Membrane Interactions, Ligand-Dependent Dynamics, and Stability of Cytochrome P4503A4 in Lipid Nanodiscs

Biochemistry. 2016 Feb 23;55(7):1058-69. doi: 10.1021/acs.biochem.5b01313. Epub 2016 Feb 8.

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Apolipoprotein A-I / chemistry
  • Apolipoprotein A-I / genetics
  • Apolipoprotein A-I / metabolism
  • Calorimetry, Differential Scanning
  • Cytochrome P-450 CYP3A / chemistry*
  • Cytochrome P-450 CYP3A / genetics
  • Cytochrome P-450 CYP3A / metabolism
  • Cytochrome P-450 CYP3A Inhibitors / pharmacology
  • Deuterium Exchange Measurement
  • Enzyme Stability / drug effects
  • Humans
  • Ketoconazole / pharmacology
  • Ligands
  • Lipid Bilayers / chemistry
  • Lipid Bilayers / metabolism
  • Membrane Microdomains / chemistry*
  • Membrane Microdomains / drug effects
  • Membrane Microdomains / metabolism
  • Membrane Proteins / chemistry
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism
  • Models, Molecular*
  • Peptide Fragments / chemistry
  • Peptide Fragments / genetics
  • Peptide Fragments / metabolism
  • Phosphatidylcholines / chemistry
  • Phosphatidylcholines / metabolism
  • Protein Conformation
  • Protein Engineering
  • Protein Structure, Tertiary
  • Protein Unfolding / drug effects
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / metabolism
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism

Substances

  • APOA1 protein, human
  • Apolipoprotein A-I
  • Cytochrome P-450 CYP3A Inhibitors
  • Ligands
  • Lipid Bilayers
  • Membrane Proteins
  • Peptide Fragments
  • Phosphatidylcholines
  • Recombinant Fusion Proteins
  • Recombinant Proteins
  • Cytochrome P-450 CYP3A
  • CYP3A4 protein, human
  • Ketoconazole
  • 1-palmitoyl-2-oleoylphosphatidylcholine