Probing Ligand Dynamics in Membrane-Bound Cytochrome P450 3A4 to Characterize Substrate Access and Product Egress Pathways

Abstract

Cytochrome P450 3A4 (CYP3A4) is the most abundant P450 isoform in the human body, and is responsible for the metabolism of more than 50% of clinically used drugs. Recently, we developed a model for the membrane-bound form of CYP3A4, employing a concerted simulation/experimental approach. From the simulations, we observed that the interaction of CYP3A4 with the membrane favors the opening of pathways leading to the active site, which might play a role in the recruitment of lipophilic ligands from the membrane.Using this membrane-bound model, we performed a microsecond-long MD simulation, providing extended conformational sampling for the protein in the presence of the membrane. From this simulation, 850 frames were then used to perform molecular docking to identify potential access/egress pathways for progesterone, representing a substrate of CYP3A4, and its 6b-hydroxy- form, representing an oxidation product. The combination of the long-time scale MD simulation with molecular docking resulted in 8500 docking poses for each ligand.The results indicate that membrane-bound CYP3A4 can accommodate both ligands in regions connecting the active site to peripheral binding sites close to the membrane, suggesting two putative access pathways from the membrane for progesterone. A series of MD simulations, performed on the most representative docking poses for both ligands, revealed that progesterone is more mobile than the product in the peripheral binding sites, and can reach the active site through the suggested pathways. Once in the active site, progesterone freely rotates and adopts different orientations, which might account for the ability of CYP3A4 to oxidize progesterone at different sites. Finally, the results of the MD simulations of 6b-hydroxy-progesterone suggest a putative egress pathway connecting the active site to the aqueous phase.