Allosteric effects on substrate dissociation from cytochrome P450 3A4 in nanodiscs observed by ensemble and single-molecule fluorescence spectroscopy

Abstract

Cytochrome P450 (CYP) 3A4 is a major human drug-metabolizing enzyme and displays pharmacologically relevant allosteric kinetics caused by multiple substrate and/or effector binding. Here, in the first single-molecule (SM) fluorescence studies of CYPs, we use total internal reflection fluorescence microscopy to measure residence times of the fluorescent dye Nile Red in CYP3A4 incorporated in surface-immobilized lipid Nanodiscs, with and without the effector alpha-naphthoflavone. We find direct evidence that CYP3A4 effectors can decrease substrate off-rates, providing a possible mechanism for effector-mediated enhancement of substrate metabolism. These interesting results highlight the potential of SM methods in studies of CYP allosteric mechanisms.

Figure 1

(a) NR (0.5 μM; λ_ex = 550 nm) fluorescence increasing with [Nanodiscs] yields a K_D of 1 μM for a singly-occupied disc. (b) NR fluorescence increases and red-shifts with increasing [NR], at three different Nanodisc concentrations, 0.5, 1.0, and 2.0 μM. Solid lines are global fits with 5 sequential NR binding modes and a negative cooperativity factor of 2.3. (Inset) Basis spectra used in singular value decomposition to deconvolute the spectral shift.

Figure 2

(a) NR binding CYP3A4-Nanodiscs (0.8 μM) also shows a fluorescence increase and red-shift. (Inset) Difference in fluorescence between NR binding CYP3A4-Nanodiscs and Nanodiscs shows decreased intensity at low [NR] and an increase at high [NR], suggesting multiple binding to CYP3A4. (b) UV-Vis spectroscopy of the NR-induced spin shift indicates the K_D of the lower-affinity site is ∼10 μM. (Inset) Difference spectrum of the spin-shift in CYP3A4-Nanodiscs. CYP3A4 was expressed, purified and incorporated into Nanodiscs as previously described.

Figure 3

Binding model for NR/CYP3A4-Nanodiscs; global fits to binding curves gave K₁ = 0.97 μM, α=2.3, K₂ ≈ 0.3 μM and K₃ ≈10 μM

Figure 4

Dwell-time histograms for NR bound to Nanodiscs, with k_off = 30 s^-1; CYP3A4-Nanodiscs, with k_off = 1.5 s^-1; and CYP3A4-Nanodiscs + 5 μM ANF, with k_off = 0.32 s^-1, show the dramatic effect of ANF on NR dissociation kinetics. (Inset) Typical photon trajectory of CYP3A4-Nanodiscs in the presence of NR only. Dwell-times were binned manually, and histograms were fit to single- or double-exponentials to yield off-rates.