Focusing the computational microscope on the dynamics of mammalian cytochrome P450s and their redox partners

Jonathan Teuffel, Goutam Mukherjee, Sungho Bosco Han, Sophia Ber, Marcus Elstner, Rebecca C. Wade

Rebecca.wade@h-its.org

Mammalian class II cytochrome P450 (CYP) enzymes are membrane-bound and rely on NADPH-cytochrome P450 reductase (CPR) and cytochrome b5 (CYb5) for the provision of electrons for catalysis. Multiresolution simulations reveal dynamic conformational ensembles of the membrane-bound proteins (1). For the highly flexible CYP 2B4, simulations show how the open conformation of the globular domain facilitates the binding of substrates from and release of products to the membrane, whereas the closed conformation prolongs the residence time of substrates or inhibitors and selectively allows the passage of smaller reactants via the solvent and water channels (2). CYP 17A1 plays a key role in the regulation of steroid hormone biosynthesis that is dependent on both CPR and CYb5 whether CYb5 plays a reductive or an allosteric regulatory role or both remains unclear. We modelled and simulated complexes of CYP 17A1 with CPR and CYb5 to investigate the determinants of electron transfer. We found that the electron transfer reorganization energy (l), a critical parameter for the determination of electron transfer rates according to Marcus theory, could be computed by combining quantum mechanics calculations for the redox-active cofactors with and molecular dynamics simulations of the protein complexes (3). Leveraging this information, we computed interprotein electron transfer rates for simulated ensembles of binary and ternary complexes of CYP 17A1 with CPR and CYb5. Our results indicate how, through rearrangements of the protein complexes on the lipid bilayer, CYb5 may fulfill both regulatory and reductive roles in the catalysis of CYP 17A1.

(1) Mukherjee et al. Comm Biol. (2021) 4(1),55

(2) Han et al. Protein Science (2024) 33(10), e5165

(3) Teuffel et al. J Chem Phys (2025) 162(19),195101.

Cytochrome P450 2U1 (CYP2U1) is an extrahepatic lipid-metabolizing enzyme that is abundantly expressed in the limbic region of the brain and thymus. Sequence alignment of CYP2U1 shows high similarity with CYP2D6 and 2J2, as well as a bulky N-terminus formed by eight proline residues. CYP2U1 is previously known to metabolize long-chain polyunsaturated fatty acids, arachidonic acid and docosahexaenoic acid, as well as lipidated neurotransmitters such as N-arachidonoylserotonin. Its involvement in various diseases and pathological conditions has drawn significant attention. Mutations in the CYP2U1 gene have been linked to hereditary spastic paraplegia (HSP), a genetic neurodegenerative disorder. Despite for the initial characterization, there has been limited knowledge on the biochemistry that governs the enzyme’s activity, as well as its physiological roles in the brain and thymus. Unraveling the biochemistry of CYP2U1 in terms of its role in lipid and drug metabolism hence holds great promise for potential therapeutic interventions in these disease states. Our hypothesis is that there is a potential interaction between CYP2U1 and drugs used for thymus cancer treatment and lipids presented in the brain, where such interactions yield bioactive metabolites that may be involved in neuro-and immunoregulation. To address this, we recombinantly expressed and purified CYP2U1 in a bacterial expression system and incorporated them into nanoscale lipid bilayers called Nanodiscs. By employing UV-vis spectroscopy, fluorescence spectroscopy, and liquid chromatography coupled with mass spectrometry (LC-MS/MS) methodologies, we explored the metabolism of drugs such as sorafenib and sunitinib (targeting thymus tumors) and lipids presented in the brain. Our findings reveal that CYP2U1 metabolizes both thymus cancer drugs into bioactive metabolites via dealkylation and oxidation. Further exploration using cellular assays also identified dealkylated sunitinib and oxidized sorafenib are capable of inhibiting cancer cell migration and tyrosine kinase functions. Through modeling of CYP2U1 in Nanodiscs, we identify novel protein-small molecule facilitated by CYP2U1. These findings shed light on the potential biochemical roles of CYP2U1, offering valuable insights for further exploration.

The flexibility of the P450 active site is essential to explain the differences in substrate interactions for different compounds. Computational approaches for the investigation of P450 substrate interactions have been hamstrung by limited predictivity. Substrates can bind to the flexible active site in multiple binding modes, and selection of the predominant ligand and active site conformation through sampling can be bottlenecked by computational resources. While recent progress in flexible backbone protein docking has been promising, their reliability has yet to be proven.
In this study, we evaluated the capabilities and limitations of flexible and rigid protein docking methods on more than a hundred experimental P450 structures with bound ligands. We also investigated the effects of overfitting and induced fit on docking performance through a comparison between re-docking and cross-docking.
While some docking methods systematically overestimate the distance between heme and ligand, for one docking method we found low overestimation bias. For the predicted site of metabolism, accuracy more than doubled with a flexible protein docking method. Flexible ligands showed the largest improvement, while for rigid ligands the improvement due to flexible protein docking was limited. The protein-ligand interaction prediction in terms of shape and pharmacophoric overlap showed almost double improvement, yet only for a small fraction of compounds was the binding pose entirely correct. These results indicate partial progress in the prediction of P450 active site interactions while pointing out several current limitations.