Membrane-embedded substrate recognition by cytochrome P450 3A4

John C Hackett

doi:10.1074/jbc.ra117.000961

Abstract

Cytochrome P450 3A4 (CYP3A4) is the dominant xenobiotic-metabolizing enzyme in the liver and intestine and is involved in the disposition of more than 50% of drugs. Because of its ability to bind multiple substrates, its reaction kinetics are complex, and its association with the microsomal membrane confounds our understanding of how this enzyme recognizes and recruits diverse substrates. Testosterone (TST) hydroxylation is the prototypical CYP3A4 reaction, displaying positive homotropic cooperativity with three binding sites. Here, exploiting the capability of accelerated molecular dynamics (aMD) to sample events in the millisecond regime, I performed >25-μs aMD simulations in the presence of three TST molecules. These simulations identified high-occupancy surface-binding sites as well as a pathway for TST ingress into the CYP3A4 active site originating in the membrane. Adaptive biasing force analysis of the latter pathway revealed a metastable intermediate that could constitute a third binding site at high TST concentrations. Prompted by the observation that interactions between TST and the G'-helix mobilize the ligand into the active site, a free-energy analysis of TST distribution in the membrane was conducted and revealed that the depth of the G'-helix is optimal for extracting TST. In summary, these simulations confirm separate, but adjacent substrate-binding sites within the enzyme and the existence of an auxiliary TST-binding site. The broader impact of these simulations is that they support a mechanism in which cytochromes P450 directly recruit membrane-solubilized substrates.

Highlights

The cytochromes P450 metabolize most drugs and environmental xenobiotics to which humans are exposed
Following the initial 50 ns of conventional molecular dynamics simulations, three TST molecules were randomly added to the water surrounding the Cytochrome P450 3A4 (CYP3A4) catalytic domain, and the system was equilibrated for an additional 100 ns in the constant temperature and pressure (NPT) ensemble
The results presented represent the first simulation of ligand binding to a cytochrome P450 from solution

Summary

Introduction

The cytochromes P450 metabolize most drugs and environmental xenobiotics to which humans are exposed. In view of the evidence that membrane interactions modulate CYP3A4 function, it is important that they be included in models, experimental or theoretical, aimed at understanding the functional dynamics of this important enzyme. The amphipathic nature of CYP3A4 substrates has led to speculation that the enzyme harvests them from the membrane; neither experimental nor theoretical evidence in direct support of this mechanism are available. The ideal computational approach to help fill this knowledge gap would permit simulation of the necessary time scales for a ligand to sufficiently explore the protein surface to identify optimal binding site(s). AMD is extended to CYP3A4 to simulate TST binding to the enzyme free of a predefined reaction coordinate. AMD successfully identifies probable locations of auxiliary binding sites that reflect experimental observations and reveals a membrane-embedded pathway for TST entrance into the active site. Freeenergy analyses of the ingress pathway provides a structural basis for the kinetic behavior of CYP3A4 mutants and insight into the mechanism for exclusion of progesterone from the active site in CYP3A4 crystal structures

Methods

Results

Conclusion