Molecular dynamics simulations elucidate the role of the F–F′ loop in substrate entry into CYP3A4

CYP3A4 metabolizes a significant proportion of all approved drugs in the human body. However, the mechanism by which substrates access the catalytic center in concert with conformational changes remains unresolved. Here, we captured spontaneous substrate-binding events from outside the enzyme to the catalytic center of CYP3A4 using long-timescale, unbiased MD simulations. During each entry process, the ligand initially resided on the surface of CYP3A4 with the F-F' loop oriented downward for over 5 μs. The F-F' loop then moved upward to permit substrate entry and restricts its exit. After entry, the substrate underwent a conformational rearrangement, while the F-F' loop began to move downward again. One of the final bound poses closely resembled the experimentally determined conformation. The dissociation constant derived from a Markov state model agreed well with experimental data. Overall, our work provides an atomistic, dynamic view of the substrate entry mechanism in CYP3A4.