Active-State Models of Ternary GPCR Complexes: Determinants of Selective Receptor-G-Protein Coupling

Based on the recently described crystal structure of the β2 adrenergic receptor - Gs-protein complex, we report the first molecular-dynamics simulations of ternary GPCR complexes designed to identify the selectivity determinants for receptor-G-protein binding. Long-term molecular dynamics simulations of agonist-bound β2AR-Gαs and D2R-Gαi complexes embedded in a hydrated bilayer environment and computational alanine-scanning mutagenesis identified distinct residues of the N-terminal region of intracellular loop 3 to be crucial for coupling selectivity. Within the G-protein, specific amino acids of the α5-helix, the C-terminus of the Gα-subunit and the regions around αN-β1 and α4-β6 were found to determine receptor recognition. Knowledge of these determinants of receptor-G-protein binding selectivity is essential for designing drugs that target specific receptor/G-protein combinations

An automated Calculation Pipeline for Differential Pair Interaction Energies with Molecular Force Fields using the Tinker Molecular Modeling Package

An automated pipeline for comprehensive calculation of intermolecular interaction energies based on molecular force-fields using the Tinker molecular modelling package is presented. Starting with non-optimized chemically intuitive monomer structures, the pipeline allows the approximation of global minimum energy monomers and dimers, configuration sampling for various monomer-monomer distances, estimation of coordination numbers by molecular dynamics simulations, and the evaluation of differential pair interaction energies. The latter are used to derive Flory-Huggins parameters and isotropic particle-particle repulsions for Dissipative Particle Dynamics (DPD). The computational results for force fields MM3, MMFF94, OPLS-AA and AMOEBA09 are analyzed with Density Functional Theory (DFT) calculations and DPD simulations for a mixture of the non-ionic polyoxyethylene alkyl ether surfactant C10E4 with water to demonstrate the usefulness of the approach

Active-State Model of a Dopamine D2 Receptor - Gai Complex Stabilized by Aripiprazole-Type Partial Agonists

Partial agonists exhibit a submaximal capacity to enhance the coupling of one receptor to an intracellular binding partner. Although a multitude of studies have reported different ligand-specific conformations for a given receptor, little is known about the mechanism by which different receptor conformations are connected to the capacity to activate the coupling to G-proteins. We have now performed molecular-dynamics simulations employing our recently described active-state homology model of the dopamine D2 receptor-Gai protein-complex coupled to the partial agonists aripiprazole and FAUC350, in order to understand the structural determinants of partial agonism better. We have compared our findings with our model of the D2R-Gai-complex in the presence of the full agonist dopamine. The two partial agonists are capable of inducing different conformations of important structural motifs, including the extracellular loop regions, the binding pocket and, in particular, intracellular G-protein-binding domains. As G-protein-coupling to certain intracellular epitopes of the receptor is considered the key step of allosterically triggered nucleotide-exchange, it is tempting to assume that impaired coupling between the receptor and the G-protein caused by distinct ligand-specific conformations is a major determinant of partial agonist efficacy

In Silico Adoption of an Orphan Nuclear Receptor NR4A1

A 4.1μs molecular dynamics simulation of the NR4A1 (hNur77) apo-protein has been undertaken and a previously undetected druggable pocket has become apparent that is located remotely from the ‘traditional’ nuclear receptor ligand-binding site. A NR4A1/bisindole ligand complex at this novel site has been found to be stable over 1 μs of simulation and to result in an interesting conformational transmission to a remote loop that has the capacity to communicate with a NBRE within a RXR-α/NR4A1 heterodimer. Several features of the simulations undertaken indicate how NR4A1 can be affected by alternate-site modulators

<i>In Silico</i> adoption of an orphan nuclear receptor NR4A1

A 4.1 μs molecular dynamics simulation of the NR4A1 (hNur77) apo-protein has been undertaken and a previously undetected druggable pocket has become apparent that is located remotely from the 'traditional' nuclear receptor ligand-binding site. A NR4A1/bis-indole ligand complex at this novel site has been found to be stable over 1 μs of simulation and to result in an interesting conformational transmission to a remote loop that has the capacity to communicate with a NBRE within a RXR-α/NR4A1 heterodimer. Several features of the simulations undertaken indicate how NR4A1 can be affected by alternate-site modulators

Alignment of the amino-acid contacts between receptors and G-proteins.

<p>Individual alignments for the receptors and the G-proteins are shown. A colored background indicates that the residue forms contacts to other amino acids (yellow: 1 or 2 contacts; green: 3 or 4 contacts; blue: at least 5 contacts). Red letters indicate residues involved in ionic interactions, whereas dotted underlines indicate contacts present in the crystal structure of β2AR-Gα_s.</p

Initial conformation of dopamine in the D2R-Gα_i-complexes.

<p>The backbone of D2R is shown as green ribbon, with important amino acids (indicated as green sticks) that stabilize the ligand dopamine in its initial conformation. Dopamine is represented as orange sticks and stabilized by ionic interactions to D114^3.32 and hydrogen bonds to S193^5.42 and S197^5.46. The second conformation of residue H393^6.55 is shown as red sticks.</p

Dihedral angle of His393^6.55 in the D2R-Gα_i-complexes.

<p>On the left side of the figure, the dihedral angle of residue His393^6.55 (atoms: C-CA-CB-CG) is depicted as green and red lines for the D2^DownR-Gα_i- and the D2^UpR-Gα_i-simulations, respectively. The right column shows representative snapshots taken from the D2R-Gα_i-simulations and visualizes the interactions of residue His393^6.55 with amino acids S193^5.43 and Y408^7.35 depending on its dihedral angle (orange: state 1; purple: state 2; dark-cyan: state 3). Helices 5, 6 and 7 are shown as ribbons, whereas the amino acids are represented as sticks. Additionally, state 2 shows the conformation of residue His^6.55 taken from the crystal structure of the dopaminergic D₃ receptor, as grey sticks.</p

Alignment of contacts areas to G-proteins of aminergic GPCRs.

<p>Amino acids of the receptors supposed to determine selective coupling between β2AR-Gα_s and D2R-Gα_i are highlighted in dark-blue and dark-green, respectively. A brighter color, light-blue or light-green, is attributed to amino acids, which show an identical sequence compared to β2AR and D2R, or, in the case of arginine and lysine residues, a similar sequence, whereas a grey color points to sequence differences. Amino acids, which appear in the interface of β2AR-Gα_s and D2R-Gα_i, but are not supposed to determine selective coupling, are colored in yellow.</p