Interactions of Cytochrome P450 3A4 with Phospholipid Bilayer Nanodiscs

Thesis (Ph.D.)--University of Washington, 2016-08

Cytochrome P450s (CYPs) are the major drug-metabolizing enzyme in humans. Cytochrome P450 3A4 (CYP3A4), in particular, is responsible for approximately half of all CYP-mediated drug metabolism. In mammals, drug-metabolizing CYPs are membrane proteins, and molecular dynamics simulations have been performed by several groups in order to probe how the membrane influences the dynamics of the CYP structure. However, the crystal structures used in these simulations were obtained in the absence of the membrane. As such, simulations using the resolved crystal structures may not accurately represent how the membrane affects CYP dynamics and thermal stability in vivo. Experimental techniques, therefore, are necessary in order to arrive at a complete understanding of CYP-membrane interactions. Using phospholipid nanodiscs as a model membrane, stopped-flow spectroscopy was performed in order to investigate the role of the membrane in modulating the dynamics of ligand binding to CYP3A4. Evidence is presented which suggests that some hydrophobic drugs may not be required to partition into the membrane in order to bind the CYP3A4 active site. Differential scanning calorimetry (DSC) was used to probe the effects of lipid composition on CYP3A4 thermal stability. Results indicate that the stability of CYP3A4 is increased upon monomerization by nanodiscs. Furthermore, it is demonstrated that nanodisc lipid composition affects both the thermal stability and ligand binding dynamics of CYP3A4. These results highlight the utility of nanodiscs for studying interactions between mammalian CYPs and the membrane.