Pathways and Mechanism of Caffeine Binding to Human Adenosine A2A Receptor

Abstract

Caffeine (CFF) is a common antagonist to the four subtypes of adenosine G-protein-coupled receptors (GPCRs), which are critical drug targets for treating heart failure, cancer, and neurological diseases. However, the pathways and mechanism of CFF binding to the target receptors remain unclear. In this study, we have performed all-atom-enhanced sampling simulations using a robust Gaussian-accelerated molecular dynamics (GaMD) method to elucidate the binding mechanism of CFF to human adenosine A_2A receptor (A_2AAR). Multiple 500-1,000 ns GaMD simulations captured both binding and dissociation of CFF in the A_2AAR. The GaMD-predicted binding poses of CFF were highly consistent with the x-ray crystal conformations with a characteristic hydrogen bond formed between CFF and residue N6.55 in the receptor. In addition, a low-energy intermediate binding conformation was revealed for CFF at the receptor extracellular mouth between ECL2 and TM1. While the ligand-binding pathways of the A_2AAR were found similar to those of other class A GPCRs identified from previous studies, the ECL2 with high sequence divergence serves as an attractive target site for designing allosteric modulators as selective drugs of the A_2AAR.

Keywords: Gaussian accelerated molecular dynamics; adenosine A2A receptor; caffeine; ligand binding; mechanism; pathways.

FIGURE 1

Gaussian-accelerated molecular dynamics (GaMD) simulations successfully captured both binding and dissociation of caffeine (CFF) in the A_2AAR. (A) Computational model used for simulations of the A_2AAR (blue ribbons) with 10 CFF molecules (orange spheres) placed far away in the solvent. The receptor was inserted in a POPC lipid bilayer (cyan sticks) and solvated in an aqueous solution (cyan) of 0.15 M NaCl. (B) X-ray structure of CFF-bound A_2AAR (PDB: 5MZP). A hydrogen bond is formed between either O11 or O13 atom of CFF with the ND2 atom of the receptor residue N6.55 in two X-ray conformations of the ligand (CFF-A and CFF-B), in which the distance between the N1 atom that connects atoms O11 and O13 in CFF and the ND2 atom of residue N6.55 stays at 5.1 Å. The seven transmembrane (TM) helices I–VII and three extracellular loops (ECL) 1–3 are labeled in the A_2AAR. (C–F) Time courses of the N6.55:ND2–CFF:N1 distance calculated from 63 ns GaMD equilibration and three independent 500–1,000 ns GaMD simulations.

FIGURE 2

2D potential of mean force (PMF) free energy profiles of the A_2AAR–caffeine (CFF) interactions. (A) Two-dimensional (2D) PMF of the distance between receptor residue N6.55 atom ND2 and CFF atom N1 and the ionic lock distance between charge centers of receptor residues R3.50 and E6.30. The low-energy states are labeled in the PMF profile, including the unbound (U1 and U2), intermediate (I), and bound (B1 and B2). (B) 2D PMF of the distance between atom CZ in receptor residue R3.50 and the hydroxyl oxygen atom of residue Y7.53 and the ionic lock distance between charge centers of receptor residues R3.50 and E6.30. The low-energy inactive and intermediate conformational states are labeled. The 3RFM and 5MZP PDB structures of inactive A_2AAR are mapped to the free energy surface as hexagons and stars.

FIGURE 3

Binding and dissociation pathways of caffeine (CFF) in the A_2AAR revealed from the Gaussian-accelerated molecular dynamics (GaMD) simulations. (A) Trace of CFF (orange) binding to the A_2AAR observed in the GaMD equilibration. Starting from free diffusion in the solvent, CFF bound to the orthosteric site of the A_2AAR receptor. (B) Binding of the second CFF (orange) to orthosteric pocket of the A_2AAR observed in GaMD Sim2. The first bound CFF is shown in red. (C) Pathway of CFF that dissociated from orthosteric site of the A_2AAR to the bulk solvent observed in GaMD Sim3. The A_2AAR receptor is shown in blue ribbons and the CFF traces are shown as orange beads. (D) The B1-bound conformational state of CFF was located between ECL2, TM3, TM5, and TM6 with interacting residues F45.52^ECL2, V3.32, M5.38, N5.42, W6.48, L6.51, H6.52, and N6.55. (E) The B2-bound conformational state of CFF was located between ECL2, TM3, TM5, and TM6 with interacting residues V3.32, L3.33, F45.52^ECL2, M5.38, N5.42, and N6.55. CFF formed a hydrogen bond with the receptor residue N6.55 in both the B1 and B2 states. (F) The intermediate (I) conformational state of CFF that was located between ECL2, N-terminus of TM1, and TM2 with interacting residues P1.28, I1.29, S2.65, K153^ECL2, S156^ECL2, Q157^ECL2, and L45.51^ECL2. CFF formed hydrogen bonds with both receptor residues I1.29 and Q157^ECL2. The CFF ligand is represented by sticks with carbon atoms colored in orange for simulation-derived low-energy conformations and pink and purple for two x-ray conformations in the 5MZP PDB structure. The receptor-interacting residues are highlighted in green.

FIGURE 4

Representative “inactive” (green) and “intermediate” (orange) low-energy conformations of the A_2AAR compared with the 5MZP PDB structure (blue). (A) The ionic lock distance between charge centers of residues R3.50 and E6.30 in the 5MZP, inactive, and intermediate conformations are 6.0, 4.4, and 4.3 Å, respectively. (B) The distance between atom CZ in residue R3.50 and hydroxyl oxygen atom of Y7.53 in the 5MZP, inactive, and intermediate conformations are 13.2, 12.4, and 6.0 Å, respectively.