contoured either at (C) 0.8σ or (D) 0.7σ (gray mesh), the minimum distance between a nitrogen atom of the guanidi- 3 50 6 30 along with the Cα trace (red). nium group of Arg139 . and a carboxylate oxygen of Glu285 .

8230 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1100185108 Moukhametzianov et al. Downloaded by guest on September 30, 2021 (the N-O distance) is consistently between 3.7–3.9 Å in the bent β1AR is much higher than rhodopsin and there is a significantly conformation, which is significantly longer than seen in dark-state longer distance between the equivalent side chains of 3.7–3.8 Å. rhodopsin (2.8–3.2 Å; Table S4). An additional potential hydro- The difference in the length of the ionic interaction will undoubt- gen bond is formed between His286 and Gln237 in H5. All these edly affect its strength and hence the conformational flexibility polar interactions are absent in the straight conformation of H6, of the region, which could contribute to the higher basal activity with a minimum N-O distance for the ionic lock of 6.2 Å. In the of β1AR. However, clearly this is not the only structural feature straight conformation of H6, there are no hydrogen bonds appar- in GPCRs that affects basal activity. For example, the naturally 4.56 ent between the cytoplasmic extension of H6 and the neighboring occurring polymorphism in human β2AR, T164I , is at the H5, although there are van der Waals contacts between Leu282 in H4–H5 interface and significantly reduces both basal activity H6 and Ile241 in H5. and the agonist-induced activation of the receptor (31). In addi- tion, the recent structure of the dopamine (D3) receptor (D3R) Discussion shows that it contains an intact ionic lock, with the N-O distance Crystallization of β1AR–M23 in different crystal forms with three of 2.5 Å (13). There is also no perceptible kink in the cytoplasmic different ligands has resulted in receptor structures that are end of H6 of D3R equivalent to the kink observed in the β1AR virtually identical except for the different conformations of the structures presented here. Thus, although the region of the ionic cytoplasmic end of H6. The fact that only two conformations lock is conserved, there are clearly subtle variations in the struc- of H6 are observed, either bent or straight, despite the range ture between different receptors. of different crystallization conditions and crystal packing interac- The ability of the HKAL sequence to have two structurally tions, suggests that these reflect unbiased conformations that different conformations constitutes a ligand-independent me- could be adopted in the native cell membranes. This idea is con- chanism that could affect the basal activity of β1AR and other sistent with current thinking that GPCRs are in a finely balanced GPCRs, depending upon where the equilibrium between the dif- dynamic equilibrium between multiple inactive states and multi- ferent conformations of the cytoplasmic extension of H6 lies. If ple activated states, even in the absence of ligands (18, 19). the equilibrium in one GPCR favors the bent conformation with β Recent molecular dynamics simulations performed on the 2AR an intact ionic lock (Rinv), as is found in rhodopsin, then the basal also indicate the coexistence of two different inactive conforma- activity of the receptor is likely to be low; a strong salt bridge tions with the ionic lock either intact or disrupted (30). The con- between R3.50−D∕E6.30 will produce this situation. If the salt formation corresponding to an intact ionic lock in the β2AR bridge is weak or absent, the equilibrium will favor formation simulation is broadly similar to that observed in the crystal struc- of the straight conformation where the ionic lock is open. Once tures, except that H6 was described as kinking slightly at this state is attained, there are no strong interactions between the 6 38 Gly276 . compared with the more marked kink observed at cytoplasmic ends of H3 and H6 that will hinder the movement of 6.32 6.33 Lys287 ∕Ala288 in β1AR. Given that the bent conformation H6 to adopt the conformation (R*) capable of coupling to G is very similar to that observed in rhodopsin (Figs. S2 and S3), this proteins; i.e., the probability of R* formation increases once the structure probably represents the most inactive state of receptor ionic lock is open. Thus the dynamics of the HKAL region can —i.e., the conformation that would be expected if an inverse potentially have an effect on the level of basal activity of a recep- agonist were bound (Rinv). In this conformation, the cytoplasmic tor and in this regard it is noteworthy that many constitutively

extension of H6 blocks the G-protein peptide binding site ob- activating mutations are found here, particularly in position BIOCHEMISTRY served in the low-pH structure of opsin (7). In the straight con- 6.34 (Fig. 4). Thus the HKAL region has probably evolved to formation, there is still insufficient room for the C-terminal maintain a level of basal activity that is compatible with the phy- peptide of the G protein to insert into the receptor, so further siological function of the receptor, with some receptors having conformational changes upon agonist binding are required for higher levels of basal activity than others. However, there are productive G-protein coupling. Thus transformation of H6 by many sites that can affect the basal activity of receptors, and the conformational dynamics of the H2866.31KAL2896.34 region the importance of the HKAL sequence might not be general is one of the essential steps required for G-protein signaling. for all rhodopsin-like GPCRs. For example, the M5 muscarinic Rhodopsin is unusual amongst GPCRs because its basal activ- receptor, when mutated in the same 6.34 site, was devoid of con- ity is virtually zero, which has been ascribed in part to the ionic stitutive activity although this site was still found to be the deter- lock between R3.50 and E6.30 with the distance between the N-O of minant of M5 receptor affinity to the G protein (32). In contrast, the side chains being 2.8–3.2 Å. In contrast, the basal activity of all 19 amino acids substitutions in this site in the α1B adrenergic

Fig. 4. Sites of constitutively activating mutations (CAMs) in the CL3 region for different GPCRs. The secondary structure of the bent conformation with the ionic lock present is shown (Left) along with the CL3 region including the ends of TM5 and TM6 enlarged (Middle). The corresponding amino sequence of β36–m23 is shown for H6, with residues corresponding to CAMs in the β2 adrenergic receptor shown in yellow and the thermostabilizing mutation A282L shown in red. GPCRs with CAMs at the 6.34 site are shown on the right with reference to their evolutionary relationships. Amino acid residues corresponding to Leu289 (6.34) in the turkey β1AR are shown in parentheses.

Moukhametzianov et al. PNAS ∣ May 17, 2011 ∣ vol. 108 ∣ no. 20 ∣ 8231 Downloaded by guest on September 30, 2021 receptor have shown different degrees of constitutive activity (t1118), or 35% PEG600 and 5% glycerol (t468). No additional cryoprotectant (33). In the rat μ-opioid receptor, different substitutions of was added to crystal t756 (iodocyanopindolol). Thr6.34 had different effects; the mutant was constitutively active, whereas the aspartate mutant had lower basal activity and Crystallography. Diffraction data for crystals t148 and t756 were collected at decreased ability to activate G proteins (34). Several human dis- 100 K with a MarCCD detector on beamline ID23-2 at the European Synchro- λ ¼ 0 873 eases have also been caused by mutations in this region. Mutation tron Radiation Facility ( . Å). Data for crystals t468 and t1118 were of this site in the luteinizing hormone receptor gene causes male collected at 100 K with a Pilatus detector on beamline X06SA at the Swiss Light Source (λ ¼ 0.98 and 1.00 Å, respectively). Each dataset was collected gonadotropin-independent precocious puberty (35) and the ana- from multiple positions on a single crystal, following extensive screening. logous mutation in the thyrotropin receptor causes hyperfunc- Diffraction data were processed with MOSFLM and SCALA (38). Because tioning thyroid adenomas (36). Increased activity of these the diffraction was anisotropic, different wedges of data were processed receptors could be due to the perturbing effect of the mutations to different resolutions and consequently the completeness of the highest on the structure and stabilization of the inactive state. resolution shell is low for crystals t148, t468, and t1118 (Table S1). All struc- tures were obtained with molecular replacement using PHASER (39) starting Methods from the 2VT4 model (11). Refinement was carried out in PHENIX (40) and β – Purification and Crystallization. For the preparation of 1AR36 M23 with REFMAC (41) with application of strict noncrystallographic symmetry aver- either cyanopindolol or iodocyanopindolol bound (crystals t148, t468, and aging between appropriate regions of the two independent monomers in t756), baculovirus-expressed receptor was solubilized in β-decylmaltoside 2þ the asymmetric unit. Structure validation was performed using the MolProb- and purified by Ni -affinity chromatography and sepharose ity Web server. ligand affinity chromatography, with detergent exchange to octylthiogluco- side (0.35%) on the ligand affinity column (28, 37). Crystallization was by vapor diffusion with addition of receptor (5.5 mg∕mL) to an equal volume ACKNOWLEDGMENTS. We thank F. Gorrec for his help with crystallization robotics and F. Marshall, M. Weir, and R. Henderson for helpful comments (0.5–1.0 μL) 0.1 M N-(2-acetamido)iminodiacetate-NaOH, pH 6.9–7.3, 29–33% on the manuscript. This work was supported by core funding from the Med- PEG 600. ical Research Council (MRC) and a grant jointly funded by Pfizer Global For crystallization of β1AR36–M23 with carazolol bound (crystal t1118), Research and Development and MRC Technology. Additional financial sup- detergent was exchanged to Hega-10 (0.35%) and receptor was eluted with port was from a Human Frontier Science Project Program Grant (RG/0052), μ 50 M carazolol. Prior to crystallization, ligand and detergent concentrations a European Commission FP6 specific targeted research project (LSH-2003- were increased to 0.5 mM and 0.5%, respectively. Crystallization was by va- 1.1.0-1), a European Synchrotron Radiation Facility (ESRF) long-term proposal por diffusion with addition of 200 nL receptor (9 mg∕mL) to an equal volume and a Biotechnology and Biological Sciences Research Council Grant (BB/ of precipitant solution (0.1 M Tris • HCl, pH 8.1, 40% PEG 400). Crystals were G003653/1). We would like to thank beamline staff at the ESRF beamline cryoprotected by a very short soak in either 60% PEG600 (t148), 40% PEG400 ID23-2 and at the Swiss Light Source beamline X06SA.

1. Fredriksson R, Schioth HB (2005) The repertoire of G-protein-coupled receptors in fully 24. Ballesteros JA, Weinstein H (1995) Integrated methods for the construction of three sequenced genomes. Mol Pharmacol 67:1414–1425. dimensional models and computational probing of structure function relations in G 2. Foord SM, et al. (2005) International Union of Pharmacology. XLVI. G protein-coupled protein-coupled receptors. Methods Neurosci 25:366–428. receptor list. Pharmacol Rev 57:279–288. 25. Ballesteros JA, et al. (2001) Activation of the β2-adrenergic receptor involves disrup- 3. Li J, Edwards PC, Burghammer M, Villa C, Schertler GF (2004) Structure of bovine tion of an ionic lock between the cytoplasmic ends of transmembrane segments 3 and rhodopsin in a trigonal crystal form. J Mol Biol 343:1409–1438. 6. J Biol Chem 276:29171–29177. 4. Okada T, et al. (2004) The retinal conformation and its environment in rhodopsin in 26. Costa T, Cotecchia S (2005) Historical review: Negative efficacy and the constitutive light of a new 2.2 Å crystal structure. J Mol Biol 342:571–583. activity of G-protein-coupled receptors. Trends Pharmacol Sci 26:618–624. 5. Palczewski K, et al. (2000) Crystal structure of rhodopsin: A G protein-coupled recep- 27. Wacker D, et al. (2010) Conserved binding mode of human β2 adrenergic receptor tor. Science 289:739–745. inverse agonists and antagonist revealed by X-ray crystallography. J Am Chem Soc 6. Park JH, Scheerer P, Hofmann KP, Choe HW, Ernst OP (2008) Crystal structure of the 132:11443–11445. – ligand-free G-protein-coupled receptor opsin. Nature 454:183 187. 28. Warne T, Serrano-Vega MJ, Tate CG, Schertler GF (2009) Development and crystalliza- 7. Scheerer P, et al. (2008) Crystal structure of opsin in its G-protein-interacting confor- tion of a minimal thermostabilised G protein-coupled receptor. Protein Expr Purif – mation. Nature 455:497 502. 65:204–213. 8. Cherezov V, et al. (2007) High-resolution crystal structure of an engineered Human 29. Serrano-Vega MJ, Magnani F, Shibata Y, Tate CG (2008) Conformational thermostabi- β2-adrenergic G protein coupled receptor. Science 318:1258–1265. lization of the β1-adrenergic receptor in a detergent-resistant form. Proc Natl Acad Sci β 9. Rasmussen SG, et al. (2007) Crystal structure of the human 2 adrenergic USA 105:877–882. – G-protein-coupled receptor. Nature 450:383 387. 30. Dror RO, et al. (2009) Identification of two distinct inactive conformations of the 10. Hanson MA, et al. (2008) A specific cholesterol binding site is established by the 2.8 A β2-adrenergic receptor reconciles structural and biochemical observations. Proc Natl structure of the human β2-adrenergic receptor. Structure 16:897–905. Acad Sci USA 106:4689–4694. 11. Warne T, et al. (2008) Structure of a β1-adrenergic G-protein-coupled receptor. 31. Green SA, Rathz DA, Schuster AJ, Liggett SB (2001) The Ile164 β2-adrenoceptor Nature 454:486–491. polymorphism alters exosite binding and conventional agonist coupling 12. Jaakola VP, et al. (2008) The 2.6 angstrom crystal structure of a human A2A adenosine to G(s). Eur J Pharmacol 421:141–147. receptor bound to an antagonist. Science 322:1211–1217. 32. Spalding TA, Burstein ES (2006) Constitutive activity of muscarinic acetylcholine recep- 13. Chien EY, et al. (2010) Structure of the human dopamine D3 receptor in complex with a tors. J Recept Signal Transduct Res 26:61–85. D2/D3 selective antagonist. Science 330:1091–1095. 33. Kjelsberg MA, Cotecchia S, Ostrowski J, Caron MG, Lefkowitz RJ (1992) Constitutive 14. Wu B, et al. (2010) Structures of the CXCR4 chemokine GPCR with small-molecule and activation of the alpha 1B-adrenergic receptor by all amino acid substitutions at a cyclic peptide antagonists. Science 330:1066–1071. single site. Evidence for a region which constrains receptor activation. J Biol Chem 15. Kobilka B, Schertler GF (2008) New G-protein-coupled receptor crystal structures: 267:1430–1433. insights and limitations. Trends Pharmacol Sci 29:79–83. 16. Schwartz TW, Hubbell WL (2008) Structural biology: A moving story of receptors. 34. Huang P, et al. (2001) Functional role of a conserved motif in TM6 of the rat mu opioid Nature 455:473–474. receptor: Constitutively active and inactive receptors result from substitutions of – 17. Tate CG, Schertler GF (2009) Engineering G protein-coupled receptors to facilitate their Thr6.34(279) with Lys and Asp. Biochemistry 40:13501 13509. structure determination. Curr Opin Struct Biol 19:386–395. 35. Schulz A, Schoneberg T, Paschke R, Schultz G, Gudermann T (1999) Role of the third 18. Deupi X, Kobilka B (2007) Activation of G protein-coupled receptors. Adv Protein intracellular loop for the activation of gonadotropin receptors. Mol Endocrinol – Chem 74:137–166. 13:181 190. 19. Kobilka BK, Deupi X (2007) Conformational complexity of G-protein-coupled recep- 36. Parma J, et al. (1993) Somatic mutations in the thyrotropin receptor gene cause – tors. Trends Pharmacol Sci 28:397–406. hyperfunctioning thyroid adenomas. Nature 365:649 651. 20. Bond RA, Ijzerman AP (2006) Recent developments in constitutive receptor activity 37. Warne T, Chirnside J, Schertler GF (2003) Expression and purification of truncated, and inverse agonism, and their potential for GPCR drug discovery. Trends Pharmacol non-glycosylated turkey β-adrenergic receptors for crystallization. Biochim Biophys Sci 27:92–96. Acta 1610:133–140. 21. Costa T, Herz A (1989) Antagonists with negative intrinsic activity at delta opioid 38. Evans P (2006) Scaling and assessment of data quality. Acta Crystallogr D Biol Crystal- receptors coupled to GTP-binding proteins. Proc Natl Acad Sci USA 86:7321–7325. logr 62:72–82. 22. Hofmann KP, et al. (2009) A G protein-coupled receptor at work: The rhodopsin model. 39. McCoy AJ, et al. (2007) Phaser crystallographic software. J Appl Crystallogr 40:658–674. Trends Biochem Sci 34:540–552. 40. Adams PD, et al. (2002) PHENIX: Building new software for automated crystallographic 23. Altenbach C, Kusnetzow AK, Ernst OP, Hofmann KP, Hubbell WL (2008) High-resolu- structure determination. Acta Crystallogr D Biol Crystallogr 58:1948–1954. tion distance mapping in rhodopsin reveals the pattern of helix movement due to 41. Murshudov GN, Vagin AA, Dodson EJ (1997) Refinement of macromolecular structures activation. Proc Natl Acad Sci USA 105:7439–7444. by the maximum-likelihood method. Acta Crystallogr D Biol Crystallogr 53:240–255.

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