Spontaneous Ligand Access Events to Membrane-Bound Cytochrome P450 2D6 Sampled at Atomic Resolution

André Fischer,Martin Smieško

doi:10.1038/s41598-019-52681-w

Abstract

The membrane-anchored enzyme Cytochrome P450 2D6 (CYP2D6) is involved in the metabolism of around 25% of marketed drugs and its metabolic performance shows a high interindividual variation. While it was suggested that ligands access the buried active site of the enzyme from the membrane, no proof from unbiased simulations has been provided to support this hypothesis. Laboratory experiments fail to capture the access process which is suspected to influence binding kinetics. Here, we applied unbiased molecular dynamics (MD) simulations to investigate the access of ligands to wild-type CYP2D6, as well as the allelic variant CYP2D6*53. In multiple simulations, substrates accessed the active site of the enzyme from the protein-membrane interface to ultimately adopt a conformation that would allow a metabolic reaction. We propose the necessary steps for ligand access and the results suggest that the increased metabolic activity of CYP2D6*53 might be caused by a facilitated ligand uptake.

Highlights

Cytochrome P450 enzymes (CYPs) are essential proteins involved in the detoxification of foreign compounds reaching the human body
The results presented here revealed the atomic mechanism of ligand uptake to the buried active site of membrane-anchored Cytochrome P450 2D6 (CYP2D6) from the protein-membrane interface
We show that the access process is linked to conformational adaptations of the protein backbone that can occur either in close proximity or in significant distance from the ligand molecule

Summary

Introduction

Cytochrome P450 enzymes (CYPs) are essential proteins involved in the detoxification of foreign compounds reaching the human body. The active site of CYPs is located in a buried cavity inside the enzyme that is connected to the surrounding environment by tunnels[2,8,9,10,11,12,13,14]. These tunnels are believed to influence both the poorly understood substrate specificity and binding kinetics of CYPs1,11,13,15. Access of ligands to mammalian CYPs is poorly understood and could not yet be observed in its full complexity in unbiased simulations. No unbiased MD protocol was applied to confirm this hypothesis in a mammalian CYP, let alone in CYP2D6

Methods

Results

Conclusion