Nanodisc-embedded cytochrome P450 P3A4 binds diverse ligands by distributing conformational dynamics to its flexible elements

Cytochrome P450 3A4 (CYP3A4) metabolizes a wide range of drugs and toxins. Interactions of CYP3A4 with ligands are difficult to predict due to promiscuity and conformational flexibility. To better understand CYP3A4 conformational responses to ligands we use hydrogen deuterium exchange mass spectrometry (HDX-MS) to investigate the effect of ligands on nanodisc-embedded CYP3A4. For a subset of CYP3A4-ligand complexes, differences in the low-frequency modes derived by principal component analyses of molecular dynamics trajectories mirrored the HDX-MS results. The effects of ligands are distributed to flexible elements of CYP3A4 between stretches of secondary structure. The largest effects occur in the F- and G-helices, where most ligands increase the flexibility of the F-helix and connecting loops and decrease the flexibility of the C-term of the Ghelix. Most ligands affect the E-F-G, C-D and H-I regions of the protein. Ligand-dependent differences are observed in the A-A' loop, B-C region, E-helix, K-beta 1 region, proximal loop, and C-term loop. Correlated HDX responses were observed in the C-D region and the C-term of the G-helix that were most pronounced for Type II ligands. Collectively, the HDX and molecular dynamics results suggest that CYP3A4 accommodates diverse binding partners by propagating local backbone fluctuations from the binding site onto the flexible regions of the enzyme via long-range interactions that are differentially modulated by ligands. In contrast to the paradigm wherein ligands decrease protein dynamics at their binding site, a wide range of ligands modestly increase CYP3A4 dynamics throughout the protein including effects remote from the active site.

Automated summary

Cytochrome P450 3A4 (CYP3A4) is involved in the metabolism of various drugs and toxins. The interactions between CYP3A4 and ligands are difficult to predict due to its promiscuity and conformational flexibility. Through the use of hydrogen deuterium exchange mass spectrometry (HDX-MS), we investigated the effect of ligands on nanodisc-embedded CYP3A4, and found that ligands have differential effects on the flexibility of different regions of the enzyme. Molecular dynamics simulations showed that ligands can modulate long-range interactions and propagate local backbone fluctuations from the binding site to flexible regions of CYP3A4. Contrary to the common paradigm, a wide range of ligands actually increase the overall dynamics of CYP3A4, including regions remote from the active site.