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The activation process of G protein-coupled receptors (GPCRs) has been extensively studied, both experimentally and computationally. In particular, Molecular Dynamics (MD) simulations have proven useful in exploring GPCR conformational space. The typical behaviour of class A GPCRs, when subjected to unbiased MD simulations from their crystallized inactive state, is to fluctuate between inactive and intermediate(s) conformations, even with bound agonist. Fully active conformation(s) are rarely stabilized unless a G protein is also bound. Despite several crystal structures of the adenosine A2a receptor (A2aR) having been resolved in complex with co-crystallized agonists and Gs protein, its agonist-mediated activation process is still not completely understood. In order to thoroughly examine the conformational landscape of A2aR activation, we performed unbiased microsecond-length MD simulations in quadruplicate, starting from the inactive conformation either in apo or with bound agonists: endogenous adenosine or synthetic NECA, embedded in two homogeneous phospholipid membranes: 1,2-dioleoyl-sn-glycerol-3-phosphoglycerol (DOPG) or 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC). In DOPC with bound adenosine or NECA, we observe transition to an intermediate receptor conformation consistent with the known adenosine-bound crystal state. In apo state in DOPG, two different intermediate conformations are obtained. One is similar to that observed with bound adenosine in DOPC, while the other is closer to the active state but not yet fully active. Exclusively, in DOPG with bound adenosine or NECA, we reproducibly identify receptor conformations with fully active features, which are able to dock Gs protein. These different receptor conformations can be attributed to the action/absence of agonist and phospholipid-mediated allosteric effects on the intracellular side of the receptor. Author summary: Unbiased microsecond-length MD simulations of the adenosine A2a receptor (A2aR) were performed in quadruplicate, starting from the inactive conformation either in apo or with bound agonists: adenosine or NECA, each of them embedded in two different homogeneous phospholipid membranes. Different intermediate or active receptor conformations were found depending on the presence/absence of bound agonist and type of lipid environment. Exclusively, in DOPG with bound agonist, we reproducibly identify receptor conformations with fully active features, which are able to dock Gs protein. These different receptor conformations can be attributed to the action/absence of agonist and phospholipid-mediated allosteric effects on the intracellular side of the receptor. Dynamic structural data are key for the understanding of agonist-mediated GPCR activation simulated in realistic membrane environments. [ABSTRACT FROM AUTHOR]

Background: G-protein-coupled receptors (GPCRs) are important drug targets and a better understanding of their molecular mechanisms would be desirable. The crystallization rate of GPCRs has accelerated in recent years as techniques have become more sophisticated, particularly with respect to Class A GPCRs interacting with G-proteins. These developments have made it possible for a quantitative analysis of GPCR geometrical features and binding-site conformations, including a statistical comparison between Class A GPCRs in active (agonist-bound) and inactive (antagonist-bound) states. Results: Here we implement algorithms for the analysis of interhelical angles, distances, interactions and binding-site volumes in the transmembrane domains of 25 Class A GPCRs (7 active and 18 inactive). Two interhelical angles change in a statistically significant way between average inactive and active states: TM3-TM6 (by -9°) and TM6-TM7 (by +12°). A third interhelical angle: TM5-TM6 shows a trend, changing by -9°. In the transition from inactive to active states, average van der Waals interactions between TM3 and TM7 significantly increase as the average distance between them decreases by >2 Å. Average H-bonding between TM3 and TM6 decreases but is seemingly compensated by an increase in H-bonding between TM5 and TM6. In five Class A GPCRs, crystallized in both active and inactive states, increased H-bonding of agonists to TM6 and TM7, relative to antagonists, is observed. These protein-agonist interactions likely favour a change in the TM6-TM7 angle, which creates a narrowing in the binding pocket of activated receptors and an average ∼200 Å3 reduction in volume. Conclusions: In terms of similar conformational changes and agonist binding pattern, Class A GPCRs appear to share a common mechanism of activation, which can be exploited in future drug development. [ABSTRACT FROM AUTHOR]

Molecular dynamics (MD) is the common computational technique for assessing efficacy of GPCR-bound ligands. Agonist efficacy measures the capability of the ligand-bound receptor of reaching the active state in comparison with the free receptor. In this respect, agonists, neutral antagonists and inverse agonists can be considered. A collection of MD simulations of both the ligand-bound and the free receptor are needed to provide reliable conclusions. Variability in the trajectories needs quantification and proper statistical tools for meaningful and non-subjective conclusions. Multiple-factor (time, ligand, lipid) ANOVA with repeated measurements on the time factor is proposed as a suitable statistical method for the analysis of agonist-dependent GPCR activation MD simulations. Inclusion of time factor in the ANOVA model is consistent with the time-dependent nature of MD. Ligand and lipid factors measure agonist and lipid influence on receptor activation. Previously reported MD simulations of adenosine A2a receptor (A2aR) are reanalyzed with this statistical method. TM6–TM3 and TM7–TM3 distances are selected as dependent variables in the ANOVA model. The ligand factor includes the presence or absence of adenosine whereas the lipid factor considers DOPC or DOPG lipids. Statistical analysis of MD simulations shows the efficacy of adenosine and the effect of the membrane lipid composition. Subsequent application of the statistical methodology to NECA A2aR agonist, with resulting P values in consistency with its pharmacological profile, suggests that the method is useful for ligand comparison and potentially for dynamic structure-based virtual screening. [ABSTRACT FROM AUTHOR]

GPCRs play critical roles in cell communication. Although GPCRs can form heteromers, their role in signaling remains elusive. Here we used rat metabotropic glutamate (mGlu) receptors as prototypical dimers to study the functional interaction between each subunit. mGluRs can form both constitutive homo- and heterodimers. Whereas both mGlu2 and mGlu4 couple to G proteins, G protein activation is mediated by mGlu4 heptahelical domain (HD) exclusively in mGlu2-4 heterodimers. Such asymmetric transduction results from the action of both the dimeric extracellular domain, and an allosteric activation by the partially-activated non-functional mGlu2 HD. G proteins activation by mGlu2 HD occurs if either the mGlu2 HD is occupied by a positive allosteric modulator or if mGlu4 HD is inhibited by a negative modulator. These data revealed an oriented asymmetry in mGlu heterodimers that can be controlled with allosteric modulators. They provide new insight on the allosteric interaction between subunits in a GPCR dimer. [ABSTRACT FROM AUTHOR]