Peripheral Ligand-binding Site in Cytochrome P450 3A4 Located with Fluorescence Resonance Energy Transfer (FRET)

Dmitri R Davydov,Jessica A.O Rumfeldt,Elena V Sineva,Harshica Fernando,Nadezhda Y Davydova,James R Halpert

doi:10.1074/jbc.m111.325654

Abstract

The mechanisms of ligand binding and allostery in the major human drug-metabolizing enzyme cytochrome P450 3A4 (CYP3A4) were explored with fluorescence resonance energy transfer (FRET) using a laser dye, fluorol-7GA (F7GA), as a model substrate. Incorporation into the enzyme of a thiol-reactive FRET probe, pyrene iodoacetamide, allowed us to monitor the binding by FRET from the pyrene donor to the F7GA acceptor. Cooperativity of the interactions detected by FRET indicates that the enzyme possesses at least two F7GA-binding sites that have different FRET efficiencies and are therefore widely separated. To probe spatial localization of these sites, we studied FRET in a series of mutants bearing pyrene iodoacetamide at different positions, and we measured the distances from each of the sites to the donor. Our results demonstrate the presence of a high affinity binding site at the enzyme periphery. Analysis of the set of measured distances complemented with molecular modeling and docking allowed us to pinpoint the most probable peripheral site. It is located in the vicinity of residues 217-220, similar to the position of the progesterone molecule bound at the distal surface of the CYP3A4 in a prior x-ray crystal structure. Peripheral binding of F7GA causes a substantial spin shift and serves as a prerequisite for the binding in the active site. This is the first indication of functionally important ligand binding outside of the active site in cytochromes P450. The findings strongly suggest that the mechanisms of CYP3A4 cooperativity involve a conformational transition triggered by an allosteric ligand.

Highlights

Cytochrome P450 3A4 (CYP3A4) can bind several substrate molecules simultaneously and exhibits cooperativity
We demonstrate that the first high affinity substrate binding event takes place outside of the active site
Fluorol-7GA as a Model Fluorescent Substrate of cytochrome P450 3A4 (CYP3A4)— In our search for an appropriate high affinity fluorescent ligand for fluorescence resonance energy transfer (FRET)-based studies of CYP3A4 cooperativity, we identified the laser dye F7GA as an allosteric ligand and potential substrate of CYP3A4

Summary

Introduction

Cytochrome P450 3A4 (CYP3A4) can bind several substrate molecules simultaneously and exhibits cooperativity. Analysis of the set of measured distances complemented with molecular modeling and docking allowed us to pinpoint the most probable peripheral site. It is located in the vicinity of residues 217–220, similar to the position of the progesterone molecule bound at the distal surface of the CYP3A4 in a prior x-ray crystal structure. Peripheral binding of F7GA causes a substantial spin shift and serves as a prerequisite for the binding in the active site This is the first indication of functionally important ligand binding outside of the active site in cytochromes P450. The findings strongly suggest that the mechanisms of CYP3A4 cooperativity involve a conformational transition triggered by an allosteric ligand

Methods

Results

Conclusion