Induced Fit Describes Ligand Binding to Membrane-Associated Cytochrome P450 3A4

Area of Science:

• Biochemistry
• Enzymology
• Pharmacology

Background:

• Cytochrome P450 3A4 (CYP3A4) is crucial for metabolizing drugs and xenobiotics.
• Understanding CYP3A4 ligand binding is key to predicting and preventing adverse drug-drug interactions.
• The precise mechanisms of CYP3A4's ligand binding, specifically conformational changes, remain incompletely understood.

Purpose of the Study:

• To investigate whether conformational selection (CS) or induced fit (IF) best describes ligand binding to CYP3A4.
• To determine the kinetic mechanisms underlying the binding of four irreversible inhibitors to CYP3A4.

Main Methods:

• Utilized highly monodisperse CYP3A4 embedded in anionic lipoprotein nanodiscs.
• Employed stopped-flow UV-visible absorption spectroscopy to monitor binding kinetics of azamulin, retapamulin, pleuromutilin, and mibrefadil.
• Applied global fitting and kinetic analyses to differentiate between CS and IF models.

Main Results:

• Ligand binding to CYP3A4 nanodiscs followed a single-site binding model with multiple kinetic steps.
• Observed trends in relaxation rates were ambiguous, but global fitting favored an induced fit (IF) mechanism.
• A transient complex formation with mibrefadil provided definitive evidence for IF in CYP3A4 ligand binding.

Conclusions:

• Kinetic data strongly support an induced fit (IF) mechanism for ligand binding to CYP3A4.
• CYP3A4's ability to accommodate diverse substrates likely involves conformational changes post-ligand binding.
• This study provides critical kinetic insights into the functional flexibility of CYP3A4.