Human Cytochrome P450 3A4

  Wyatt Cole '19 and Tim Lewis '18


I. Introduction

Cytochromes P450 are a family of enzymes, which play critical role in the body by metabolizing endogenous compounds, and many xenobiotics or substances foreign to the body. Cytochrome P450 34A (CYP3A4) is one isoform of Cytochromes P450. CYP3A4 is a heme containing enzyme which metabolizes many marketed drugs. Cytochromes P450's many isoforms are responsible for the metabolism of more than 90% of marketed drugs, and CYP3A4 metabolizes more marketed drugs than all the other P450 isoforms combined.Despite CYP3A4’s critical role in drug metabolism our understanding of how it recognizes these varying chemical structures has been limited, without an accurate three-dimensional structure. Researchers have previously been forced to use homology models sharing less than 25% sequence identity with CYP3A4. Williams et al. seek to correct this problem by reporting the crystal structure of both unlinganded CYP3A4 and CYP3A4 bound to either the inhibitor metyrapone of the substrate progesterone. 

II. General Structure

CYP3A4 conforms to many of the patterns seen in P450’s many isoforms such as a small N-terminal domain and a larger helical C-terminal domain. The C-terminal domain contains the enzymes active site with the . This heme iron is ligated by a conserved cysteine Cys-442 and interacts with Arg-105, Trp-126, Arg-130, Arg-375, and Arg-440 .There are two channels in this protein one formed by the and the other formed by the . The B’ and C helices separate these channels; this region can change its conformation depending on the presence or absence of a ligand. CYP34A also has several unique features such as a hydrophobic region located around the loop following the . CYP3A4 also has a strikingly small helix without any secondary structure, in the region following .CYP3A4 also has a , consisting of Phe-108, Phe-213, Phe-215, Phe-219, Phe-220 , Phe-241 and Phe-304, located above the active site and forming a hydrophobic core through pi stacking. Changes in CYP3A4 have been shown to occur in the presence of a substrate effector molecule bound in the active site. The active site of CYP3A4 lies in the helical C-terminal domain and is composed of three subpockets. This active site is surprisingly small particularly when the large diversity of CYP3A4 substrates is taken into account. This small active site is predicted to undergo substantial conformational changes including the movement of the Phe cluster and the to accommodate larger substrates. It is also predicted that movement of the region could also open up the channel and increase the volume of the active site. 

III. Metyrapone and Progesterone Binding

Metyrapone’s binding results in no fundamental protein conformational changes. The crystal structure of CYP34A shows metyrapone bound by an alkyl-pyridine nitrogen to the heme iron. Metyrapone is a small substrate and its binding in the active site leaves room for other molecules to potentially bind. Progesterone also induced very little conformational change binding at a peripheral site close to the Phe cluster and away from the heme iron. Progesterone nestles against the Phe cluster on the side chains of and interacts with the protein through a hydrogen bond between its acetyl oxygen and the amide nitrogen. Although progesterone could potentially bind the active site of CYP3A4 there is no evidence that this actually occurs. Progesterone’s binding in the peripheral binding pocket is supported by the residues, , implicated in progesterone’s homotrophic and heterotrophic cooperativity existing in the vicinity of this pocket.

Peripheral binding of Progesterone to CYP3A4

IV. Ketoconazole Binding

The large antifungal drug ketoconazole induces dramatic conformational changes in CYP3A4. The Crystal structure determined by Ekroos et. al. shows bond in the active site. The keto group of the first ketoconazole molecule is located in the polar pocket, lined by the side chains , Ketoconazole binding is further stabilized in CYP3A4 by chlorobenzyl ring pi-stacking with the side chain of . The other ketoconazole molecule is stacked in an antiparallel orientation above the first. This ketoconazole’s keto group hydrogen bonds with the side chain of . This simultaneous binding of two ketoconazole ligands could be an artifact of high concentrations of ketoconazole used in crystallization. There were notable conformational changes observed in the F and G helices and intervening loops upon ketoconazole binding. In ketoconazole-CYP3A4 complex the is also broken up with some of its hydrophobic chains exposed to the surrounding medium. When ketoconazole is bound Arg-212, which is usually found in CYP3A4’s active site is found on CYP3A4’s surface, which is usually found on the protein surface is found in the active site. There are also distortions seen in the loop connecting the C-terminal and beta-sheets and in the I helix cleft when ketoconazole is bound. 

Phe cluster in unbound CYP3A4

VI. References

Ekroos M, Sjogren T. Structural basis for ligand promiscuity in cytochrome P450 3A4. Proc Natl Acad Sci USA. 2006; 103(37):13682-13687. DOI:10.1073/pnas.0603236103

Williams AP, Cosme J, Vinkovic´ DM, Ward A, Angrove H, Day P, Vonrhein, Tickle I, Jhoti H. Crystal Structures of Human Cytochrome P450 3A4 Bound to Metyrapone and Progesterone. 2004; 5684:683-686. DOI: 10.1126/science.1099736

Poulos T, Finzel BC, Howard AJ. High-resolution Crystal Structure of Cytochrome P450cam. J. Mol. Biol. 1986;195;687-700. PMID: 365642828:4568-4574.

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