Crystal structures of ligand complexes of P450eryF exhibiting homotropic cooperativity

Abstract

Several mammalian cytochrome P450 (P450) isoforms demonstrate homotropic cooperativity with a number of substrates, including steroids and polycyclic aromatic hydrocarbons. To identify structural factors contributing to steroid and polycyclic aromatic hydrocarbon binding to P450 enzymes and to determine the location of the allosteric site, we investigated interactions of the macrolide hydroxylase P450eryF from Saccharopolyspora erythraea with androstenedione and 9-aminophenanthrene. Spectroscopic binding assays indicate that P450eryF binds androstenedione with an affinity of 365 microM and a Hill coefficient of 1.31 +/- 0.6 and coordinates with 9-aminophenanthrene with an affinity of 91 microM and a Hill coefficient of 1.38 +/- 0.2. Crystals of complexes of androstenedione and 9-aminophenanthrene with P450eryF were grown and diffracted to 2.1 A and 2.35 A, respectively. Electron density maps indicate that for both complexes two ligand molecules are simultaneously present in the active site. The P450eryF/androstenedione model was refined to an r = 18.9%, and the two androstenedione molecules have similar conformations. The proximal androstenedione is positioned such that the alpha-face of carbon-6 is closest to the heme iron, and the second steroid molecule is positioned 5.5 A distal in the active site. The P450eryF/9-aminophenanthrene model was refined to an r = 19.7% with the proximal 9-aminophenanthrene coordinated with the heme iron through the 9-amino group and the second ligand positioned approximately 6 A distal in the active site. These results establish that homotropic cooperativity in ligand binding can result from binding of two substrate molecules within the active site pocket without major conformational changes in the protein.

Figure 1

Ligand-induced spectral shifts of P450eryF. (A) Effects of androstenedione. Duplicate assays were performed by using 19 substrate concentrations ranging from 40–880 μM; difference spectra from a single assay are shown. Data from two experiments (open and closed symbols) are presented in the form of a Hill plot: the Hill coefficient observed, n = 1.31 ± 0.6, is shown by a solid line, and plots for n = 1 and 2 are shown as dashed lines for comparison. (B) Spectral shifts induced by 9-aminophenanthrene. Duplicate assays were performed by using 19 ligand concentrations ranging from 20 μM-560 μM. Data from two experiments (open and closed symbols) are presented in the form of a Hill plot: the Hill coefficient observed, n = 1.38 ± 0.2, is shown by a solid line, and plots for n = 1 and 2 are shown as dashed lines for comparison.

Figure 2

The final 2F_o − F_c electron density map and model for the P450eryF/androstenedione structure. Two views are shown (A and B), and electron density is displayed only for the region surrounding the active site. Figures were generated by using the program setor (30).

Figure 3

Position of androstenedione in the active site of P450eryF. Two views are shown (A and B). Residues within 4 Å of the ligands that may contact the ligands are shown.

Figure 4

The final 2F_o − F_c electron density map and model for the P450eryF/9-aminophenanthrene structure. Two views are shown (A and B), and electron density is displayed only for the region surrounding the active site.

Figure 5

9-Aminophenanthrene in the active site of P450eryF. Two views are shown (A and B). Residues within 4 Å of the ligands that may contact the ligands are shown.

Figure 6

Superposition of the P450eryF/6-DEB structure with P450eryF/androstenedione (A and B) or P450eryF/9-aminophenanthrene (C and D). Two views of each structure are presented. The heme group and 6-DEB are shown as stick models, and androstenedione and 9-aminophenanthrene are shown as solid ball-and-stick models. The numbering systems of androstenedione and 9-aminophenanthrene are indicated, and the distances (Å) to the heme iron of carbons 4–7 of Andro1 are given.