Structural basis for ligand promiscuity in cytochrome P450 3A4

Abstract

Cytochrome P450 (CYP) 3A4 is the most promiscuous of the human CYP enzymes and contributes to the metabolism of approximately 50% of marketed drugs. It is also the isoform most often involved in unwanted drug-drug interactions. A better understanding of the molecular mechanisms governing CYP3A4-ligand interaction therefore would be of great importance to any drug discovery effort. Here, we present crystal structures of human CYP3A4 in complex with two well characterized drugs: ketoconazole and erythromycin. In contrast to previous reports, the protein undergoes dramatic conformational changes upon ligand binding with an increase in the active site volume by >80%. The structures represent two distinct open conformations of CYP3A4 because ketoconazole and erythromycin induce different types of coordinate shifts. The binding of two molecules of ketoconazole to the CYP3A4 active site and the clear indication of multiple binding modes for erythromycin has implications for the interpretation of the atypical kinetic data often displayed by CYP3A4. The extreme flexibility revealed by the present structures also challenges any attempt to apply computational design tools without the support of relevant experimental data.

Fig. 1.

Chemical structures of ketoconazole (A) and erythromycin (B).

Fig. 2.

Overall structures of CYP3A4 in complex with ketoconazole (A) and erythromycin (B). Structures are shown in dark gray with color highlighting of helices F to G (residues 202–260) in red, helix I (residues 291–323) in green, and the C-terminal loop (residues 464–498) in blue. The complex structures are superimposed on the ligand-free structure (Protein Data Bank ID code 1TQN) shown in light colors. Orange arrows indicate the direction of coordinate shifts in the F-G region relative the ligand-free structure.

Fig. 3.

Ketoconazole binding to CYP3A4. Ketoconazole molecules are shown in orange stick representation. The heme group is shown in magenta. Secondary structure and stick representations of side chains within 4 Å from the ligands are shown in green. The C-terminal loop (residues 464–498) was omitted for clarity. A superposition of the ligand-free structure (Protein Data Bank ID code 1TQN) is shown in gray. The mesh represents a F_o − F_c difference map contoured at 4.5 σ calculated in the absence of ligands by using the program AutoBUSTER (19).

Fig. 4.

Erythromycin binding to CYP3A4. Erythromycin is shown in orange stick representation. The heme is shown in magenta. Secondary structure and stick representations of side chains within 4 Å from the ligand are shown in green. A possible conformation for the disordered loop connecting helices F and F′ is shown in yellow. The mesh represents a F_o − F_c difference map contoured at 4.5 σ calculated in the absence of ligands by using the program AutoBUSTER (19).

Fig. 5.

Comparison of observed structural flexibility in CYP3A4 (A) and CYP2B4 (B). For each isoform, the secondary structure of the most compact conformation (Protein Data Bank ID codes 1TQN for CYP3A4 and 1SUO for CYP2B4) is shown colored according to the maximum observed difference in C^α position in an optimal superposition of all available structures (Protein Data Bank ID codes 1TQN, 2J0C, and 2J0D for CYP3A4 and 1PO5, 1SUO, and 2BDM for CYP2B4). Maximum observed C^α differences >6 Å are shown in red. Each isoform is shown in two orientations related by a 180° rotation about a vertical axis in the plane of the paper. Heme groups are shown in stick representation.