GDP Release from the Open Conformation of Gα Requires Allosteric Signaling from the Agonist-Bound Human ß2 Adrenergic Receptor.

G-protein-coupled receptors (GPCRs) transmit signals into the cell in response to ligand binding at its extracellular domain, which is characterized by the coupling of agonist-induced receptor conformational change to guanine nucleotide (GDP) exchange with guanosine triphosphate on a heterotrimeric (αßγ) guanine nucleotide-binding protein (G-protein), leading to the activation of the G-protein. The signal transduction mechanisms have been widely researched in vivo and in silico. However, coordinated communication from stimulating ligands to the bound GDP still remains elusive. In the present study, we used microsecond (µS) molecular dynamic (MD) simulations to directly probe the communication from the ß2 adrenergic receptor (ß2AR) with an agonist or an antagonist or no ligand to GDP bound to the open conformation of the Gα protein. Molecular mechanism-general Born surface area calculation results indicated either the agonist or the antagonist destabilized the binding between the receptor and the G-protein but the agonist caused a higher level of destabilization than the antagonist. This is consistent with the role of agonist in the activation of the G-protein. Interestingly, while GDP remained bound with the Gα-protein for the two inactive systems (antagonist-bound and apo form), GDP dissociated from the open conformation of the Gα protein for the agonist activated system. Data obtained from MD simulations indicated that the receptor and the Gα subunit play a big role in coordinated communication and nucleotide exchange. Based on residue interaction network analysis, we observed that engagement of agonist-bound ß2AR with an α5 helix of Gα is essential for the GDP release and the residues in the phosphate-binding loop, α1 helix, and α5 helix play very important roles in the GDP release. The insights on GPCR-G-protein communication will facilitate the rational design of agonists and antagonists that target both active and inactive GPCR binding pockets, leading to more precise drugs.

Activation Mechanism of Corticotrophin Releasing Factor Receptor Type 1 Elucidated Using Molecular Dynamics Simulations.

The corticotropin-releasing factor receptor type 1 (CRF1R), a member of class B G-protein-coupled receptors (GPCRs), is a good drug target for treating depression, anxiety, and other stress-related neurodisorders. However, there is no approved drug targeting the CRF1R to date, partly due to inadequate structural information and its elusive activation mechanism. Here, by use of the crystal structures of its transmembrane domain (TMD) and the N-terminal extracellular domain (ECD) as a template, a full-length homology model of CRF1R was built and its complexes with peptide agonist urocortin 1 or small molecule antagonist CP-376395 were subjected to all-atom molecular dynamics simulations. We observed well preserved helical contents in the TMD through simulations, while the transmembrane (TM) helices showed clear rearrangements. The TM rearrangement is especially pronounced for the TM6 in the agonist-bound CRF1R system. The observed conformational changes are likely due to breakage of interhelical/inter-regional hydrogen bonds in the TMD. Dynamical network analysis identifies communities with high connections to TM6. Simulations reveal three key residues, Y3566.53, Q3847.49, and L3957.60, which corroborate experimental mutagenesis data, implying the important roles in the receptor activation. The observed large-scale conformational changes are related to CRF1R activation by agonist binding, providing guidance for ligand design.

Binding of agonist WAY-267,464 and antagonist WAY-methylated to oxytocin receptor probed by all-atom molecular dynamics simulations.

AIMS: Non-peptide ligands of oxytocin receptor (OTR) have promising potentialities as therapeutic agents with improved pharmacological properties. WAY-267,464 is a non-peptide agonist which loses its agonist activity when its resorcinol moiety is methylated, yielding a partial antagonist (denoted here, WAY-Methylated). This study attempts to rationalize these opposing activities by comparative analyses of structural dynamicsof OTR in complex with these ligands. MAIN METHODS: Glide extra precision (XP) docking with and without positional constraints was employed to probe alternative binding poses of both WAY-267,464 and WAY-Methylated. The more preferred configuration of each system was subjected to an extended 2 µs MD simulation and the physics-based Molecular Mechanics-Generalized Born Surface Area (MM-GBSA) binding energy was used to rank the complexes with improved accuracy, in addition to empirical-based Glide docking score. Network analysis was performed, and the identified critical residues were cross-referenced with the experimental mutagenesis data. KEY FINDINGS: The added methyl groups in the antagonist WAY-Methylated enhanced hydrophobicity, resulting in a flipped binding pose deeper in the binding pocket. Interestingly, OTR responded to the methylation by stabilizing the initial inactive conformation, decreasing fluctuations and increasing the overall secondary structural composition. Conversely, the agonist WAY-267,464 produced larger fluctuations to allow the receptor to change from the default inactive state to a state of partial activation. These transitions were further supported by the identified critical residues overlapping with experimental mutagenesis data. SIGNIFICANCE: These findings provide insights into the activation mechanism of OTR by WAY-267.464 and its antagonism by WAY-Methylated.