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• W2914322496 abstract "•GPCR conformation dynamics reveals the forward and backward allosteric mechanism•Agonist binding increases the entropy in the intracellular region of the GPCR•G protein binding shrinks the receptor-ligand contacts in the extracellular region•Increased allostery between G protein and agonist in the GPCR-G protein complex Agonist binding in the extracellular region of the G protein-coupled adenosine A2A receptor increases its affinity to the G proteins in the intracellular region, and vice versa. The structural basis for this effect is not evident from the crystal structures of A2AR in various conformational states since it stems from the receptor dynamics. Using atomistic molecular dynamics simulations on four different conformational states of the adenosine A2A receptor, we observed that the agonists show decreased ligand mobility, lower entropy of the extracellular loops in the active-intermediate state compared with the inactive state. In contrast, the entropy of the intracellular region increases to prime the receptor for coupling the G protein. Coupling of the G protein to A2AR shrinks the agonist binding site, making tighter receptor agonist contacts with an increase in the strength of allosteric communication compared with the active-intermediate state. These insights provide a strong basis for structure-based ligand design studies. Agonist binding in the extracellular region of the G protein-coupled adenosine A2A receptor increases its affinity to the G proteins in the intracellular region, and vice versa. The structural basis for this effect is not evident from the crystal structures of A2AR in various conformational states since it stems from the receptor dynamics. Using atomistic molecular dynamics simulations on four different conformational states of the adenosine A2A receptor, we observed that the agonists show decreased ligand mobility, lower entropy of the extracellular loops in the active-intermediate state compared with the inactive state. In contrast, the entropy of the intracellular region increases to prime the receptor for coupling the G protein. Coupling of the G protein to A2AR shrinks the agonist binding site, making tighter receptor agonist contacts with an increase in the strength of allosteric communication compared with the active-intermediate state. These insights provide a strong basis for structure-based ligand design studies. The adenosine A2A receptor (A2AR) is a G protein-coupled receptor (GPCR) that is activated in vivo by the agonist adenosine (Fredholm et al., 2011Fredholm B.B. IJzerman A.P. Jacobson K.A. Linden J. Muller 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 (1008) Google Scholar). Subsequent coupling of the G protein, Gs, leads to the eventual increase in intracellular (IC) cyclic AMP through activation of adenylate cyclase and the modulation of downstream signaling pathways. A2AR is a validated drug target for the treatment of Parkinson's disease and cancer, which has resulted in its well-characterized pharmacology and a wide variety of synthetic agonists and antagonists (de Lera Ruiz et al., 2014de Lera Ruiz M. Lim Y.H. Zheng J. Adenosine A2A receptor as a drug discovery target.J. Med. Chem. 2014; 57: 3623-3650Crossref PubMed Scopus (203) Google Scholar). The structure of A2AR has been determined in the antagonist-bound inactive state (Cheng et al., 2017Cheng R.K.Y. Segala E. Robertson N. Deflorian F. Dore A.S. Errey J.C. Fiez-Vandal C. Marshall F.H. Cooke R.M. Structures of human A1 and A2A adenosine receptors with xanthines reveal determinants of selectivity.Structure. 2017; 25: 1275-1285.e4Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, Dore et al., 2011Dore A.S. Robertson N. Errey J.C. Ng I. Hollenstein K. Tehan B. Hurrell E. Bennett K. Congreve M. Magnani F. et al.Structure of the adenosine A(2A) receptor in complex with ZM241385 and the xanthines XAC and caffeine.Structure. 2011; 19: 1283-1293Abstract Full Text Full Text PDF PubMed Scopus (432) Google Scholar, 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 (1558) Google Scholar, Liu et al., 2012Liu W. Chun E. Thompson A.A. Chubukov P. Xu F. Katritch V. Han G.W. Roth C.B. Heitman L.H. IJzerman A.P. et al.Structural basis for allosteric regulation of GPCRs by sodium ions.Science. 2012; 337: 232-236Crossref PubMed Scopus (722) Google Scholar, Segala et al., 2016Segala E. Guo D. Cheng R.K. Bortolato A. Deflorian F. Dore A.S. Errey J.C. Heitman L.H. IJzerman A.P. et al.Controlling the dissociation of ligands from the adenosine a2a receptor through modulation of salt bridge strength.J. Med. Chem. 2016; 59: 6470-6479Crossref PubMed Scopus (115) Google Scholar, Sun et al., 2017Sun B. Bachhawat P. Chu M.L. Wood M. Ceska T. Sands Z.A. Mercier J. Lebon F. Kobilka T.S. Kobilka B.K. et al.Crystal structure of the adenosine A2A receptor bound to an antagonist reveals a potential allosteric pocket.Proc. Natl. Acad. Sci. U S A. 2017; 114: 2066-2071Crossref PubMed Scopus (88) Google Scholar), the agonist-bound active-intermediate state (Lebon et al., 2011Lebon 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 (695) Google Scholar, Lebon et al., 2015Lebon G. Edwards P.C. Leslie A.G. Tate C.G. Molecular determinants of CGS21680 binding to the human adenosine A2A receptor.Mol. Pharmacol. 2015; 87: 907-915Crossref PubMed Scopus (93) 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 (685) Google Scholar), and in the fully active state bound to either mini-Gs (Carpenter et al., 2016Carpenter B. Nehme R. Warne T. Leslie A.G. Tate C.G. Structure of the adenosine A(2A) receptor bound to an engineered G protein.Nature. 2016; 536: 104-107Crossref PubMed Scopus (285) Google Scholar) or to heterotrimeric Gs (Tate et al., 2018Tate C.G. García-Nafría J. Lee Y. Bai X. Carpenter B. Cryo-EM structure of the adenosine A2A receptor coupled to an engineered heterotrimeric G protein.bioRxiv. 2018; : 267674https://doi.org/10.1101/267674Crossref Google Scholar). The mechanism of activation conforms to the canonical paradigm (Rasmussen et al., 2011aRasmussen S.G. Choi H.J. Fung J.J. Pardon E. Casarosa P. Chae P.S. Devree B.T. Rosenbaum D.M. Thian F.S. Kobilka T.S. et al.Structure of a nanobody-stabilized active state of the beta(2) adrenoceptor.Nature. 2011; 469: 175-180Crossref PubMed Scopus (1311) Google Scholar) where agonist binding results in a slight contraction of the orthosteric binding pocket, rotamer changes of the hydrophobic gating residues Pro5.50-Ile3.40-Phe6.44 and opening of a cleft on the cytoplasmic face of the receptor primarily through an outward movement of transmembrane helix 6 (TM6). The C-terminal helix of Gs, known as the α5 helix, binds in this cleft, resulting in nucleotide exchange and activation of the G protein (Carpenter et al., 2016Carpenter B. Nehme R. Warne T. Leslie A.G. Tate C.G. Structure of the adenosine A(2A) receptor bound to an engineered G protein.Nature. 2016; 536: 104-107Crossref PubMed Scopus (285) Google Scholar, Rasmussen et al., 2011bRasmussen S.G. DeVree B.T. Zou Y. Kruse A.C. Chung K.Y. Kobilka T.S. Thian F.S. Chae P.S. Pardon E. Calinski D. et al.Crystal structure of the beta2 adrenergic receptor-Gs protein complex.Nature. 2011; 477: 549-555Crossref PubMed Scopus (2267) Google Scholar). The structure of A2AR in three defined conformations provides a series of snapshots during activation, but they do not provide information regarding receptor dynamics or the allosteric effect of G protein binding. The β2-adrenergic receptor (β2AR) is the most studied GPCR in terms of the dynamics of activation (Nygaard et al., 2013Nygaard R. Zou Y. Dror R.O. Mildorf T.J. Arlow D.H. Manglik A. Pan A.C. Liu C.W. Fung J.J. Bokoch M.P. et al.The dynamic process of beta(2)-adrenergic receptor activation.Cell. 2013; 152: 532-542Abstract Full Text Full Text PDF PubMed Scopus (610) Google Scholar) and there are many similarities between A2AR and β2AR, but also some differences. The architecture of A2AR and β2AR is similar, they both couple to Gs and they both exist in an ensemble of conformations in the absence of an agonist (Manglik et al., 2015Manglik A. Kim T.H. Masureel M. Altenbach C. Yang Z. Hilger D. Lerch M.T. Kobilka T.S. Thian F.S. Hubbell W.L. et al.Structural insights into the dynamic process of beta2-adrenergic receptor signaling.Cell. 2015; 161: 1101-1111Abstract Full Text Full Text PDF PubMed Scopus (437) Google Scholar, Ye et al., 2016Ye L. Van Eps N. Zimmer M. Ernst O.P. Prosser R.S. Activation of the A2A adenosine G-protein-coupled receptor by conformational selection.Nature. 2016; 533: 265-268Crossref PubMed Scopus (218) Google Scholar). In addition, coupling of Gs to the agonist-bound A2AR or β2AR increases the affinity of agonists at both receptors (Carpenter et al., 2016Carpenter B. Nehme R. Warne T. Leslie A.G. Tate C.G. Structure of the adenosine A(2A) receptor bound to an engineered G protein.Nature. 2016; 536: 104-107Crossref PubMed Scopus (285) Google Scholar, Chung et al., 2011Chung K.Y. Rasmussen S.G. Liu T. Li S. DeVree B.T. Chae P.S. Calinski D. Kobilka B.K. Woods Jr., V.L. Sunahara R.K. Conformational changes in the G protein Gs induced by the beta2 adrenergic receptor.Nature. 2011; 477: 611-615Crossref PubMed Scopus (290) Google Scholar). In β2AR, it has been proposed that this increase in agonist affinity is a consequence of a closure of the entrance to the orthosteric binding pocket, resulting in a steric block to the exit of the agonist from the receptor (DeVree et al., 2016DeVree B.T. Mahoney J.P. Velez-Ruiz G.A. Rasmussen S.G. Kuszak A.J. Edwald E. Fung J.J. Manglik A. Masureel M. Du Y. et al.Allosteric coupling from G protein to the agonist-binding pocket in GPCRs.Nature. 2016; 535: 182-186Crossref PubMed Scopus (171) Google Scholar). The reason for the increase in agonist affinity in A2AR upon G protein coupling is unclear, because of the different energy landscapes of the respective receptors (Lebon et al., 2012Lebon G. Warne T. Tate C.G. Agonist-bound structures of G protein-coupled receptors.Curr. Opin. Struct. Biol. 2012; 22: 482-490Crossref PubMed Scopus (82) Google Scholar). Crystal structures show that the agonist binding to β-AR stabilizes them in an inactive-like state (Sato et al., 2015Sato T. Baker J. Warne T. Brown G.A. Leslie A.G. Congreve M. Tate C.G. Pharmacological analysis and structure determination of 7-methylcyanopindolol-bound beta1-adrenergic receptor.Mol. Pharmacol. 2015; 88: 1024-1034Crossref PubMed Scopus (17) Google Scholar). In contrast, agonist binding to A2AR results in conformational changes throughout the receptor into an active-intermediate state (Lebon et al., 2011Lebon 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 (695) Google Scholar) and only the outward bending of the cytoplasmic end of TM6 accompanies G protein binding (Carpenter et al., 2016Carpenter B. Nehme R. Warne T. Leslie A.G. Tate C.G. Structure of the adenosine A(2A) receptor bound to an engineered G protein.Nature. 2016; 536: 104-107Crossref PubMed Scopus (285) Google Scholar). This is different compared with the receptor-wide changes observed in β2AR during the transition from the agonist-bound inactive state to the G protein-coupled state. Interestingly, the transition in A2AR from the active-intermediate to the fully active state does not involve any significant structural changes in the extracellular (EC) half of the receptor that defines the conformation of the ligand binding pocket (Carpenter et al., 2016Carpenter B. Nehme R. Warne T. Leslie A.G. Tate C.G. Structure of the adenosine A(2A) receptor bound to an engineered G protein.Nature. 2016; 536: 104-107Crossref PubMed Scopus (285) Google Scholar). The similarities and differences in the various states of these two Gs-coupled receptors depend on the dynamics and the energy landscape of these two receptors. Although the influence of the agonist in shifting the GPCR conformational ensemble has been studied (Manglik et al., 2015Manglik A. Kim T.H. Masureel M. Altenbach C. Yang Z. Hilger D. Lerch M.T. Kobilka T.S. Thian F.S. Hubbell W.L. et al.Structural insights into the dynamic process of beta2-adrenergic receptor signaling.Cell. 2015; 161: 1101-1111Abstract Full Text Full Text PDF PubMed Scopus (437) Google Scholar, Niesen et al., 2013Niesen M.J. Bhattacharya S. Grisshammer R. Tate C.G. Vaidehi N. Thermostabilization of the beta1-adrenergic receptor correlates with increased entropy of the inactive state.J. Phys. Chem. B. 2013; 117: 7283-7291Crossref PubMed Scopus (12) Google Scholar, Nygaard et al., 2013Nygaard R. Zou Y. Dror R.O. Mildorf T.J. Arlow D.H. Manglik A. Pan A.C. Liu C.W. Fung J.J. Bokoch M.P. et al.The dynamic process of beta(2)-adrenergic receptor activation.Cell. 2013; 152: 532-542Abstract Full Text Full Text PDF PubMed Scopus (610) Google Scholar), studies on the “reverse” influence of the ensemble of the GPCR-G protein complex on the agonist binding and the GPCR dynamics have been sparse. In addition, the role of allosteric effects in the GPCR when bound to both agonist and G protein is unclear. The crystal structures available in three different conformation states of A2AR offers a unique opportunity for studying the consequences of G protein coupling to a GPCR without the difficulties of decoupling the effects of the agonist from the effects of the G protein. We have therefore used atomistic molecular dynamics (MD) simulations on A2AR to understand the dynamic ensemble of conformations of the receptor in the inactive state, active-intermediate state, and the G protein-coupled fully active state to study the effects of the agonist and G protein on the receptor. Our results show that agonist binding to A2AR decreases the ligand mobility and entropy of the EC regions in the agonist-bound active-intermediate state compared with the agonist-bound inactive state. Importantly, the entropy of the IC regions increases upon agonist binding in the active-intermediate state compared with the inactive state, probably priming the receptor to bind to the G protein. Stabilization of the G protein-bound fully active conformation of A2AR shows increase in allosteric communication between the EC regions and the G protein-coupling IC regions. This reverse allosteric effect from the G protein to the ligand binding site explains the observed increase in agonist binding affinity to the G protein-coupled GPCR (DeVree et al., 2016DeVree B.T. Mahoney J.P. Velez-Ruiz G.A. Rasmussen S.G. Kuszak A.J. Edwald E. Fung J.J. Manglik A. Masureel M. Du Y. et al.Allosteric coupling from G protein to the agonist-binding pocket in GPCRs.Nature. 2016; 535: 182-186Crossref PubMed Scopus (171) Google Scholar, Carpenter et al., 2016Carpenter B. Nehme R. Warne T. Leslie A.G. Tate C.G. Structure of the adenosine A(2A) receptor bound to an engineered G protein.Nature. 2016; 536: 104-107Crossref PubMed Scopus (285) Google Scholar). Atomistic MD simulations were performed on A2AR bound to the agonists adenosine (ADO) or 5-N-ethylcarboxamidoadenosine (NECA), each in four different conformational states: (1) the inactive state of the receptor, R; (2) the active-intermediate state, R′; (3) the mini-Gs bound fully active state, R∗·G; and (4) a metastable state, R∗·G−, formed in silico by removal of mini-Gs from R∗·G. We also performed MD simulations on the inverse agonist ZM241385 bound inactive state and active-intermediate state of the receptor. To study the effect of Na+ ions on receptor dynamics, we performed MD simulations with Na+ ion in the ZM241385-bound inactive state, NECA-bound inactive, active-intermediate, and fully active states as detailed in Table S1 of the Supplemental Information. The initial structures for the simulations on R′ and R∗·G were from the crystal structures of A2AR bound to either NECA (PDB: 2YDV; Lebon et al., 2011Lebon 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 (695) Google Scholar) or NECA and mini-Gs (PDB: 5G53; Carpenter et al., 2016Carpenter B. Nehme R. Warne T. Leslie A.G. Tate C.G. Structure of the adenosine A(2A) receptor bound to an engineered G protein.Nature. 2016; 536: 104-107Crossref PubMed Scopus (285) Google Scholar). The inactive state with NECA bound was generated from the crystal structure of A2AR bound to the inverse agonist ZM241385 (Dore et al., 2011Dore A.S. Robertson N. Errey J.C. Ng I. Hollenstein K. Tehan B. Hurrell E. Bennett K. Congreve M. Magnani F. et al.Structure of the adenosine A(2A) receptor in complex with ZM241385 and the xanthines XAC and caffeine.Structure. 2011; 19: 1283-1293Abstract Full Text Full Text PDF PubMed Scopus (432) Google Scholar) by replacement of the ligand with NECA followed by an equilibration protocol and MD production runs (see STAR Methods). The R∗·G− state was generated by removing mini-Gs from the R∗·G state followed by equilibration and production runs. We used the crystal structure with Na+ ion bound for ZM241385-bound R state (PDB: 4EIY; Liu et al., 2012Liu W. Chun E. Thompson A.A. Chubukov P. Xu F. Katritch V. Han G.W. Roth C.B. Heitman L.H. IJzerman A.P. et al.Structural basis for allosteric regulation of GPCRs by sodium ions.Science. 2012; 337: 232-236Crossref PubMed Scopus (722) Google Scholar). The conformation for the R′ state of the wild-type A2AR generated above, was used to transfer the ZM241385 from the R state to the R′ state for further simulations. The list of systems simulated in this study, the notations used to represent different conformational states, and other details of these systems are given in Table S1. To analyze the conformation ensembles of A2AR in the different states, MD simulations totaling 1 μs (5 separate simulations of 200 ns each) were performed on the ligand-receptor or the ligand-receptor complex, with mini-Gs, placed in explicit water and a lipid bilayer composed of palmitoyloleoylphosphatidylcholine. The results for A2AR bound to NECA are shown in the main text; similar results were obtained using adenosine and are all shown in the Supplemental Information (Figures S1–S5). The conformations from the MD simulation trajectories were clustered by the Cα-Cα distances between residues R1023.50 and E2286.30 on TM3 and TM6 and between R1023.50 and Y2887.53 on TM3 and TM7 (Figure 1), which are indicative of the receptor conformational changes upon activation (Tehan et al., 2014Tehan B.G. Bortolato A. Blaney F.E. Weir M.P. Mason J.S. Unifying family A GPCR theories of activation.Pharmacol. Ther. 2014; 143: 51-60Crossref PubMed Scopus (147) Google Scholar). It should be noted that these two distances are not the only measures of receptor activation. However, we use these two distances only to assess the breadth of the conformational sampling during MD simulations and not as a measure of receptor activation. An analysis of these distances for RNECA and R∗·GNECA showed well-defined values, whereas the equivalent distances in R′NECA and R∗·G−NECA exhibited a large spread of values. These data are consistent with increased flexibility and conformational heterogeneity of R′NECA and R∗·G−NECA compared with RNECA and R∗·GNECA. The inverse agonist ZM241385-bound R and R′ states show narrow variations in the TM3-TM6 and TM3-TM7 distances (Figure S1C), consistent with the receptor being close to the starting R and R′ states respectively. Mapping the receptor flexibility calculated as root-mean-square fluctuation (RMSF) in Cartesian coordinates following superimposition of all frames onto the structures of A2AR showed that the most flexible region varies between the different conformational states. When NECA is bound to the inactive state (RNECA), the EC surface of A2AR is highly mobile, whereas the IC surface shows little variation in structure. In contrast, the R′ state is characterized by reduced mobility of the EC region and increased mobility of the IC region. G protein binding decreases the flexibility of the IC region, while the flexibility of the EC region remains the same. However, removal of the G protein to generate the R∗·G−NECA state results in a highly flexible metastable state. The conformation ensemble for ADO bound to different states of A2AR shows a similar trend (Figures S1A and S1B). Using the Bennett Acceptance Ratio method (see the STAR Methods) for calculating the difference in free energies between two systems, the free energy of binding was calculated for the agonists NECA and ADO to R, R′, R∗·G, and R∗·G− conformations of A2AR. The binding free energy of both NECA and ADO is more favorable by 9.6 ± 0.8 and 8.3 ± 0.7 kcal/mol, respectively (Figure 2) in the fully active state, R∗·G, compared with the inactive state, R. The binding free energy of these two agonists is also more favorable in R∗·G compared with the R′ state, suggesting that the G protein coupling influences agonist affinity. This is corroborated by the decrease in affinity observed upon removal of the G protein in the R∗·G− state (Table S2 of the Supplemental Information). It should be noted that the difference in calculated binding free energy is higher than the measured differences in binding affinity (Table S2 of the Supplemental Information). This discrepancy could be because the experimental binding affinity manifests from an equilibrium among multiple conformational states such as inactive and active-intermediate states, for example. Even the thermostabilized receptor will be in equilibrium between various conformational states in the presence of an agonist, including the inactive state and active-intermediate state. On the other hand, the MD simulations sample a smaller ensemble of states close to the starting conformational state. Therefore, the binding free energies calculated from the MD simulations reflect the ligand affinity to the specific conformational state of the receptor. The average interaction energy of the ligand (agonist and inverse agonist) with the receptor, averaged over the MD trajectories, for each receptor conformational state is shown in Figure S2. Details of the calculation of the interaction energies are given in the STAR Methods. Both the agonists NECA and adenosine show highest interaction energy in the R∗·G state, while inverse agonist ZM241385 shows the highest interaction energy in the inactive R state. It is interesting to note that the agonist and inverse agonist do not show significant difference in their interaction energy in the active-intermediate R′ state. The dynamics of agonists within the orthosteric binding site was assessed during the MD simulations to provide possible insights into why the affinity of NECA increases upon G protein coupling. The agonist movement and flexibility in the receptor was assessed by calculating the spatial distribution function for particular atoms in the agonists during the MD simulation (Figure 3A for NECA top panel, and Figure S3 for ADO; Table S3 of the Supplemental Information has a complete list of all the ligand-receptor contacts in all the conformational states and their relative populations) and also the RMSF from the average structure calculated from the MD simulations (Figure S4A). By both criteria, NECA shows high levels of movement within the RNECA and R∗·G−NECA states. In contrast, in the R′NECA and R∗·GNECA states, there appears to be far less movement of the agonists, suggestive of the orthosteric binding pocket being more rigid forming tighter ligand-protein contacts. The average volume of the agonist binding site remains similar in the R′ and R∗·G and R∗·G− states, but there is a significant decrease in the volume upon transition from R to R′ (Figure S4C). Thus there is not a simple relationship between the volume of the orthosteric binding site and the degree of ligand movement. Similar trends in ADO flexibility was observed in different conformational states of A2AR (compare Figures 3 with S3). The number of residues making sustained contacts with the agonist (present in greater than 40% of the MD snapshots) was significantly less in the R and R∗·G− states compared with R′ and R∗·G (Figure 3B for NECA and Figure S3B for ADO), which might be expected given the different levels of ligand motion. Most of the A2AR-agonist contacts in the R′ and R∗·G state are preserved, although there are slight differences in contacts with V843.32, M1775.38, and S2777.42, which show a low frequency of interaction below 40% of the MD snapshots in at least one of the structures. The torsional entropy was calculated for all the residues in the EC loops and the IC loops, including two turns of the adjacent TM helices (see the STAR Methods) for all the agonist-bound conformational states (Figure 4). The entropy of the EC region is highest in the R state, and this entropy decreases when the agonist stabilizes the R′ state. The entropy in the EC region in the R′, R∗·G and, R∗·G− states are all similar. In contrast, the entropy of the IC regions is highest in the R′ and R∗·G− states. As expected, G protein coupling in the R∗·G state significantly reduces the entropy of the IC region, especially in the IC loop 2 (ICL2) region (Figure S4D of the Supplemental Information), to a level similar to that observed in the R state. It should be noted that the reduced entropy could simply stem from the G protein binding to the IC regions of the receptor in the R∗·G state. The fluctuations in the RMSF of the Cα atoms of A2AR in the EC regions for the four conformational states of A2AR bound to NECA, reflect this trend in entropy (Figure 4B). The entropy of the individual EC and IC loops shown in Figure S4D of the Supplemental Information exhibits the same trend as the total entropy of the loops. The residues in the ICL3 loop are missing in the present simulations and we examined if this could contribute to the increased RMSF in ICL3 loop in the R′ state. We had previously published results on the dynamics of NECA-bound R′ state in which we modeled the entire ICL3 loop conformation (Lee et al., 2014Lee S. Bhattacharya S. Grisshammer R. Tate C.G. Vaidehi N. Dynamic Behavior of the Active and Inactive States of the Adenosine A2A Receptor.J. Phys. Chem. B. 2014; 118: 3355-3365Crossref PubMed Scopus (20) Google Scholar). Comparison of the flexibility (RMSF) of the ICL3 loop residues from the present simulations of NECA-bound R′ state without the ICL3 loop, to the RMSF of the ICL3 loop residues from our previous work shows that the change in RMSF is minimal with or without the ICL3 loop residues (Figure S4B). This reaffirms our finding in Figure S4D that the flexibility of the IC loops is influenced by agonist binding in the R′ state compared with the R state. Allosteric effects play an important role in communicating the effect of agonist binding to the G protein coupling region and vice versa. Therefore delineating the residues in the allosteric communication pipelines will provide vital information for designing drugs with selectivity. The strength of the allosteric communication pipelines was calculated in the agonist-bound R, R′, R∗·G, and R∗·G− conformations using the program Allosteer (Bhattacharya et al., 2016Bhattacharya S. Salomon-Ferrer R. Lee S. Vaidehi N. Conserved mechanism of conformational stability and dynamics in G-protein-coupled receptors.J. Chem. Theory Comput. 2016; 12: 5575-5584Crossref PubMed Scopus (18) Google Scholar, Bhattacharya and Vaidehi, 2014Bhattacharya S. Vaidehi N. Differences in allosteric communication pipelines in the inactive and active states of a GPCR.Biophys. J. 2014; 107: 422-434Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, Vaidehi and Bhattacharya, 2016Vaidehi N. Bhattacharya S. Allosteric communication pipelines in G-protein-coupled receptors.Curr. Opin. Pharmacol. 2016; 30: 76-83Crossref PubMed Scopus (25) Google Scholar) (see the STAR Methods). Binding of agonist or the G protein modifies the strength of the allosteric communication pipelines. The agonist-bound R′ state shows a strong allosteric coupling between the IC G protein coupling regions and the EC loop regions compared with the R state. However, G protein coupling to the receptor results in a dramatic increase in the strength of the allosteric coupling in R∗·G state (Figures 5 and S5 for ADO). The strength of allosteric communication reflects the level of correlated motion between residues in the EC and IC regions. Therefore, binding of both NECA and mini-Gs shows increased correlated motion in the receptor, thus stabilizing the fully active state. An elegant structural study on the effect of Na+ ion on the inverse agonist-bound A2AR structure (Liu et al., 2012Liu W. Chun E. Thompson A.A. Chubukov P. Xu F. Katritch V. Han G.W. Roth C.B. Heitman L.H. IJzerman A.P. et al.Structural basis for allosteric regulation of GPCRs by sodium ions.Science. 2012; 337: 232-236Crossref PubMed Scopus (722) Google Scholar) showed that the Na+ ion attracts a cluster of well-ordered water molecules in its binding site near D522.50 and S913.39. This crystal structure also shows a water" @default.
• W2914322496 created "2019-02-21" @default.
• W2914322496 creator A5019715172 @default.
• W2914322496 creator A5048652542 @default.
• W2914322496 creator A5060670368 @default.
• W2914322496 creator A5087200267 @default.
• W2914322496 date "2019-04-01" @default.
• W2914322496 modified "2023-10-15" @default.
• W2914322496 title "Dynamic Role of the G Protein in Stabilizing the Active State of the Adenosine A2A Receptor" @default.
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