Incorporation of charged residues in the CYP2J2 F-G loop disrupts CYP2J2–lipid bilayer interactions
| Autor: | Javier L. Baylon, Emad Tajkhorshid, Aditi Das, Yelena V. Grinkova, Jared A. Hammernik, Daryl D. Meling, Daniel R. McDougle, Amogh Kambalyal |
|---|---|
| Jazyk: | angličtina |
| Předmět: |
Stereochemistry
Protein Conformation Mutant Lipid Bilayers Static Electricity Biophysics Cytochrome P450 Molecular Dynamics Simulation 010402 general chemistry 01 natural sciences Biochemistry Cytochrome P-450 CYP2J2 Article 03 medical and health sciences chemistry.chemical_compound Molecular dynamics Structure-Activity Relationship Cytochrome P-450 Enzyme System F-G loop Amino Acids Lipid bilayer POPC 030304 developmental biology 0303 health sciences Quenching (fluorescence) Binding Sites biology Nanodiscs Cell Biology 0104 chemical sciences Enzyme Activation Membrane Epoxygenase chemistry Amino Acid Substitution Models Chemical Membrane topology biology.protein Eicosanoids CYP2J2 Protein Binding |
| Zdroj: | Biochimica et Biophysica Acta (BBA) - Biomembranes. (10):2460-2470 |
| ISSN: | 0005-2736 |
| DOI: | 10.1016/j.bbamem.2015.07.015 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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