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• W2142322475 abstract "Methylxanthines, including caffeine and theophylline, are among the most widely consumed stimulant drugs in the world. These effects are mediated primarily via blockade of adenosine receptors. Xanthine analogs with improved properties have been developed as potential treatments for diseases such as Parkinson's disease. Here we report the structures of a thermostabilized adenosine A2A receptor in complex with the xanthines xanthine amine congener and caffeine, as well as the A2A selective inverse agonist ZM241385. The receptor is crystallized in the inactive state conformation as defined by the presence of a salt bridge known as the ionic lock. The complete third intracellular loop, responsible for G protein coupling, is visible consisting of extended helices 5 and 6. The structures provide new insight into the features that define the ligand binding pocket of the adenosine receptor for ligands of diverse chemotypes as well as the cytoplasmic regions that interact with signal transduction proteins. Methylxanthines, including caffeine and theophylline, are among the most widely consumed stimulant drugs in the world. These effects are mediated primarily via blockade of adenosine receptors. Xanthine analogs with improved properties have been developed as potential treatments for diseases such as Parkinson's disease. Here we report the structures of a thermostabilized adenosine A2A receptor in complex with the xanthines xanthine amine congener and caffeine, as well as the A2A selective inverse agonist ZM241385. The receptor is crystallized in the inactive state conformation as defined by the presence of a salt bridge known as the ionic lock. The complete third intracellular loop, responsible for G protein coupling, is visible consisting of extended helices 5 and 6. The structures provide new insight into the features that define the ligand binding pocket of the adenosine receptor for ligands of diverse chemotypes as well as the cytoplasmic regions that interact with signal transduction proteins. Structure of adenosine A2A receptor in ground state conformation with ionic lock Complete third intracellular loop present consists of extended helices 5 and 6 Complexes with xanthines and ZM241385 show overlapping binding interactions Thermostabilization enables a structure bound with the low affinity ligand caffeine Plant-derived methylxanthines that include caffeine (from the coffee bean), theophylline (from the tea leaf), and theobromine (from the cocoa bean) are among the most widely consumed stimulant substances in the world with Americans consuming an average of 200 mg of caffeine per day (Daly, 2007Daly J.W. Caffeine analogs: biomedical impact.Cell. Mol. Life Sci. 2007; 64: 2153-2169Crossref PubMed Scopus (189) Google Scholar). In 1981, it was demonstrated that the behavioral stimulant effects of methylxanthines were mediated by blockade of adenosine receptors (Snyder et al., 1981Snyder S.H. Katims J.J. Annau Z. Bruns R.F. Daly J.W. Adenosine receptors and behavioral actions of methylxanthines.Proc. Natl. Acad. Sci. USA. 1981; 78: 3260-3264Crossref PubMed Scopus (645) Google Scholar), although at higher concentrations methylxanthines also have effects on several other target proteins such as phosphodiesterases (Daly, 2007Daly J.W. Caffeine analogs: biomedical impact.Cell. Mol. Life Sci. 2007; 64: 2153-2169Crossref PubMed Scopus (189) Google Scholar). There are four receptors for adenosine (A1, A2A, A2B, A3) (Fredholm et al., 2011Fredholm B.B. IJzerman A.P. Jacobson K.A. Linden J. Müller C.E. International Union of Basic and Clinical Pharmacology. LXXXI. Nomenclature and classification of adenosine receptors—an update.Pharmacol. Rev. 2011; 63: 1-34Crossref PubMed Scopus (895) Google Scholar) that are all members of the G protein-coupled receptor (GPCR) family of membrane spanning proteins. The receptors are widely expressed in the brain, cardiovascular, and immune system and there is growing evidence that drugs acting at adenosine receptors represent promising approaches in a wide range of diseases (Jacobson and Gao, 2006Jacobson K.A. Gao Z.G. Adenosine receptors as therapeutic targets.Nat. Rev. Drug Discov. 2006; 5: 247-264Crossref PubMed Scopus (1081) Google Scholar). Activation of adenosine receptors results in a conformational change propagated to the intracellular surface where the receptors interact either with heterotrimeric G proteins (Gilman, 1987Gilman A.G. G proteins: transducers of receptor-generated signals.Annu. Rev. Biochem. 1987; 56: 615-649Crossref PubMed Scopus (4616) Google Scholar) or through β-arrestin (DeWire et al., 2007DeWire S.M. Ahn S. Lefkowitz R.J. Shenoy S.K. Beta-arrestins and cell signaling.Annu. Rev. Physiol. 2007; 69: 483-510Crossref PubMed Scopus (1080) Google Scholar) to regulate signaling to ion channels and enzyme pathways. The naturally occurring methylxanthines such as caffeine are nonselective and have micromolar affinities at adenosine receptors (Müller and Jacobson, 2011Müller C.E. Jacobson K.A. Xanthines as adenosine receptor antagonists.Handb. Exp. Pharmacol. 2011; 200: 151-199Crossref PubMed Scopus (86) Google Scholar). A large number of derivatives and analogs of these compounds have been made, with the aim of obtaining higher affinity and more selective ligands as research tools to characterize the function of adenosine receptors as well as for therapeutic purposes. Xanthine-based drugs have been evaluated clinically in diseases including asthma, Parkinson's disease, and pain (Daly, 2007Daly J.W. Caffeine analogs: biomedical impact.Cell. Mol. Life Sci. 2007; 64: 2153-2169Crossref PubMed Scopus (189) Google Scholar). 8-Aryl derivatives of the xanthine core include the amine congener 8-[4-[[[[(2-aminoethyl)amino]carbonyl]methyl]oxy]phenyl]-l,3-dipropylxanthine (XAC), which has a greatly enhanced affinity for adenosine receptors and an increased solubility compared to caffeine. This compound has proved a very useful tool in the study of adenosine receptors because it has been radiolabeled for use as a tracer and has been immobilized for affinity purification of adenosine receptors (Müller and Jacobson, 2011Müller C.E. Jacobson K.A. Xanthines as adenosine receptor antagonists.Handb. Exp. Pharmacol. 2011; 200: 151-199Crossref PubMed Scopus (86) Google Scholar). The adenosine A2A receptor is of particular interest as a drug target for Parkinson's disease, with the drug preladenant currently in clinical trials (Salamone, 2010Salamone J.D. Preladenant, a novel adenosine A(2A) receptor antagonist for the potential treatment of parkinsonism and other disorders.IDrugs. 2010; 13: 723-731PubMed Google Scholar). The design of drugs for GPCRs is hampered by the lack of structural information and hence obtaining the structure of this receptor in complex with a range of different ligand chemotypes was required to assist in the discovery of novel drugs targeted at this receptor. Obtaining high resolution structures of GPCRs is hampered by their intrinsic flexibility and their instability when removed from the plasma membrane (Tate, 2010Tate C.G. Practical considerations of membrane protein instability during purification and crystallization.Methods Mol. Biol. 2010; 601: 187-203Crossref PubMed Scopus (84) Google Scholar). A number of approaches have recently been developed to overcome this problem. The first non-rhodopsin GPCR structure to be determined was the β2-adrenergic receptor (Rasmussen et al., 2007Rasmussen S.G. Choi H.J. Rosenbaum D.M. Kobilka T.S. Thian F.S. Edwards P.C. Burghammer M. Ratnala V.R. Sanishvili R. Fischetti R.F. et al.Crystal structure of the human β2 adrenergic G-protein-coupled receptor.Nature. 2007; 450: 383-387Crossref PubMed Scopus (1589) Google Scholar) in complex with an antibody fragment bound to the third intracellular loop (ICL)—a critical domain of the receptor that mediates G protein coupling, but also contributes to receptor flexibility. A higher resolution structure of the β2AR was obtained by fusing T4 lysozyme into ICL3 (Cherezov et al., 2007Cherezov V. Rosenbaum D.M. Hanson M.A. Rasmussen S.G. Thian F.S. Kobilka T.S. Choi H.J. Kuhn P. Weis W.I. Kobilka B.K. Stevens R.C. High-resolution crystal structure of an engineered human β2-adrenergic G protein-coupled receptor.Science. 2007; 318: 1258-1265Crossref PubMed Scopus (2614) Google Scholar) and the same methodology was used to obtain the first structure of the adenosine A2A receptor (A2A-T4L) (Jaakola et al., 2008Jaakola V.P. Griffith M.T. Hanson M.A. Cherezov V. Chien E.Y. Lane J.R. Ijzerman A.P. Stevens R.C. The 2.6 angstrom crystal structure of a human A2A adenosine receptor bound to an antagonist.Science. 2008; 322: 1211-1217Crossref PubMed Scopus (1498) Google Scholar) in complex with ZM241385, as well as more recently the structures of the chemokine receptor CXCR4 (Wu et al., 2010Wu B. Chien E.Y. Mol C.D. Fenalti G. Liu W. Katritch V. Abagyan R. Brooun A. Wells P. Bi F.C. et al.Structures of the CXCR4 chemokine GPCR with small-molecule and cyclic peptide antagonists.Science. 2010; 330: 1066-1071Crossref PubMed Scopus (1374) Google Scholar) and the dopamine D3 receptor (Chien et al., 2010Chien E.Y. Liu W. Zhao Q. Katritch V. Han G.W. Hanson M.A. Shi L. Newman A.H. Javitch J.A. Cherezov V. Stevens R.C. Structure of the human dopamine D3 receptor in complex with a D2/D3 selective antagonist.Science. 2010; 330: 1091-1095Crossref PubMed Scopus (911) Google Scholar). The A2A-T4L receptor has also been crystallized in the presence of the high affinity agonist UK-432097 (Xu et al., 2011Xu F. Wu H. Katritch V. Han G.W. Jacobson K.A. Gao Z.G. Cherezov V. Stevens R.C. Structure of an agonist-bound human A2A adenosine receptor.Science. 2011; 332: 322-327Crossref PubMed Scopus (652) Google Scholar). The conformational state of these receptors can be difficult to determine because insertion of the T4 lysozyme can alter the pharmacology and prevents signaling (Rosenbaum et al., 2007Rosenbaum D.M. Cherezov V. Hanson M.A. Rasmussen S.G. Thian F.S. Kobilka T.S. Choi H.J. Yao X.J. Weis W.I. Stevens R.C. Kobilka B.K. GPCR engineering yields high-resolution structural insights into β2-adrenergic receptor function.Science. 2007; 318: 1266-1273Crossref PubMed Scopus (1122) Google Scholar). Nevertheless clear differences can be seen between the two A2A-T4L structures bound to the inverse agonist ZM241385 and the agonist UK-432097 that resemble some of the changes associated with receptor activation (Xu et al., 2011Xu F. Wu H. Katritch V. Han G.W. Jacobson K.A. Gao Z.G. Cherezov V. Stevens R.C. Structure of an agonist-bound human A2A adenosine receptor.Science. 2011; 332: 322-327Crossref PubMed Scopus (652) Google Scholar). An alternative approach to obtaining structures is conformational thermostabilization. This method involves the introduction of a small number of point mutations into the receptor, which increases the thermostability while altering the equilibrium between the agonist and antagonist conformation. The thermostabilization of a particular conformation is directed by the pharmacology of the ligand used during the selection of residues for mutation (Tate and Schertler, 2009Tate C.G. Schertler G.F. Engineering G protein-coupled receptors to facilitate their structure determination.Curr. Opin. Struct. Biol. 2009; 19: 386-395Crossref PubMed Scopus (147) Google Scholar). The first structure obtained using this approach was the β1-adrenergic receptor (β1AR) (Warne et al., 2008Warne T. Serrano-Vega M.J. Baker J.G. Moukhametzianov R. Edwards P.C. Henderson R. Leslie A.G. Tate C.G. Schertler G.F. Structure of a beta1-adrenergic G-protein-coupled receptor.Nature. 2008; 454: 486-491Crossref PubMed Scopus (1198) Google Scholar). This was the first non-rhodopsin structure to clearly show features of the cytoplasmic regions of the receptor and revealed the presence of a short well-defined helix in ICL2. However, in this structure ICL3 was truncated to assist in crystallization. A thermostabilized neurotensin receptor has also been described (Shibata et al., 2009Shibata Y. White J.F. Serrano-Vega M.J. Magnani F. Aloia A.L. Grisshammer R. Tate C.G. Thermostabilization of the neurotensin receptor NTS1.J. Mol. Biol. 2009; 390: 262-277Crossref PubMed Scopus (128) Google Scholar). Such receptors are known as StaRs for “stabilized receptors” (Robertson et al., 2011Robertson N. Jazayeri A. Errey J. Baig A. Hurrell E. Zhukov A. Langmead C.J. Weir M. Marshall F.H. The properties of thermostabilised G protein-coupled receptors (StaRs) and their use in drug discovery.Neuropharmacology. 2011; 60: 36-44Crossref PubMed Scopus (114) Google Scholar). The adenosine A2A receptor was previously thermostabilized in both agonist and inverse agonist conformations (Magnani et al., 2008Magnani F. Shibata Y. Serrano-Vega M.J. Tate C.G. Co-evolving stability and conformational homogeneity of the human adenosine A2a receptor.Proc. Natl. Acad. Sci. USA. 2008; 105: 10744-10749Crossref PubMed Scopus (167) Google Scholar), however, these engineered proteins, were not considered stable enough for crystallization. A structure of the A2A receptor stabilized in an agonist conformation bound to adenosine and adenosine-5′-(N-ethylcarboxamide (NECA) has recently been obtained (Lebon et al., 2011aLebon G. Bennett K. Jazayeri A. Tate C.G. Thermostabilisation of an agonist-bound conformation of the human adenosine A(2A) receptor.J. Mol. Biol. 2011; 409: 298-310Crossref PubMed Scopus (94) Google Scholar, Lebon et al., 2011bLebon G. Warne T. Edwards P.C. Bennett K. Langmead C.J. Leslie A.G. Tate C.G. Agonist-bound adenosine A2A receptor structures reveal common features of GPCR activation.Nature. 2011; 474: 521-525Crossref PubMed Scopus (641) Google Scholar). Here we report the stabilization of the A2A receptor in the inverse agonist conformation and subsequent X-ray structures of this receptor in complex with ZM241385 and the xanthines XAC and caffeine. The structures of the adenosine A2A receptor described here provide new insight into the binding mode of ligands of different chemical classes. Furthermore this is the first structure of the adenosine A2A receptor in the fully inactive state with the ionic lock present and the complete intracellular loop permitting for the first time a detailed view of the structural activation spectrum for this receptor. The adenosine A2A receptor was previously stabilized in both agonist and inverse agonist conformations (Magnani et al., 2008Magnani F. Shibata Y. Serrano-Vega M.J. Tate C.G. Co-evolving stability and conformational homogeneity of the human adenosine A2a receptor.Proc. Natl. Acad. Sci. USA. 2008; 105: 10744-10749Crossref PubMed Scopus (167) Google Scholar), however, the stabilized inverse agonist receptor known as Rant21 or A2A-StaR1 (containing the stabilizing mutations A54L2.52, T88A3.36, K122A4.43, V239A6.41; superscripts refer to Ballesteros-Weinstein numbering17) was not considered of sufficient stability for structural studies. Further mutagenesis in the presence of the inverse agonist ligand ZM241385 (Poucher et al., 1995Poucher S.M. Keddie J.R. Singh P. Stoggall S.M. Caulkett P.W. Jones G. Coll M.G. The in vitro pharmacology of ZM 241385, a potent, non-xanthine A2a selective adenosine receptor antagonist.Br. J. Pharmacol. 1995; 115: 1096-1102Crossref PubMed Scopus (315) Google Scholar) resulted in the identification of an additional four stabilizing mutations (R107A3.55, L202A5.63, L235A6.37, S277A7.42) giving an apparent thermostability of 47°C in 0.1% decylmaltoside (Figure 1A ) resulting in A2A-StaR2. For crystallization, A2A-StaR2 was truncated at the C terminus by 96 amino acids up to Ala316 and included a C-terminal decameric His-tag for purification. An N154A mutation was introduced to remove the glycosylation site (Figure 1B). The engineered receptor A2A-StaR2 bound ZM241385 (KD = 1.9 nM) and also a range of structurally diverse antagonists with a similar affinity to the wild-type receptor (Figure 2A ; see Table S1 available online). In contrast, the affinities of agonists including NECA and CGS21860 were reduced by >100-fold and the receptor no longer activated G proteins. This pharmacology is consistent with trapping of the inverse agonist conformation and is similar to the change in pharmacology observed for the stabilized β1AR-m23 (Serrano-Vega et al., 2008Serrano-Vega M.J. Magnani F. Shibata Y. Tate C.G. Conformational thermostabilization of the β1-adrenergic receptor in a detergent-resistant form.Proc. Natl. Acad. Sci. USA. 2008; 105: 877-882Crossref PubMed Scopus (344) Google Scholar). The thermostabilizing residues Thr883.36 and Ser2777.42 lie at the bottom of the agonist binding pocket and have been shown to play a role in binding of the ribose ring of the agonist and in agonist activation (Ivanov et al., 2009Ivanov A.A. Barak D. Jacobson K.A. Evaluation of homology modeling of G-protein-coupled receptors in light of the A(2A) adenosine receptor crystallographic structure.J. Med. Chem. 2009; 52: 3284-3292Crossref PubMed Scopus (88) Google Scholar, Jiang et al., 1996Jiang Q. Van Rhee A.M. Kim J. Yehle S. Wess J. Jacobson K.A. Hydrophilic side chains in the third and seventh transmembrane helical domains of human A2A adenosine receptors are required for ligand recognition.Mol. Pharmacol. 1996; 50: 512-521PubMed Google Scholar, Xu et al., 2011Xu F. Wu H. Katritch V. Han G.W. Jacobson K.A. Gao Z.G. Cherezov V. Stevens R.C. Structure of an agonist-bound human A2A adenosine receptor.Science. 2011; 332: 322-327Crossref PubMed Scopus (652) Google Scholar, Lebon et al., 2011bLebon G. Warne T. Edwards P.C. Bennett K. Langmead C.J. Leslie A.G. Tate C.G. Agonist-bound adenosine A2A receptor structures reveal common features of GPCR activation.Nature. 2011; 474: 521-525Crossref PubMed Scopus (641) Google Scholar). Mutation of these residues is highly stabilizing toward the inverse agonist bound but not the agonist bound conformation suggesting that they play a key role in the conformational selection of the receptor. In order to determine whether the loss of agonist affinity was directly related to mutation of these residues both T88A3.36 and S277A7.42 in A2A-StaR2 were mutated back to their wild-type amino acids, both individually and together. The stabilized receptor containing the six mutations A54L2.52, K122A4.43, V239A6.41, R107A3.55, L202A5.63, L235A6.37 in the absence of T88A3.36, and/or S277A7.42 still had identical pharmacology with respect to loss of agonist binding (Table S2). This suggests that the loss of agonist affinity is not just a direct result of these mutations but is also due to conformational selection as previously observed for the stabilized β1AR-m23 (Serrano-Vega et al., 2008Serrano-Vega M.J. Magnani F. Shibata Y. Tate C.G. Conformational thermostabilization of the β1-adrenergic receptor in a detergent-resistant form.Proc. Natl. Acad. Sci. USA. 2008; 105: 877-882Crossref PubMed Scopus (344) Google Scholar). Although T88A3.36 and S277A7.42 have little effect on inverse agonist/antagonist pharmacology we cannot rule out the possibility that the presence of these mutations has an effect on the binding of the ligands presented here, however, the consistency of the binding interactions described here with previous extensive mutagenesis data (Dal Ben et al., 2010Dal Ben D. Lambertucci C. Marucci G. Volpini R. Cristalli G. Adenosine receptor modeling: what does the A2A crystal structure tell us?.Curr. Top. Med. Chem. 2010; 10: 993-1018Crossref PubMed Scopus (38) Google Scholar) and the structures of A2A-T4L in complex with ZM241385 (Jaakola et al., 2008Jaakola V.P. Griffith M.T. Hanson M.A. Cherezov V. Chien E.Y. Lane J.R. Ijzerman A.P. Stevens R.C. The 2.6 angstrom crystal structure of a human A2A adenosine receptor bound to an antagonist.Science. 2008; 322: 1211-1217Crossref PubMed Scopus (1498) Google Scholar), UK-432097 (Xu et al., 2011Xu F. Wu H. Katritch V. Han G.W. Jacobson K.A. Gao Z.G. Cherezov V. Stevens R.C. Structure of an agonist-bound human A2A adenosine receptor.Science. 2011; 332: 322-327Crossref PubMed Scopus (652) Google Scholar) and adenosine, and NECA (Lebon et al., 2011bLebon G. Warne T. Edwards P.C. Bennett K. Langmead C.J. Leslie A.G. Tate C.G. Agonist-bound adenosine A2A receptor structures reveal common features of GPCR activation.Nature. 2011; 474: 521-525Crossref PubMed Scopus (641) Google Scholar) suggest that this is not the case. Additionally, all of the inverse agonist ligand structures presented here are found positioned over 5 Å from either of these residues. Prior to the structure determination of A2A-StaR2 bound to the ligands ZM241385, XAC and caffeine, their pharmacological profile was characterized. Stable inducible cell lines expressing the native adenosine A2A receptor were constructed using the Flp-In TREx system (Invitrogen). Induction of receptor expression resulted in gradually increasing basal levels of signaling, in the presence of adenosine deaminase that removes endogenous adenosine, indicative of constitutive activity. All the compounds used for subsequent structural studies ZM241385, XAC, and caffeine were found to equally inhibit constitutive activity indicative of them being inverse agonists whereas istradefylline (Jenner, 2005Jenner P. Istradefylline, a novel adenosine A2A receptor antagonist, for the treatment of Parkinson's disease.Expert Opin. Investig. Drugs. 2005; 14: 729-738Crossref PubMed Scopus (123) Google Scholar) was found to be a weak partial inverse agonist (Figure 2B). The purified A2A-StaR2 in complex with ZM241385 was crystallized by vapor diffusion in sitting drops and data collected on the microfocus beamline I24 at the Diamond Light Source (Oxfordshire, UK) corresponding to a 99.9% complete data set to 3.29 Å. The structure was solved by molecular replacement with 3EML with one copy of the A2A-StaR2 per crystallographic asymmetric unit. Statistics for data collection and refinement are given in Table 1.Table 1Crystallographic Table of StatisticsData CollectionA2A StaR2-ZM241385A2A StaR2-XACbNote the XAC- and caffeine-bound diffraction data exhibited significant anisotropy.A2A StaR2-CaffeinebNote the XAC- and caffeine-bound diffraction data exhibited significant anisotropy.Space groupI222I222I222Cell dimensions a, b, c, (Å)111.93, 112.55, 125.68111.86, 113.06, 126.80112.33, 113.33, 129.30Cell angles α, β, γ (°)90.0, 90.0, 90.090.0, 90.0, 90.090.0, 90.0, 90.0Resolution (Å)83.95–3.2948.69–3.3050.0–3.60RmergeaStatistics in parentheses throughout refer to outer resolution shell.0.11 (0.88)0.09 (0.69)0.13 (0.83)I/σ I10.3 (2.2)7.7 (1.5)6.8 (1.5)Completeness (%)99.9 (100.0)89.8 (90.2)91.9 (93.5)Redundancy10.7 (10.8)5.3 (5.2)6.7 (6.8)Refinement Resolution (Å)20.00–3.2919.93–3.3020.00–3.60 Reflections (n)12,01010,6278939 Rwork/Rfree (%)27.6/31.529.8/31.929.7/31.1Atoms (n) Protein225022502250 Ligand242014B-factors Å2 Protein138.2157.1148.9 Ligand148.3136.8146.4Rmsd Bond lengths (Å)0.0010.0010.002 Bond angles (°)0.3840.3940.389Ramachandran plot Preferred (%)90.9493.7393.73 Allowed (%)8.365.576.27 Outlier (%)0.700.700.00Rmsd, root-mean-square deviations.a Statistics in parentheses throughout refer to outer resolution shell.b Note the XAC- and caffeine-bound diffraction data exhibited significant anisotropy. Open table in a new tab Rmsd, root-mean-square deviations. The overall structure (Figure 3A ) includes residues 7–149 and 158–305 and the ligand ZM241385. A striking feature of the A2A-StaR2 structure is the extended nature of transmembrane helix (TM)5 and TM6 that project ∼15 Å into the cytoplasm. TM5 extends through to Ser2135.74 and is connected to TM6 by six residues to the helix of TM6 commencing at Arg2206.22 (Figure 3B; Figure S1). The ordered and extended nature of the ICL3, which is also seen in squid rhodopsin (Murakami and Kouyama, 2008Murakami M. Kouyama T. Crystal structure of squid rhodopsin.Nature. 2008; 453: 363-367Crossref PubMed Scopus (400) Google Scholar) is contributed to by Pro215 and Pro217 and a network of potential hydrogen bonds involving Arg2226.24, the main chain carbonyl of Met2115.72, and between Gln2145.75 and the main chain carbonyl of Pro217 and Leu216. This is the first time structures of the same GPCR have been obtained using the different crystallization strategies of thermostabilization and T4L fusions and so provides a useful comparison of the techniques. The similarity of the structures provides confidence that engineering receptors through either fusion proteins or mutagenesis provides an effective approach to GPCR crystallization and does not result in major abnormalities of the structures. A comparison of the position of the thermostabilizing mutations in the A2A-StaR2 with the position of these residues in the A2A-T4L structure shows no obvious perturbation of the structure around the mutations (Figure S2), however, we cannot rule out the possibility that individual mutations may alter the structure in some way. In the case of all GPCR structures, care needs to be taken with more detailed interpretation of the structures, preferably in the context of pharmacological analysis of the crystallization constructs. The closest structural agreement between the A2A-StaR2-ZM241385 and the A2A-T4L-ZM241385 structure occurs between TMs 1, 2, 3, 4, and 7 (Cα root-mean-square deviation [rmsd] = 0.51 Å) whereas superposition of TM5 and TM6 (residues 174–203 and 222–258) reveals significant differences (Cα rmsd = 1.62 Å). In A2A-T4L the C-terminal portion of TM5 is displaced out of the helical bundle and moves laterally toward TM6 compared to A2A-StaR2 (Figure 4A ). The intracellular end of TM6 in A2A-T4L is rotated toward TM5 by pivoting ∼42° at Val2296.31 away from the helical bundle, whereas TM6 of A2A-StaR2 continues to pack with TM5. The global position of TM5 and TM6 of the A2A-StaR2 are in closer agreement to the ground state of rhodopsin (Protein Data Bank [PDB] code: 1F88) (Figure 4B) than A2A-T4L. Although both structures of the A2A receptor bind the same inverse agonist ligands ZM241385 there are significant differences in the pharmacology. The A2A-StaR2 has a pharmacology consistent with the inverse agonist state whereas A2A-T4L has a more agonist-like pharmacology with respect to agonist binding (Jaakola et al., 2008Jaakola V.P. Griffith M.T. Hanson M.A. Cherezov V. Chien E.Y. Lane J.R. Ijzerman A.P. Stevens R.C. The 2.6 angstrom crystal structure of a human A2A adenosine receptor bound to an antagonist.Science. 2008; 322: 1211-1217Crossref PubMed Scopus (1498) Google Scholar). Differences are also seen in the receptor structures that are consistent with the two structures representing different conformational states of the receptor. One of the most highly conserved sequence motifs in GPCRs is the E/DRY motif in TM3. In bovine rhodopsin the side chain of Arg1353.50 within the E/DRY motif hydrogen bonds to the side chain of Glu2476.30 at the N terminus of TM6 to form the so-called “ionic lock”(Vogel et al., 2008Vogel R. Mahalingam M. Lüdeke S. Huber T. Siebert F. Sakmar T.P. Functional role of the “ionic lock”—an interhelical hydrogen-bond network in family A heptahelical receptors.J. Mol. Biol. 2008; 380: 648-655Crossref PubMed Scopus (129) Google Scholar), part of a network of interactions bridging TM3 and TM6 and stabilizing the inactive-state conformation. Inverse agonists are considered to preferentially bind to and stabilize this inactive conformation thus reducing any basal activity (Kenakin, 2004Kenakin T. Principles: receptor theory in pharmacology.Trends Pharmacol. Sci. 2004; 25: 186-192Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar). During activation the ionic lock is broken allowing the outward movement of TM6. A2A-StaR2 has the potential ionic lock in place with the side chains of Glu2286.30 and Arg1023.50 in a similar conformation to that found in dark-state rhodopsin (Figures 4A and 4B; Figure S3A). The presence of the ionic lock in the A2A-StaR2 is similar to that in dark-state rhodopsin and is consistent with this structure representing the ground state of the receptor. The absence of the ionic lock in the A2A-T4L structure (Figure 4A) appears to be the result of an outward movement and rotation in TM6 resulting from the T4 lysozyme fusion in ICL3. This effect of the T4L fusion is receptor specific because the ionic lock is present in the dopamine D3 receptor-T4L structure (Chien et al., 2010Chien E.Y. Liu W. Zhao Q. Katritch V. Han G.W. Hanson M.A. Shi L. Newman A.H. Javitch J.A. Cherezov V. Stevens R.C. Structure of the human dopamine D3 receptor in complex with a D2/D3 selective antagonist.Science. 2010; 330: 1091-1095Crossref PubMed Scopus (911) Google Scholar). The impact of the T4L on the structure may also depend it's the position within ICL3 and the length of the truncated ICL3 loop. The extracellular surface of the A2A receptor consists primarily of the second and third extracellular loops (ECL2 and ECL3) with ECL2 ordered through disulfide linkages to ECL1 (Jaakola et al., 2008Jaakola V.P. Griffith M.T. Hanson M.A. Cherezov V. Chien E.Y. Lane J.R. Ijzerman A.P. Stevens R.C. The 2.6 angstrom crystal structure of a human A2A adenosine receptor bound to an antagonist.Science. 2008; 322: 1211-1217Crossref PubMed Scopus (1498) Google Scholar). Interestingly, changes at the extracellular surface of the ligand binding site involving a different rotamer conformation in His2646.66 and a displacement of the disulfide bonded CysProAspCys motif (still maintaining the disulfide link between Cys2596.61 and Cys2626.64) away from the entrance of the ligand binding cavity may facilitate a more “open” entrance in comparison to A2A-T4L, facilitating access of the ligand to the binding pocket of the inactive receptor. Data from agonist bound structures of the adrenergic receptors suggests th" @default.
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