G-Protein-Coupled Receptor-Membrane Interactions Depend on the Receptor Activation State

Abstract

G-protein-coupled receptors (GPCRs) are the largest family of human membrane proteins and serve as primary targets of approximately one-third of currently marketed drugs. In particular, adenosine A₁ receptor (A₁ AR) is an important therapeutic target for treating cardiac ischemia-reperfusion injuries, neuropathic pain, and renal diseases. As a prototypical GPCR, the A₁ AR is located within a phospholipid membrane bilayer and transmits cellular signals by changing between different conformational states. It is important to elucidate the lipid-protein interactions in order to understand the functional mechanism of GPCRs. Here, all-atom simulations using a robust Gaussian accelerated molecular dynamics (GaMD) method were performed on both the inactive (antagonist bound) and active (agonist and G-protein bound) A₁ AR, which was embedded in a 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) lipid bilayer. In the GaMD simulations, the membrane lipids played a key role in stabilizing different conformational states of the A₁ AR. Our simulations further identified important regions of the receptor that interacted distinctly with the lipids in highly correlated manner. Activation of the A₁ AR led to differential dynamics in the upper and lower leaflets of the lipid bilayer. In summary, GaMD enhanced simulations have revealed strongly coupled dynamics of the GPCR and lipids that depend on the receptor activation state. © 2019 Wiley Periodicals, Inc.

Keywords: G-protein-coupled receptors; Gaussian accelerated molecular dynamics; adenosine A1 receptor; enhanced sampling; protein-lipid interactions.

Figure 1:

Comparison of structural flexibility of the inactive and active A1AR systems obtained from dihedral GaMD simulations: (A) Root-mean-square fluctuations (RMSFs) of the inactive PSB36-A1AR complex. (B) RMSFs of the active ADO-A1AR-Gi protein complex. A color scale of 0 Å (blue) to 3 Å (red) was used.

Figure 2:

The -S_CD order parameters calculated for sn-2 acyl chains of POPC lipids in different simulation systems: (A) Inactive A1AR using dihedral-boost GaMD, (B) Active A1AR using dihedral-boost GaMD, (C) Inactive A1AR using dual-boost GaMD and (D) Active A1AR using dual-boost GaMD. Red diamond lines represent the average -S_CD order parameters for the cytoplasmic lower leaflet and blue diamond lines for the extracellular upper leaflet.

Figure 3:

Free energy profiles of the extracellular upper leaflet of membrane in different simulation systems regarding the number of lipids within 5 Å of the receptor TM6 and the receptor R3.50 – E6.30 distance: (A) Inactive A₁AR using dihedral-boost GaMD, (B) Active A₁AR using dihedral-boost GaMD, (C) Inactive A₁AR using dual-boost GaMD and (D) Active A₁AR using dual-boost GaMD. The R3.50 – E6.30 distance is ~7 Å in the inactive A1AR and increases to ~17 Å in the active A1AR due to outward movement of TM6.

Figure 4:

Free energy profiles of the cytoplasmic lower leaflet of membrane in different simulation systems regarding the number of lipids within 5 Å of the receptor TM6 and the receptor R3.50 – E6.30 distance: (A) Inactive A₁AR using dihedral-boost GaMD, (B) Active A₁AR using dihedral-boost GaMD, (C) Inactive A₁AR using dual-boost GaMD and (D) Active A₁AR using dual-boost GaMD. The R3.50 – E6.30 distance is ~7 Å in the inactive A1AR and increases to ~17 Å in the active A1AR due to outward movement of TM6.

Figure 5:

Minimum energy states of POPC lipid interacting with the positively-charged lysine residues in TM6 of the receptor obtained from dihedral GaMD simulations. (A) One POPC molecule in the upper leaflet interacts with one lysine residue (K265^ECL3) of the inactive A₁AR. (B) Three POPC molecules (POPC1, POPC2, POPC3) in the lower leaflet interact with four Lysine residues (K263^6.25, K267^6.29, K270^6.32 and K273^6.35) of the active A₁AR. The receptor TM6 is colored in gray.

Figure 6:

Dynamic correlation matrices calculated for lipids in the extracellular upper leaflet with residues in the A1AR in different simulation systems: (A) Inactive A₁AR using dihedral-boost GaMD, (B) Active A₁AR using dihedral-boost GaMD, (C) Inactive A₁AR using dual-boost GaMD and (D) Active A₁AR using dual-boost GaMD. The Cα atoms of the receptor and phosphorous atoms in the lipid head groups were used for calculating the correlation matrices here. Similar results were obtained using the C₈ and C₁₈ atoms in the lipid hydrophobic tails as shown in Figure S5. The receptor ICL1, ICL2 and ICL3 represent intracellular loops between TM helices 1-2, 3-4, and 5-6 respectively. Similarly, the receptor ECL1, ECL2 and ECL3 represent extracellular loops between TM helices 2-3, 4-5, and 6-7 respectively.

Figure 7:

Dynamic correlation matrices calculated for lipids in the intracellular lower leaflet with residues in the A1AR in different simulation systems: (A) Inactive A₁AR using dihedral-boost GaMD, (B) Active A₁AR using dihedral-boost GaMD, (C) Inactive A₁AR using dual-boost GaMD and (D) Active A₁AR using dual-boost GaMD. The Cα atoms of the receptor and phosphorous atoms in the lipid head groups were used for calculating the correlation matrices here. Similar results were obtained using the C₈ and C₁₈ atoms in the lipid hydrophobic tails as shown in Figure S5. The receptor ICL1, ICL2 and ICL3 represent intracellular loops between TM helices 1-2, 3-4, and 5-6 respectively. Similarly, the receptor ECL1, ECL2 and ECL3 represent extracellular loops between TM helices 2-3, 4-5, and 6-7 respectively.