Cytochrome P450 Prefers to be in Liquid-Ordered Domains in the Endoplasmic Reticulum

Abstract

High-resolution mapping of protein/lipid interactions is essential to fully understand the biological function of a membrane protein, but poses tremendous challenges to most biophysical and biochemical approaches. These challenges are more severe for membrane proteins like cytochrome P450 that contain a very large soluble domain and exhibit a completely different time scale of dynamics as compared to its transmembrane domain [Durr et al., BBA Biomemb, 1768(12), 2007; Yamamoto et al., Sci Rep, 3, 2013; Yamamoto et al., Sci Rep, 7, 2017]. In this study, we use peptide-based lipid nanodiscs to “trap” the microsomal cytochrome P450 2B4 alone or also in complex with its redox partners [Zhang et al., Angew Chem, 55(14), 2016]. We effectively utilized the unique nature of peptide-based nanodiscs, that allows for the lipid exchange among nanodiscs, in order to characterize the immediate membrane environment preferred/stabilized by cytochrome P450. We report the first evidence that CYP2B4 is able to induce the formation of “raft”-like domains in a biomimetic of the endoplasmic reticulum (ER). NMR experiments were used to identify and quantitatively determine the lipids present in the nanodiscs. In addition, biophysical experiments were supported by molecular dynamics simulations to identify a sphingomyelin binding motif in P450, which is largely conserved among microsomal P450s. The protein-induced raft formation increased the thermal stability of P450 and also dramatically altered the ligand binding kinetics. These results unveil lipid/protein dynamics which can possibly contribute to the delicate mechanism of redox catalysis in lipid membrane systems [Barnaba et al., F1000Research, 6, 2017].