title: Conformational Mobility in Cytochrome P450 3A4 Explored by Pressure-Perturbation EPR Spectroscopy.
abstract: We used high hydrostatic pressure as a tool for exploring the conformational landscape of human cytochrome P450 3A4 (CYP3A4) by electron paramagnetic resonance and fluorescence spectroscopy. Site-directed incorporation of a luminescence resonance energy transfer donor-acceptor pair allowed us to identify a pressure-dependent equilibrium between two states of the enzyme, where an increase in pressure increased the spatial separation between the two distantly located fluorophores. This transition is characterized by volume change (ΔV°) and P1/2 values of -36.8 ± 5.0 mL/mol and 1.45 ± 0.33 kbar, respectively, which corresponds to a Keq° of 0.13 ± 0.06, so that only 15% of the enzyme adopts the pressure-promoted conformation at ambient pressure. This pressure-promoted displacement of the equilibrium is eliminated by the addition of testosterone, an allosteric activator. Using site-directed spin labeling, we demonstrated that the pressure- and testosterone-sensitive transition is also revealed by pressure-induced changes in the electron paramagnetic resonance spectra of a nitroxide side chain placed at position 85 or 409 of the enzyme. Furthermore, we observed a pressure-induced displacement of the emission maxima of a solvatochromic fluorophore (7-diethylamino-3-((((2-maleimidyl)ethyl)amino)carbonyl) coumarin) placed at the same positions, which suggests a relocation to a more polar environment. Taken together, the results reveal an effector-dependent conformational equilibrium between open and closed states of CYP3A4 that involves a pronounced change at the interface between the region of α-helices A/A' and the meander loop of the enzyme, where residues 85 and 409 are located. Our study demonstrates the high potential of pressure-perturbation strategies for studying protein conformational landscapes.
authors.affiliation: Department of Chemistry, Washington State University, Pullman, Washington; V. N. Orekhovich Institute of Biomedical Chemistry, Russian Academy of Medical Sciences, Moscow, Russia. Electronic address: dmrdavyd@gmail.com.
authors.forename: Dmitri R
authors.initials: DR
authors.lastname: Davydov
authors.affiliation: Jules Stein Eye Institute, University of California, Los Angeles, Los Angeles, California; Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, California.
authors.forename: Zhongyu
authors.initials: Z
authors.lastname: Yang
authors.affiliation: Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, California.
authors.forename: Nadezhda
authors.initials: N
authors.lastname: Davydova
authors.affiliation: Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, California.
authors.forename: James R
authors.initials: JR
authors.lastname: Halpert
authors.affiliation: Jules Stein Eye Institute, University of California, Los Angeles, Los Angeles, California; Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, California.
authors.forename: Wayne L
authors.initials: WL
authors.lastname: Hubbell
mesh_terms: D051544:Cytochrome P-450 CYP3A / Q000737:chemistry* / Q000235:genetics; D004578:Electron Spin Resonance Spectroscopy; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D011312:Pressure; D011487:Protein Conformation
chemical_list: D005456:Fluorescent Dyes; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human