Structure and function of cytochromes P450 2B: from mechanism-based inactivators to X-ray crystal structures and back

Abstract

This article reviews work from the author dating back to 1978 and focuses on the structural basis of cytochrome P450 (P450) function using available contemporary techniques. Early studies used mechanism-based inactivators that bound to the protein moiety of hepatic P450s to try to localize the active site. Subsequent studies used cDNA cloning, heterologous expression, site-directed mutagenesis, and homology modeling based on multiple bacterial P450 X-ray crystal structures to predict the active sites of CYP2B enzymes with considerable accuracy. Breakthroughs in engineering and expression of mammalian P450s enabled us to determine our first X-ray crystal structure of ligand-free rabbit CYP2B4. To date, we have solved 11 CYP2B4 and three human CYP2B6 structures, which represent four significantly different conformations. The plasticity of CYP2B4 has been confirmed by deuterium exchange mass spectrometry and is substantiated by molecular dynamics simulations. In addition to major movement of secondary structure elements, more subtle reorientation of active site side chains, especially Phe206, Phe297, and Glu301, contributes to the ability of CYP2B enzymes to bind various ligands. Isothermal titration calorimetry has proven to be a useful tool for studying the thermodynamics of ligand binding to CYP2B4 and CYP2B6, and NMR has enabled study of ligand binding orientation in solution as an adjunct to X-ray crystallography. A major challenge remains to harness the power of the various approaches to facilitate prediction of CYP2B specificity and inhibition.

Fig. 1.

Structural plasticity of CYP2B enzymes observed by X-ray crystallography. A, the structural diversity of CYP2B ligands and substrates is indicated by the variability of size, shape, and chemical functionality of compounds bound in CYP2B4 and CYP2B6 structures. In binding these ligands, CYP2B enzymes have been observed to adopt four markedly different conformations. B, in the absence of ligand, CYP2B4 is able to adopt an open conformation, with a U-shaped active site cleft. C, the most populated CYP2B form is the closed conformation, which corresponds to CYP2B4 complexes of 4-CPI, 1-CPI, ticlopidine, and clopidogrel, the closed tBPA-modified and closed ligand-free CYP2B4 structures, and the CYP2B6 complexes of 4-CPI, BP, and NBP. D, CYP2B4 has also been observed in a second open conformation in complex with bifonazole that is distinct from the open ligand-free structure. The open tBPA-modified CYP2B4 structure also resembles this conformation. E, the 1-PBI complex of CYP2B4 adopts a conformation that is intermediate to the closed and bifonazole-bound structures. Flexible helices of note have been labeled in B to E.

Fig. 2.

Movement of active site residues in CYP2B-ligand complexes. To accommodate their various substrates and inhibitors, the closed forms of CYP2B enzymes show a reorientation of Phe206, Phe297, and Glu301. A, an overlay of the 1-CPI (yellow) and 4-CPI (blue) CYP2B4 complexes highlights the movement of Phe206 and Phe297. In the CYP2B4 complexes of 1-CPI and clopidogrel, the closed tBPA-modified CYP2B4 structure, and the CYP2B6 complexes of NBP and BP, Phe206 enters the active site, while Phe297 rotates out. In the 4-CPI complexes of CYP2B6 and CYP2B4, and the closed ligand-free and CYP2B4-ticlopidine structures, these two residues swap positions so that Phe206 is out and Phe297 swings in. B, an overlay of the CYP2B4 complexes of 4-CPI (blue), ticlopidine (green), and clopidogrel (teal) shows how Glu301 enters the active site to hydrogen-bond with 4-CPI and moves out of the active site to varying degrees in other CYP2B structures.

Fig. 3.

Active site comparison of CYP2B4 (blue) and CYP2B6 (light green) closed structures. A, an overlay of the 4-CPI complexes of both enzymes shows a remarkably similar active site. The only difference in amino acid side chains is at position 363, which is Ile in CYP2B4 and Leu in CYP2B6. B, small changes in the active sites of these two enzymes result in significant differences in cavity volumes. Rotations of Ile101, Glu301, and Val477 result in active site volumes of 253 ų and 582 ų for CYP2B4 and CYP2B6, respectively.

Fig. 4.

Ticlopidine oxidation by CYP2B enzymes results in a complex mixture of products. CYP2B4 and CYP2B6 produce, to varying degrees, six major metabolites depending on the site of oxidation. These are M1, M2, a dihydrothienopyridinium metabolite (M3), a thienopyridinium metabolite (M4), M5, and M6, which is formed from the dimerization of ticlopidine S-oxide.