Differing Membrane Interactions of Two Highly Similar Drug-Metabolizing Cytochrome P450 Isoforms: CYP 2C9 and CYP 2C19

Abstract

The human cytochrome P450 (CYP) 2C9 and 2C19 enzymes are two highly similar isoforms with key roles in drug metabolism. They are anchored to the endoplasmic reticulum membrane by their N-terminal transmembrane helix and interactions of their cytoplasmic globular domain with the membrane. However, their crystal structures were determined after N-terminal truncation and mutating residues in the globular domain that contact the membrane. Therefore, the CYP-membrane interactions are not structurally well-characterized and their dynamics and the influence of membrane interactions on CYP function are not well understood. We describe herein the modeling and simulation of CYP 2C9 and CYP 2C19 in a phospholipid bilayer. The simulations revealed that, despite high sequence conservation, the small sequence and structural differences between the two isoforms altered the interactions and orientations of the CYPs in the membrane bilayer. We identified residues (including K72, P73, and I99 in CYP 2C9 and E72, R73, and H99 in CYP 2C19) at the protein-membrane interface that contribute not only to the differing orientations adopted by the two isoforms in the membrane, but also to their differing substrate specificities by affecting the substrate access tunnels. Our findings provide a mechanistic interpretation of experimentally observed effects of mutagenesis on substrate selectivity.

Molecular Dynamics Simulations in Drug Discovery and Pharmaceutical Development

“…For this, CYP3A4 was used as the test case for the insertion and orientation of a CYP enzyme in a POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) bilayer. We then extended the same approach to other major drug-metabolizing CYPs such as CYP2C9, CYP2C19, CYP1A1, CYP1A2, as well as to the human steroidogenic enzymes CYP17 and CYP19 that are [417][418][419]. From the results of our simulations, we have been able to identify differences in the orientations or the distance of the globular domain with respect to the membrane, and important residues at the membrane-protein interface that result in such differences.…”

“…Through our optimized multiscale simulation protocol, we identified that the two highly similar CYPs: CYP2C9 and CYP2C19 that share 94% sequence identity, can have not only distinct substrate specificities but also different membrane protein orientations and interactions [419]. Such differences were due to the primary sequence variation in the linker, beta-strand1, the B-C loop, helices F, F'-G', G regions and their turns.…”

An electron transfer competent structural ensemble of membrane-bound cytochrome P450 1A1 and cytochrome P450 oxidoreductase

“…To date, there is no experimentally determined structure of a full CYP-CPR/cyt b5 complex available, although structures of the individual globular domains of the number of mammalian CYPs and CPRs have been determined by crystallography after removal of the N-terminal transmembrane domain to facilitate expression and crystallization 2 . Various modeling and simulation procedures have been developed to predict the orientation of full-length CYPs in phospholipid bilayers [16][17][18][19][20][21][22] . Notably, T. brucei CYP 51 simulated 20 prior to the determination in 2014 of the crystal structure of yeast CYP 51 23 , the only eukaryotic CYP for which a full-length structure has been determined experimentally, indicated a very similar orientation of the protein with respect to the membrane.…”

Prevalence of five pharmacologically most important CYP2C9 and CYP2C19 allelic variants in the population from the Republic of Srpska in Bosnia and Herzegovina

“…The cytochrome P450 (CYP) superfamily consists of enzymes with highly diverse roles in the metabolism of drugs, fatty acids, steroids, and xenobiotics ( 1 , 2 ). Their genetic variants, single nucleotide polymorphisms (SNPs) in particular, can lead to therapy failure, severe toxicity, and increased susceptibility to cancer and other diseases caused by chemicals ( 3 ).…”