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.

Low pressure‐induced secondary structure transitions of regenerated silk fibroin in its wet film studied by time‐resolved infrared spectroscopy

“…In addition to chemical factors, 5,6 physical factors, especially the hydrostatic pressure in silkworm glands may also play an important role. 7 Although a large amount of literature [8][9][10][11][12][13][14][15][16][17][18][19][20][21][22] reported the influence of high hydrostatic pressure on the conformational transitions of many other proteins, no investigation on the effect of pressure on silk fibroin has been found so far.…”

“…shows the IR spectra of a RSF wet film under a constant pressure with different time. The pressure applied to the wet film is 6.895 MPa, relatively low compared with the high pressure used in many other studies on protein solutions [8][9][10][11][12][13][14][15][16][17][18][19][20][21][22]. A wet film was used to imitate the formation of silk from its high-concentration aqueous solution of silk fibroin.…”

High-Pressure EPR Spectroscopy Studies of the E. coli Lipopolysaccharide Transport Proteins LptA and LptC

“…This is a concept that has been extensively utilized in the nuclear magnetic resonance (NMR) field but with limited use by electron paramagnetic resonance (EPR) spectroscopists. Recently, the application of high pressure to the study of proteins by EPR spectroscopy (e.g., [3]) has been reintroduced by the Hubbell group at the University of California, Los Angeles [4–9]. This methodology is possible using X-band EPR spectroscopy due to a number of advances, including pressure intensifiers operating up to 6.2 kbar, ceramic sample cells withstanding up to 2 kbar pressure, and EPR resonators that can accommodate and enhance the sensitivity of samples contained in pressurizable sample cells.…”

Role of Protein-Protein Interactions in Metabolism: Genetics, Structure, Function

“…Interestingly, the CYP2C8 crystal dimer (PDB: 1PQ2) also displayed a peripheral binding site for two palmitate molecules in a similar location to that of the CYP3A4 dimer, but its functional significance was not explored. Davydov et al (2016) have now provided evidence from studies using LRET/FRET and chemical cross-linking that the CYP3A4 crystal dimer is a reasonable analog to a quaternary structure adopted in the membrane. First, it was described above how FRET/LRET studies indicated that the binding of ANF to CYP3A4 could change the oligomeric state of the enzyme.…”