title: Incorporation of charged residues in the CYP2J2 F-G loop disrupts CYP2J2-lipid bilayer interactions.
abstract: CYP2J2 epoxygenase is an extrahepatic, membrane bound cytochrome P450 (CYP) that is primarily found in the heart and mediates endogenous fatty acid metabolism. CYP2J2 interacts with membranes through an N-terminal anchor and various non-contiguous hydrophobic residues. The molecular details of the motifs that mediate membrane interactions are complex and not fully understood. To gain better insights of these complex protein-lipid interactions, we employed molecular dynamics (MD) simulations using a highly mobile membrane mimetic (HMMM) model that enabled multiple independent spontaneous membrane binding events to be captured. Simulations revealed that CYP2J2 engages with the membrane at the F-G loop through hydrophobic residues Trp-235, Ille-236, and Phe-239. To explore the role of these residues, three F-G loop mutants were modeled from the truncated CYP2J2 construct (Δ34) which included Δ34-I236D, Δ34-F239H and Δ34-I236D/F239H. Using the HMMM coordinates of CYP2J2, the simulations were extended to a full POPC membrane which showed a significant decrease in the depth of insertion for each of the F-G loop mutants. The CYP2J2 F-G loop mutants were expressed in E. coli and were shown to be localized to the cytosolic fraction at a greater percentage relative to construct Δ34. Notably, the functional data demonstrated that the double mutant, Δ34-I236D/F239H, maintained native-like enzymatic activity. The membrane insertion characteristics were examined by monitoring CYP2J2 Trp-quenching fluorescence spectroscopy upon binding nanodiscs containing pyrene phospholipids. Relative to the Δ34 construct, the F-G loop mutants exhibited lower Trp quenching and membrane insertion. Taken together, the results suggest that the mutants exhibit a different membrane topology in agreement with the MD simulations and provide important evidence towards the involvement of key residues in the F-G loop of CYP2J2.
authors.affiliation: Department of Comparative Biosciences, University of Illinois Urbana-Champaign, Urbana IL 61801.|Medical Scholars Program, University of Illinois Urbana-Champaign, Urbana IL 61801.
authors.forename: Daniel R
authors.initials: DR
authors.lastname: McDougle
authors.affiliation: Center for Biophysics and Computational Biology, University of Illinois Urbana-Champaign, Urbana IL 61801.|Beckman Institute for Advanced Science and Technology, University of Illinois Urbana-Champaign, Urbana IL 61801.
authors.forename: Javier L
authors.initials: JL
authors.lastname: Baylon
authors.affiliation: Department of Biochemistry, University of Illinois Urbana-Champaign, Urbana IL 61801.
authors.forename: Daryl D
authors.initials: DD
authors.lastname: Meling
authors.affiliation: Department of Biochemistry, University of Illinois Urbana-Champaign, Urbana IL 61801.
authors.forename: Amogh
authors.initials: A
authors.lastname: Kambalyal
authors.affiliation: Department of Biochemistry, University of Illinois Urbana-Champaign, Urbana IL 61801.
authors.forename: Yelena V
authors.initials: YV
authors.lastname: Grinkova
authors.affiliation: Department of Biochemistry, University of Illinois Urbana-Champaign, Urbana IL 61801.
authors.forename: Jared
authors.initials: J
authors.lastname: Hammernik
authors.affiliation: Department of Biochemistry, University of Illinois Urbana-Champaign, Urbana IL 61801.|Center for Biophysics and Computational Biology, University of Illinois Urbana-Champaign, Urbana IL 61801.|Beckman Institute for Advanced Science and Technology, University of Illinois Urbana-Champaign, Urbana IL 61801.
authors.forename: Emad
authors.initials: E
authors.lastname: Tajkhorshid
authors.affiliation: Department of Comparative Biosciences, University of Illinois Urbana-Champaign, Urbana IL 61801.|Department of Biochemistry, University of Illinois Urbana-Champaign, Urbana IL 61801.|Beckman Institute for Advanced Science and Technology, University of Illinois Urbana-Champaign, Urbana IL 61801.
authors.forename: Aditi
authors.initials: A
authors.lastname: Das
mesh_terms: D019943:Amino Acid Substitution; D000596:Amino Acids / Q000737:chemistry*; D001665:Binding Sites; D000090762:Cytochrome P-450 CYP2J2; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000648:ultrastructure*; D004789:Enzyme Activation; D008051:Lipid Bilayers / Q000737:chemistry*; D008956:Models, Chemical; D056004:Molecular Dynamics Simulation; D011485:Protein Binding; D011487:Protein Conformation; D055672:Static Electricity; D013329:Structure-Activity Relationship
chemical_list: D000596:Amino Acids; D008051:Lipid Bilayers; D003577:Cytochrome P-450 Enzyme System; D000090762:Cytochrome P-450 CYP2J2
keywords: CYP2J2; Cytochrome P450; Eicosanoids; Epoxygenase; F-G loop; Nanodiscs