Ligand and Protein Dynamics of CYP3A4

Abstract

Cytochrome P450 3A4 (CYP3A4) dominates drug metabolism in humans. Examples of promiscuity (binding and catalytic) and allosterism in CYP3A4 are ubiquitous but poorly understood. This work is dedicated to improving our understanding of the linkage between ligand binding and protein conformational dynamics with theoretical considerations of their impact on CYP3A4 catalysis using biophysical and theoretical methodologies. Specifically, the results herein demonstrate the complexity (requiring more than 2-states) in the ligand binding interactions for drugs and small molecules. Chapter 2 details the ligand-induced effects on conformational dynamics of CYP3A4 via hydrogen-deuterium exchange mass spectrometry for the allosteric effector, midazolam (MDZ). The MDZ-induced effects on the structural dynamics of CYP3A4 are not confined to the active site and they include regions remote from the heme active center. The results suggest the location of the allosteric MDZ binding site. Chapter 3 details theoretical aspects of multi-state ligand binding mechanisms wherein full kinetic derivations are presented with emphasis on methods for differentiating them. The results delineate the kinetic and thermodynamic signatures for various multi-state binding mechanisms through a global analytical approach. In addition, a novel experimental approach is presented wherein ligand dissociation is used to further distinguish ligand binding mechanisms when coupled with the more traditional ligand binding approach. In Chapter 4, the mechanism of ligand binding for three small molecules (1,2,3-triazole, imidazole, and cyanide) binding to CYP3A4 is characterized via UV/Vis stopped-flow spectroscopy using the analytical approach described in Chapter 3. Assignment of a ligand binding mechanism to kinetic data provides an indirect measure of the relationship between conformational dynamics and ligand binding mechanism. The results show that, even for small molecules, the binding mechanism is complex, requiring interrogation of 4-state binding mechanisms to adequately describe the data. The recovered ligand binding mechanism (4-state induced fit) is consistent with the observed heterogeneity reported by other equilibrium based spectroscopic methods. The functional implications for the ligand binding interactions will be presented.