Jose Cosme

Crystal structure of human cytochrome P450 2C9 with bound warfarin

Cytochrome P450 proteins (CYP450s) are membrane-associated haem proteins that metabolize physiologically important compounds in many species of microorganisms, plants and animals. Mammalian CYP450s recognize and metabolize diverse xenobiotics such as drug molecules, environmental compounds and pollutants. Human CYP450 proteins CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 are the major drug-metabolizing isoforms, and contribute to the oxidative metabolism of more than 90% of the drugs in current clinical use. Polymorphic variants have also been reported for some CYP450 isoforms, which has implications for the efficacy of drugs in individuals, and for the co-administration of drugs. The molecular basis of drug recognition by human CYP450s, however, has remained elusive. Here we describe the crystal structure of a human CYP450, CYP2C9, both unliganded and in complex with the anti-coagulant drug warfarin. The structure defines unanticipated interactions between CYP2C9 and warfarin, and reveals a new binding pocket. The binding mode of warfarin suggests that CYP2C9 may undergo an allosteric mechanism during its function. The newly discovered binding pocket also suggests that CYP2C9 may simultaneously accommodate multiple ligands during its biological function, and provides a possible molecular basis for understanding complex drug–drug interactions.

Mammalian microsomal cytochrome P450 monooxygenase: structural adaptations for membrane binding and functional diversity.

Microsomal cytochrome P450s participate in xenobiotic detoxification, procarcinogen activation, and steroid hormone synthesis. The first structure of a mammalian microsomal P450 suggests that the association of P450s with the endoplasmic reticulum involves a hydrophobic surface of the protein formed by noncontiguous portions of the polypeptide chain. This interaction places the entrance of the putative substrate access channel in or near the membrane and orients the face of the protein proximal to the heme cofactor perpendicular to the plane of the membrane for interaction with the P450 reductase. This structure offers a template for modeling other mammalian P450s and should aid drug discovery and the prediction of drug-drug interactions.

Engineering Microsomal Cytochrome P450 2C5 to Be a Soluble, Monomeric Enzyme MUTATIONS THAT ALTER AGGREGATION, PHOSPHOLIPID DEPENDENCE OF CATALYSIS, AND MEMBRANE BINDING

Deletion of the N-terminal membrane-spanning domain from microsomal P450s 2C5 and 2C3 generates the enzymes, 2C5dH and 2C3dH, that exhibit a salt-dependent association with membranes indicating that they retain a monofacial membrane interaction domain. The two proteins are tetramers and dimers, respectively, in high salt buffers, and only 2C5dH requires phospholipids to reconstitute fully the catalytic activity of the enzyme. Amino acid residues derived from P450 2C3dH between residues 201 and 210 were substituted for the corresponding residues in P450 2C5 to identify those that would diminish the membrane interaction, the phospholipid dependence of catalysis, and aggregation of 2C5dH. Each of four substitutions, N202H, I207L, S209G, and S210T, diminished the aggregation of P450 2C5dH and produced a monomeric enzyme. The N202H and I207L mutations also diminished the stimulation of catalytic activity by phospholipid and reduced the binding of P450 2C5dH to phospholipid vesicles. The modified enzymes exhibit rates of progesterone 21-hydroxylation that are similar to that of P450 2C5dH. These conditionally membrane-bound P450s with improved solubility in high salt buffers are suitable for crystallization and structural determination by x-ray diffraction studies.

Microsomal cytochrome P450 2C5: comparison to microbial P450s and unique features.

Although microsomal P450s represent the majority of P450s, only microbial P450s have been amenable to crystal structure solution. We have recently solved the first crystal structure of a microsomal P450, 2C5, a progesterone hydroxylase from rabbit. We discuss the features of the protein in common with existing structures of microbial P450s and limitations of homology modeling mammalian P450s based on the microbial structures. Unique features involving membrane, substrate and cytochrome P450 reductase interactions are also discussed.

Analyzing binding of N-terminal truncated, microsomal cytochrome P450s to membranes.

Publisher Summary This chapter describes two approaches for analysis of the effects of mutations on the binding of N-terminal-truncated microsomal P450s to phospholipid membranes. Detailed knowledge of the membrane association of cytochrome P450s is important for understanding conformational changes, dynamic interactions with redox partners, substrate accessibility, heme topology, and aggregation. Identification of the structural features that underlie the association of P450 enzymes with biological membranes will contribute to understanding the role of the membrane in redox partner interactions and substrate binding. The assays described in the chapter provide a basis for ascertaining the effects of protein modification on membrane binding and, by inference, the role of specific regions of the protein in membrane interactions. These assays have implicated the region between F and G helices of P450 2C5 in membrane interactions and in the aggregation of the protein when dissociated from membranes.