Cytochrome-P450-Induced Ordering of Microsomal Membranes Modulates Affinity for Drugs

Abstract

Although membrane environment is known to boost drug metabolism by mammalian cytochrome P450s, the factors that stabilize the structural folding and enhance protein function are unclear. In this study, we use peptide-based lipid nanodiscs to "trap" the lipid boundaries of microsomal cytochrome P450 2B4. We report the first evidence that CYP2B4 is able to induce the formation of raft domains in a biomimetic compound of the endoplasmic reticulum. NMR experiments were used to identify and quantitatively determine the lipids present in nanodiscs. A combination of biophysical experiments and molecular dynamics simulations revealed a sphingomyelin binding region in CYP2B4. The protein-induced lipid raft formation increased the thermal stability of P450 and dramatically altered ligand binding kinetics of the hydrophilic ligand BHT. These results unveil membrane/protein dynamics that contribute to the delicate mechanism of redox catalysis in lipid membrane.

Keywords: biophysics; hemeproteins; lipids; membranes; nanodiscs.

Figure 1.. Peptide-based nanodiscs as a tool to unveil lipid-boundary regions of microsomal CYP2B4.

(a) Schematic of the lipid-exchange experiment. CYP2B4 was reconstituted with a 2.5-fold excess of empty nanodiscs and incubated overnight to allow lipid exchange. (b) SEC profiles showing two partially overlapping peaks, representing the reconstituted and empty fractions, respectively; the protein-containing fraction was also characterized by the absorbance at the Soret maximum (417 nm).

Figure 2.. CYP2B4-induced liquid-ordered domain modulates drug affinity.

(a) ³¹P NMR on detergent-treated nanodiscs was used to assess the composition of the protein-containing fraction after overnight lipid exchange. (b) Lipids and cholesterol contents in empty, CYP2B4, and cytb₅ 4F-ER nanodiscs after lipid exchange, as measured by ³¹P NMR and GC-MS. Data are expressed as average ± standard deviation (n=3). *p<0.05, **p<0.01, ***p<0.001. (c) Fluorescence autocorrelation functions for nanodiscs. Scatter plot represent average ± standard deviation of two independent experiments; solid lines represent numerical fitting using a 3D correlation diffusion model. The lipid-probe DiI C₁₂ differently partitioned in P450-containing nanodiscs, causing changes in the autocorrelation function. (d) DSC curves. (e) Protein-induced changes in lipid boundaries alter the affinity for BHT. (f) Schematic of lipid-induced modulation of ligand affinity for BHT.

Figure 3.. Atomistic insights into the full-length CYP2B4 interaction with ER membrane.

(a) All-atom (left) and coarse-grained (right) models of CYP2B4. Protein and lipids are shown as cartoon and stick, respectively, in the all-atom model, whereas as spheres and dotted spheres in the CG-MD models. (b) CG-MD snapshots showing the TMD orientation in POPC and ER nanodiscs at 0 (cyan) and 10 (orange) μs. (c) Percentage of helicity rise per residue in CYP2B4-TMD with simulation time. (d) Root mean square fluctuation of CYP2B4. TMD and linker connecting TMD and soluble domain are highlighted in blue and cyan, respectively. (e) MD snapshot illustrating the interaction between CYP2B4 and ER, and (f) SM and cholesterol-rich ER_Ex. SM and POPS are shown in black and purple, respectively.