Comparative Study of Receptor-, Receptor State-, and Membrane-Dependent Cholesterol Binding Sites in A 2A and A 1 Adenosine Receptors Using Coarse-Grained Molecular Dynamics Simulations.

We used coarse-grained molecular dynamics (CG MD) simulations to study protein-cholesterol interactions for different activation states of the A 2A adenosine receptor (A 2A R) and the A 1 adenosine receptor (A 1 R) and predict new cholesterol binding sites indicating amino acid residues with a high residence time in three biologically relevant membranes. Compared to 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine (POPC)-cholesterol and POPC-phosphatidylinositol-bisphosphate (PIP 2 )-cholesterol, the plasma mimetic membrane best described the cholesterol binding sites previously detected for the inactive state of A 2A R and revealed the binding sites with long-lasting amino acid residues. We observed that using the plasma mimetic membrane and plotting residues with cholesterol residence time ≥2 μs, our CG MD simulations captured most obviously the cholesterol-protein interactions. For the inactive A 2A R, we identified one more binding site in which cholesterol is bound to residues with a long residence time compared to the previously detected, for the active A 1 R, three binding sites, and for the inactive A 1 R, two binding sites. We calculated that for the active states, cholesterol binds to residues with a much longer residence time compared to the inactive state for both A 2A R and A 1 R. The stability of the identified binding sites to A 1 R or A 2A R with CG MD simulations was additionally investigated with potential of mean force calculations using umbrella sampling. We observed that the binding sites with residues to which cholesterol has a long residence time in A 2A R have shallow binding free energy minima compared to the related binding sites in A 1 R, suggesting a stronger binding for cholesterol to A 1 R. The differences in binding sites in which cholesterol is stabilized and interacts with residues with a long residence time between active and inactive states of A 1 R and A 2A R can be important for differences in functional activity and orthosteric agonist or antagonist affinity and can be used for the design of allosteric modulators, which can bind through lipid pathways. We observed a stronger binding for cholesterol to A 1 R (i.e., generally higher association rates) compared to A 2A R, which remains to be demonstrated. For the active states, cholesterol binds to residues with much longer residence times compared to the inactive state for both A 2A R and A 1 R. Taken together, binding sites of active A 1 R may be considered as promising allosteric targets.