Investigating the structural plasticity of a cytochrome P450: three-dimensional structures of P450 EryK and binding to its physiological substrate

Abstract

Cytochrome P450s are heme-containing proteins that catalyze the oxidative metabolism of many physiological endogenous compounds. Because of their unique oxygen chemistry and their key role in drug and xenobiotic metabolism, particular attention has been devoted in elucidating their mechanism of substrate recognition. In this work, we analyzed the three-dimensional structures of a monomeric cytochrome P450 from Saccharopolyspora erythraea, commonly called EryK, and the binding kinetics to its physiological ligand, erythromycin D. Three different structures of EryK were obtained: two ligand-free forms and one in complex with its substrate. Analysis of the substrate-bound structure revealed the key structural determinants involved in substrate recognition and selectivity. Interestingly, the ligand-free structures of EryK suggested that the protein may explore an open and a closed conformation in the absence of substrate. In an effort to validate this hypothesis and to investigate the energetics between such alternative conformations, we performed stopped-flow absorbance experiments. Data demonstrated that EryK binds erythromycin D via a mechanism involving at least two steps. Contrary to previously characterized cytochrome P450s, analysis of double jump mixing experiments confirmed that this complex scenario arises from a pre-existing equilibrium between the open and closed subpopulations of EryK, rather than from an induced-fit type mechanism.

FIGURE 1.

Pathway of erythromycin A biosynthesis. The reaction leading to the shunt product of ErB is indicated by the dashed arrow.

SCHEME 1

SCHEME 2

FIGURE 2.

Structures of EryK. A, the structure of the open EryK is shown, the elements that move upon substrate binding are highlighted in blue. B, substrate bound EryK, the elements that close on the active site are given in green. C, superposition of closed (pink and magenta) and open ligand-free structures of EryK. D, superposition of the open (blue) and substrate-bound (light and dark green) structures of EryK. The arrows point toward the FG and BC loops, which undergo the most pronounced displacement. Heme groups are in red, ErD in orange, and capital letters indicate the helices according P450 nomenclature. Panels A and B show EryK in the standard P450 orientation, whereas panels C and D utilize an orientation that allows clear visualization of the structure transitions.

FIGURE 3.

Substrate recognition and locking within the active site of EryK. A, a close up of the helix F network in superposed open EryK (blue), ligand-free closed EryK (pink), and ErD-EryK (green). Superposition was based on secondary structure motifs as implemented in COOT (48). B, view of contacts between the EryK and its substrate ErD. The electron density omit map (2F_o − F_c) is contoured at 1.0σ around ErD. C, the HSWP network is shown for ligand-free closed EryK (pink), superposed to open EryK (blue). D, the HSWP network is shown for ErD-EryK (green), superposed to open EryK (blue). Heme groups, substrate (orange), and side chains are in sticks. Dashed lines indicate H-bonds and close contacts, the actual distances are given in supplemental Tables S1 and S2.

FIGURE 4.

Binding kinetics of EryK to ErD. The traces reported were obtained at 90 μm ErD and 7.5 μm EryK in the absence (panel A) and presence (panel B) of 2 m NaCl. Traces reported are the averages of three independent acquisitions. The residuals to a single (top) and double (bottom) exponential fit are reported below each trace. Thin lines are the fit to a single exponential decay.

FIGURE 5.

Binding kinetics of EryK to ErD. Observed rate constants are plotted as a function of [ErD]. The rate constants for the fast phase (filled circles) increase linearly with increasing [ErD], whereas the rate constants for the slow phase (empty circles) are essentially independent on [ErD]. Panels A and B refer to data recorded in the absence and presence of 2 m NaCl, respectively.

SCHEME 3

FIGURE 6.

Double jump mixing experiments monitoring the binding of EryK to ErD. The amplitudes for the two phases are reported as a function of the delay time between the first and second mix (see text for details regarding the experimental setup). The amplitudes obtained for the slow phase (filled circles) and for the fast phase (shown in the inset) were globally fitted to a single exponential function, yielding a value of k_obs = 1.8 ± 0.1 s⁻¹.

SCHEME 4

FIGURE 7.

Schematic energy diagram showing the binding reaction of EryK to ErD. Representative structures of closed-free (pink), open-free (blue) EryK, and the ligand-bound ErD-EryK (green) are also shown. It should be noted that, given that observed kinetics is well fitted by a double exponential process under all investigated conditions, the existence of a late closure step may only be invoked indirectly from crystallographic work, as well as from the slow apparent second-order rate constant. The microscopic rate constants, obtained in the presence of 2 m NaCl, were calculated as reported below. As described in the text, we designed a double jump experiment with the specific aim of shifting the equilibrium between the open and closed states, prior the encountering the ligand. Hence, the amplitude dependence of the two phases as a function of the delay time (Fig. 6) directly reports on the kinetics of the first step in Schemes 3 and 4, the observed rate constant k_obs being equal to (k₁ + k₋₁) = 1.8 ± 0.1 s⁻¹. On the other hand, by considering that the binding reaction between the open conformer of EryK and ErD occurs faster than the conformational change between the open and closed states of EryK, the rate constant for the slow phase λ₂ should tend to be k₁ at [ErD] → ∞ (see Equation 5). Data in Fig. 5 clearly reveal that λ₂ is independent on [ErD], suggesting that the limiting value of k₁ is reached at relatively low [ErD] and equal to 1.3 ± 0.1 s⁻¹. Based on these observations, we estimated a value of k₋₁ = 0.5 ± 0.1 s⁻¹. Importantly, a similar magnitude of k₁ and k₋₁ are consistent with the proposed heterogeneous conformation of EryK, the equilibrium constant of the first step being equal to k₁/k₋₁.