Structure and Function of Cytochromes P450 2B: From Mechanism-Based Inactivators to X-Ray Crystal Structures and Back

Abstract

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.

Structural and Biophysical Characterization of Human Cytochromes P450 2B6 and 2A6 Bound to Volatile Hydrocarbons: Analysis and Comparison

“…The structural overlay of CYP2A6 and CYP2B6 in complex with sabinene resulted in an RMSD of ∼0.78 Å (Fig. 3A), which is slightly larger than the RMSD observed between CYP2B4 and CYP2B6 in complex with either 4-CPI (0.65 Å) or amlodipine (0.66 Å) (Halpert, 2011;Shah et al, 2012), and CYP2A6 and CYP2A13 with pilocarpine (0.63 Å) . However, the sequence identity between CYP2A6 and CYP2B6 is only 51%, which is significantly less than that between CYP2B4 and CYP2B6 (78%) or CYP2A6 and CYP2A13 (94%), suggesting that the ability to conform to these ligands outweighs sequence differences in the amino acid sequences of CYP2B6 and CYP2A6.…”

Cytochrome P450 Architecture and Cysteine Nucleophile Placement Impact Raloxifene-Mediated Mechanism-Based Inactivation

“…At the core of P450 enzymes is a porphyrin heme, locked in place by electrostatic interactions with a number of conserved arginine residues and a ring of hydrophobic amino acids that sandwich the heme against the rigid, conserved I helix Yano et al, 2004;Sansen et al, 2007;Halpert, 2011;Shah et al, 2011). Positioning the I helix of each P450 parallel to the z-axis, each of the isoforms was divided into four quadrants to explore the location of the cysteine residues (Fig.…”

Correlating Structure and Function of Drug-Metabolizing Enzymes: Progress and Ongoing Challenges

“…Dynamic relationships between the loop-like structures that form the substrate binding cavity enable adaptations that open and close solvent access channels to facilitate access to and egress from the active site for solvent molecules, substrates, inhibitors, and products (Cojocaru et al, 2007) and that facilitate adaptations to accommodate structurally diverse drugs (Wester et al, 2003;Ekroos and Sjogren, 2006;Halpert, 2011). These adaptations can be important for positioning the substrate near the reactive intermediate and prolongation of residency during the reaction cycle.…”