title: Membrane Fluidity Modulates Thermal Stability and Ligand Binding of Cytochrome P4503A4 in Lipid Nanodiscs.
abstract: Cytochrome P4503A4 (CYP3A4) is a peripheral membrane protein that plays a major role in enzymatic detoxification of many drugs and toxins. CYP3A4 has an integral membrane N-terminal helix and a localized patch comprised of the G' and F' helix regions that are embedded in the membrane, but the effects of membrane composition on CYP3A4 function are unknown. Here, circular dichroism and differential scanning calorimetry were used to compare the stability of CYP3A4 in lipid bilayer nanodiscs with varying ratios of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine to 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). These lipids differ in the acyl-chain length and their degree of unsaturation. The thermal denaturation of CYP3A4 in nanodiscs occurs in a temperature range distinct from that of the nanodisc denaturation so it can be monitored calorimetrically. Melting temperatures (Tm), heat capacities (ΔCp), and calorimetric enthalpies (ΔHcal) for denaturation of CYP3A4 each increased with an increasing fraction of DMPC, with a maximum at 50% DMPC, before decreasing at 75% DMPC. Addition of the inhibitor ketoconazole results in increased thermal stability, and larger ΔCpand ΔHcalvalues, with different sensitivities to lipid composition. Effects of lipid composition on ligand binding dynamics were also studied. Equilibrium binding affinities of both ketoconazole (KTZ) and testosterone (TST) were minimally affected by lipid composition. However, stopped-flow analyses indicate that the rates of KTZ binding reach a maximum in membranes containing 50% DMPC, whereas the rate of TST binding decreases continuously with an increasing DMPC concentration. These results indicate that CYP3A4 is highly sensitive to the acyl-chain composition of the lipids and fluidity of the membrane in which it is embedded.
authors: Department of Medicinal Chemistry, University of Washington , Box 357610, Seattle, Washington 98195-7610, United States.
authors: Wynton D
authors: WD
authors: McClary
authors: Department of Medicinal Chemistry, University of Washington , Box 357610, Seattle, Washington 98195-7610, United States.
authors: John P
authors: JP
authors: Sumida
authors: Department of Medicinal Chemistry, University of Washington , Box 357610, Seattle, Washington 98195-7610, United States.
authors: Michele
authors: M
authors: Scian
authors: Department of Medicinal Chemistry, University of Washington , Box 357610, Seattle, Washington 98195-7610, United States.
authors: Lorela
authors: L
authors: Paço
authors: Department of Medicinal Chemistry, University of Washington , Box 357610, Seattle, Washington 98195-7610, United States.
authors: William M
authors: WM
authors: Atkins
mesh_terms: D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D051544:Cytochrome P-450 CYP3A / Q000737:chemistry* / Q000378:metabolism; D065692:Cytochrome P-450 CYP3A Inhibitors / Q000378:metabolism / Q000494:pharmacology; D004134:Dimyristoylphosphatidylcholine / Q000737:chemistry; D004795:Enzyme Stability / Q000187:drug effects; D006801:Humans; D007654:Ketoconazole / Q000378:metabolism / Q000494:pharmacology; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D008055:Lipids / Q000737:chemistry*; D008560:Membrane Fluidity; D008958:Models, Molecular; D049329:Nanostructures / Q000737:chemistry*; D010713:Phosphatidylcholines / Q000737:chemistry; D011485:Protein Binding; D011489:Protein Denaturation; D000072417:Protein Domains; D013696:Temperature; D013739:Testosterone / Q000378:metabolism; D013816:Thermodynamics
chemical_list: D065692:Cytochrome P-450 CYP3A Inhibitors; D008051:Lipid Bilayers; D008055:Lipids; D010713:Phosphatidylcholines; D013739:Testosterone; D051544:Cytochrome P-450 CYP3A; D007654:Ketoconazole; C028694:1-palmitoyl-2-oleoylphosphatidylcholine; D004134:Dimyristoylphosphatidylcholine