Comparative Study of State-Dependent Cholesterol Binding Sites in Adenosine A _2A and A ₁ Receptors Using Coarse-Grained Molecular Dynamics Simulations in Biologically Relevant Membranes

In this study 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 A ₁ adenosine receptor (A ₁ R). Three different membranes were used, of which a plasma mimetic membrane best matched cholesterol binding sites previously detected in X-ray structures and computationally for the inactive state of A _2A R. Here, cholesterol binds stably between TM5 and TM6 in the intracellular leaf and between the extracellular part of TM6 and TM7. The stability of the identified binding sites to A ₁ R with CG MD simulations were further investigated using potential of mean force calculations combined with umbrella sampling.

Cholesterol binding to certain cavities in active or inactive state can stabilize a conformation of active or inactive state respectively and is important to identify these cavities. For the active state the cholesterol binding sites have much longer residence times compared to the inactive state for both A _2A R and A ₁ R.

We observed for the active A ₁ R two cholesterol binding sites in the extracellular membrane leaf in a cavity between TM2/TM3 and along TM3 in the intracellular leaf. We found a partially overlapped binding area between TM6 and TM7 in the extracellular membrane leaf for active state and inactive A ₁ R state which can be antagonized by cholesterol molecules although the site has a much longer residence time for active A ₁ R. For the active A _2A R we predicted a high residence time cholesterol position between TM1 and TM7 in the middle region of TMD which is different from the binding sites calculated in active or inactive A ₁ R and for the inactive A _2A R. For the inactive A _2A R cholesterol binds to the same extracellular area between TM6 and TM7 as in the inactive A ₁ R.

The differences in stable, long residence time cholesterol binding sites between active and inactive states of A ₁ R and for A _2A R can be important for functional activity and orthosteric agonist or antagonist affinity and can be used for the design of allosteric modulators which can bind through lipid pathways.