Fig 1.
Structural comparison of DOPG and DOPC lipid headgroups.
DOPG (A) headgroup is composed of a neutral glycerol group (red box) bound to the phosphate group (yellow box). DOPC (B) headgroup is composed of a positively-charged choline group bound (blue box) to the phosphate group (yellow box). DOPC and DOPG contain identical 18:1 fatty-acid chains (R).
Fig 2.
Docking of adenosine in the inactive crystal structure of adenosine A2a receptor (A2aR).
A) Molecular structure of adenosine. Comparison of B) co-crystallized adenosine (lime) in agonist-bound A2aR crystal structure (PDB entry: 2YDO, light green), and C) docked adenosine (magenta) in the inactive crystal structure of A2aR (PDB entry: 4EIY, pink). Selected residues participating in ligand binding are displayed. ECL2 and TM helices 1, 5–7 are labelled.
Fig 3.
Stabilization of the inactive conformation of apo A2aR in DOPC membrane.
A) Superposition of the inactive-state crystal structure of A2aR (PDB entry: 4EIY, pink) with docked adenosine and an MD-generated apo conformation achieved within a DOPC membrane (magenta, belonging to replica #2 from 1.7 μs) showing B) and C) selected residues delineating the orthosteric pocket. ECL2 and TM helices are labelled where applicable. D) Fluctuation of the distance between TM3-TM7 (from Cα atoms of R1023.50 and Y2887.53, respectively) during MD simulations, starting from the inactive crystal structure (PDB entry: 4EIY). E) Fluctuation of the distance between TM3-TM6 (from Cα atoms of R1023.50 and E2286.30, respectively). F) and G) Vertical movement of ECL2 and TM3, respectively. MD simulations are performed in quadruplicate. Corresponding flat-lines are included to show the observed distance in the active (PDB entry: 6GDG) and inactive (PDB entry: 4EIY) A2aR crystal structures.
Fig 4.
Transition towards an intermediate conformation of adenosine-bound A2aR in DOPC membrane.
A) Superposition of the intermediate state crystal structure of A2aR (PDB entry: 2YDO, light green) and an MD-generated conformation achieved within a DOPC membrane (blue, belonging to replica #3 from 1.5 μs) bound to adenosine, showing B) and C) protein-agonist interactions in the orthosteric pocket with adenosine atoms displayed as spheres. ECL2 and TM helices are labelled where applicable. D) RMSD of bound adenosine (ADN) (calculated with respect to initial docking pose). E) Conformational fluctuation (RMSF) of adenosine. F) Distance between TM3-TM7 (from Cα atoms of R1023.50 and Y2887.53, respectively) during MD simulations starting from the inactive crystal structure (PDB entry: 4EIY). G) Distance between TM3-TM6 (from Cα atoms of R1023.50 and E2286.30, respectively). MD simulations are performed in quadruplicate. Corresponding flat-lines show the observed distance in the active (PDB entry: 6GDG) and inactive (PDB entry: 4EIY) A2aR crystal structures.
Fig 5.
Transition towards an alternative intermediate conformation of apo A2aR in a DOPG membrane.
A) Comparison of the intermediate state crystal structure of A2aR (PDB entry: 2YDO, light green) with bound adenosine and an MD-generated apo conformation achieved within a DOPG membrane (orange, belonging to replica #1 from 1.6 μs), showing B) and C) selected residues delineating the orthosteric pocket. ECL2 and TM helices are labelled where applicable. D) Distance between TM3-TM7 (from Cα atoms of R1023.50 and Y2887.53, respectively) during MD simulations, starting from the inactive crystal structure (PDB entry: 4EIY). E) Distance between TM3-TM6 (from Cα atoms of R1023.50 and E2286.30, respectively). F) and G) Vertical movement of ECL2 and TM3, respectively. MD simulations are performed in quadruplicate. Corresponding flat-lines show the observed distance in the active (PDB entry: 6GDG) and inactive (PDB entry: 4EIY) A2aR crystal structures.
Fig 6.
Membrane thickness and allosteric protein-lipid interactions during MD simulations of A2aR.
Average membrane thickness measurements across 2 μs MD simulations of A2aR in A) DOPC with bound adenosine (replica #1), B) DOPG in apo (replica #2), and C) DOPG with bound adenosine (replica #1). D-G) Specific intracellular protein-lipid interactions of adenosine-bound A2aR in a DOPG membrane during 2.0 μs MD simulation (replica #1). D) Allosteric interaction between polar residues on TM1 (W291.55 and W321.58) in A2aR (green) with a DOPG lipid (gold). Electrostatic and H-bond interactions between charged/polar residues on: E) TM4 and ICL2 (R11134.52, R1204.41), F) TM6 (Q2266.28, H2306.32, K2336.35) and G) TM5 (R1995.60, R2065.67) with DOPG lipids (gold).
Fig 7.
Protein-lipid allosteric interaction with the ionic-lock in MD simulations of A2aR in a DOPG membrane.
A) Intracellular view of A2aR where ionic-lock residue R1023.50 electrostatically interacts with a DOPG lipid, which intrudes between TM6 and TM7 into the G protein binding-site in apo state (orange) (belonging to replica #4 from 1.8 μs), and B) with bound adenosine (green) (belonging to replica #2 from 1.6 μs). Protein-lipid interaction distance over time between R1023.50 sidechain and lipid phosphate group in four replicas of A2aR in DOPG membrane in C) apo state and D) adenosine-bound (ADN), respectively.
Fig 8.
Transition towards an active-like state of A2aR in MD simulations with bound adenosine in a DOPG membrane.
A) Comparison of the MD-generated conformation of A2aR bound to adenosine (ADN) within a DOPG membrane (green, belonging to replica #2 from 0.7 μs) with the active crystal structure of A2aR with bound NECA (brown, PDB entry: 6GDG), showing B) and C) protein-agonist interactions in the orthosteric pocket with adenosine and NECA atoms displayed as spheres. ECL2 and TM helices labelled where applicable. D) RMSD of bound adenosine (calculated with respect to initial docking pose). E) Conformational fluctuation (RMSF) of adenosine. F) Distance between TM3-TM7 (from Cα atoms of R1023.50 and Y2887.53, respectively) during MD simulations starting from the inactive crystal structure (PDB entry: 4EIY). G) Distance between TM3-TM6 (from Cα atoms of R1023.50 and E2286.30, respectively). MD simulations are performed in quadruplicate. Corresponding flat-lines show the observed distance in the active (PDB entry: 6GDG) and inactive (PDB entry: 4EIY) A2aR crystal structures.
Fig 9.
Comparison of water-mediated polar networks in the core of A2aR during MD simulations.
Average water density of A) apo A2aR in DOPC membrane, and B) A2aR bound to adenosine within a DOPG membrane (green) over respective replica #1 trajectories. Respective water molecule distribution snapshots at C) 1.5 μs and D) 1.3 μs. Water-mediated hydrogen bonds are represented as dotted lines.
Fig 10.
The two-dimensional collective variable (CV) space of A2aR activation and receptor conformations during respective MD simulations: A) apo or with bound adenosine (ADN); B) with bound NECA. CVs correspond to distances between ionic lock residues R3.50 and E6.30 (TM3-TM6), and residues R3.50 and Y7.53 (TM3-TM7). The reference distances from inactive, intermediate and active crystal structures are shown as blue, cyan, and green dots, respectively. Frequency of receptor conformation is represented by a “heat” scale (low: black, high: yellow).
Fig 11.
The MD-generated receptor conformation of A2aR bound to adenosine in DOPG membrane is able to bind co-crystallized Gs-alpha protein in the same way as the active-state crystal structure (PDB id: 6GDG).
A) The crystal structure of the active state of A2aR (brown) bound to its co-crystallized Gs-alpha protein (pink, PDB id: 6GDG) and its re-docked Gs-alpha subunit superimposed (gold). B) MD-generated active-like conformation of A2aR bound to adenosine in a DOPG membrane (green, belonging to replica #2 from 1.6 μs) docks Gs-alpha protein (gold) in similar fashion to the active crystal (pink). C) Intermediate conformation of A2aR, bound to adenosine in DOPC membrane (blue, belonging to replica #4 from 1.3 μs) fails to properly dock Gs-alpha protein (gold) compared to its active crystal position (pink). D) Intermediate conformation of apo A2aR in DOPG membrane (red, belonging to replica #1 from 1.6 μs) partially docks Gs-alpha protein (gold) compared to its active crystal position (pink).