Structure and Dynamics of the Membrane-Bound Cytochrome P450 2C9

Abstract

The microsomal, membrane-bound, human cytochrome P450 (CYP) 2C9 is a liver-specific monooxygenase essential for drug metabolism. CYPs require electron transfer from the membrane-bound CYP reductase (CPR) for catalysis. The structural details and functional relevance of the CYP-membrane interaction are not understood. From multiple coarse grained molecular simulations started with arbitrary configurations of protein-membrane complexes, we found two predominant orientations of CYP2C9 in the membrane, both consistent with experiments and conserved in atomic-resolution simulations. The dynamics of membrane-bound and soluble CYP2C9 revealed correlations between opening and closing of different tunnels from the enzyme's buried active site. The membrane facilitated the opening of a tunnel leading into it by stabilizing the open state of an internal aromatic gate. Other tunnels opened selectively in the simulations of product-bound CYP2C9. We propose that the membrane promotes binding of liposoluble substrates by stabilizing protein conformations with an open access tunnel and provide evidence for selective substrate access and product release routes in mammalian CYPs. The models derived here are suitable for extension to incorporate other CYPs for oligomerization studies or the CYP reductase for studies of the electron transfer mechanism, whereas the modeling procedure is generally applicable to study proteins anchored in the bilayer by a single transmembrane helix.

Interactions among Cytochromes P450 in Microsomal Membranes

“…This conclusion is in agreement with the results of Baylon et al (55), who concluded that the membrane binding of P450 is largely independent of the presence of the N-terminal hydrophobic anchor. According to the current concepts, the interactions of the microsomal P450 enzymes with the membrane are in large part determined by the regions between the N-terminal anchor and ␣-helix A (54,56,57) as well as by the hydrophobic surface in the region of ␣-helices FЈ and GЈ (55,58,59). Furthermore, according to the x-ray structure of the full-length CYP51, the constrained orientation of the P450 catalytic domain relative to the membrane is dictated by a network of interactions of polar residues in the C-terminal part of the transmembrane helix and the following proline-rich region (54), which are completely retained in our constructs.…”

Structure–phenotype correlations of human CYP21A2 mutations in congenital adrenal hyperplasia

“…Residues 1-25 were modeled in a canonical α-helical conformation, whereas residues 26-28, which connect the anchoring transmembrane helix to the first residue in the model, were built in a random-coil conformation using MODELLER. The orientation and position of the model, with respect to the lipid bilayer, was obtained using the membrane self-assembly coarse-grained dynamics protocol by Cojocaru et al (38). This permits unbiased orientation of the protein in the membrane, which is not achievable via atomistic simulations.…”

Correlating Structure and Function of Drug-Metabolizing Enzymes: Progress and Ongoing Challenges

“…The presence of a second substrate molecule in the similarly expanded active site cavities has been reported also for the adrenal steroid 21-hydroxylase, P450 21A2, with progesterone, P450s 2B4 and 2B6 with amlodipine, and P450 2A13 with nicotine-derived nitrosamine ketone molecules bound in the active site. In contrast to the soluble P450 102A1, the dmd.aspetjournals.org entrance channels for P450 2D6 and other drug-metabolizing P450s are likely to be buried in the surface of the membrane (Berka et al, 2011;Cojocaru et al, 2011).…”