The Molecular Mechanism Underlying Ligand Binding to the Membrane-Embedded Site of a G‑Protein-Coupled Receptor

The crystal structure of P2Y₁ receptor (P2Y₁R), a class A GPCR, revealed a special extra-helical site for its antagonist, BPTU, which locates in-between the membrane and the protein. However, due to the limitation of crystallization experiments, the membrane was mimicked by use of detergents, and the information related to the binding of BPTU to the receptor in the membrane environment is rather limited. In the present work, we conducted a total of ∼7.5 μs all-atom simulations in explicit solvent using conventional molecular dynamics and multiple enhanced sampling methods, with models of BPTU and a POPC bilayer, both in the absence and presence of P2Y₁R. Our simulations revealed that BPTU prefers partitioning into the interface of polar/lipophilic region of the lipid bilayer before associating with the receptor. Then, it interacts with the second extracellular loop of the receptor and reaches the binding site through the lipid–receptor interface. In addition, by use of funnel-metadynamics simulations which efficiently enhance the sampling of bound and unbound states, we provide a statistically accurate description of the underlying binding free energy landscape. The calculated absolute ligand–receptor binding affinity is in excellent agreement with the experimental data (Δ<i>G</i>_{b0_theo} = −11.5 kcal mol^–1, Δ<i>G</i>_{b0_exp}= −11.7 kcal mol^–1). Our study broadens the view of the current experimental/theoretical models and our understanding of the protein–ligand recognition mechanism in the lipid environment. The strategy used in this work is potentially applicable to investigate ligands association/dissociation with other membrane-embedded sites, allowing identification of compounds targeting membrane receptors of pharmacological interest