title: Membrane Interactions, Ligand-Dependent Dynamics, and Stability of Cytochrome P4503A4 in Lipid Nanodiscs.
abstract: Membrane-bound cytochrome P4503A4 (CYP3A4) is the major source of enzymatic drug metabolism. Although several structural models of CYP3A4 in various ligand complexes are available, none includes a lipid bilayer. Details of the effects of the membrane on protein dynamics and solvation, and access channels for ligands, remain uncertain. H/D exchange mass spectrometry (H/DXMS) with ligand free CYP3A4 containing a deletion of residues 3-12, compared to that of the full length wild type, in lipid nanodiscs afforded 91% sequence coverage. Deuterium exchange was fast in the F- and G-helices, HI loop, and C-terminal loop. In contrast, there is very low exchange in the F'- and G'-helices. The results are consistent with the overall membrane orientation of CYP3A4 suggested by published MD simulations and spectroscopic results, and the solvent accessibility of the F/G loop suggests that it is not deeply membrane-embedded. Addition of ketoconazole results in only modest, but global, changes in solvent accessibility. Interestingly, with ketoconazole bound some peptides become less solvent accessible or dynamic, including the F- and G-helices, but several peptides demonstrate modestly increased accessibility. Differential scanning calorimetry (DSC) of CYP3A4-nanodiscs suggests membrane-induced stabilization compared to that of aggregated CYP3A4 in buffer, and this stabilization is enhanced upon addition of the ligand ketoconazole. This ligand-induced stabilization is accompanied by a very large increase in ΔH for CYP3A4 denaturation in nanodiscs, possibly due to increased CYP3A4-membrane interactions. Together, the results suggest a distinct orientation of CYP3A4 on the lipid membrane, and they highlight likely solvent access channels, which are consistent with several MD simulations.
authors.affiliation: Department of Medicinal Chemistry, University of Washington , Box 357610, Seattle, Washington 98195-7610, United States.
authors.forename: Nicholas A
authors.initials: NA
authors.lastname: Treuheit
authors.affiliation: Department of Medicinal Chemistry, University of Washington , Box 357610, Seattle, Washington 98195-7610, United States.
authors.forename: Michelle
authors.initials: M
authors.lastname: Redhair
authors.affiliation: Department of Medicinal Chemistry, University of Washington , Box 357610, Seattle, Washington 98195-7610, United States.
authors.forename: Hyewon
authors.initials: H
authors.lastname: Kwon
authors.affiliation: Department of Medicinal Chemistry, University of Washington , Box 357610, Seattle, Washington 98195-7610, United States.
authors.forename: Wynton D
authors.initials: WD
authors.lastname: McClary
authors.affiliation: Department of Medicinal Chemistry, University of Washington , Box 357610, Seattle, Washington 98195-7610, United States.
authors.forename: Miklos
authors.initials: M
authors.lastname: Guttman
authors.affiliation: Department of Medicinal Chemistry, University of Washington , Box 357610, Seattle, Washington 98195-7610, United States.
authors.forename: John P
authors.initials: JP
authors.lastname: Sumida
authors.affiliation: Department of Medicinal Chemistry, University of Washington , Box 357610, Seattle, Washington 98195-7610, United States.
authors.forename: William M
authors.initials: WM
authors.lastname: Atkins
mesh_terms: D016632:Apolipoprotein A-I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002152:Calorimetry, Differential Scanning; D051544:Cytochrome P-450 CYP3A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D065692:Cytochrome P-450 CYP3A Inhibitors / Q000494:pharmacology; D041961:Deuterium Exchange Measurement; D004795:Enzyme Stability / Q000187:drug effects; D006801:Humans; D007654:Ketoconazole / Q000494:pharmacology; D008024:Ligands; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D021962:Membrane Microdomains / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010713:Phosphatidylcholines / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D058767:Protein Unfolding / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: C505693:APOA1 protein, human; D016632:Apolipoprotein A-I; D065692:Cytochrome P-450 CYP3A Inhibitors; D008024:Ligands; D008051:Lipid Bilayers; D008565:Membrane Proteins; D010446:Peptide Fragments; D010713:Phosphatidylcholines; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D007654:Ketoconazole; C028694:1-palmitoyl-2-oleoylphosphatidylcholine
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