Crystal Structures of Human Cytochrome P450 3A4 Bound to Metyrapone and Progesterone

Cytochromes P450 (P450s) metabolize a wide range of endogenous compounds and xenobiotics, such as pollutants, environmental compounds, and drug molecules. The microsomal, membrane-associated, P450 isoforms CYP3A4, CYP2D6, CYP2C9, CYP2C19, CYP2E1, and CYP1A2 are responsible for the oxidative metabolism of more than 90% of marketed drugs. Cytochrome P450 3A4 (CYP3A4) metabolizes more drug molecules than all other isoforms combined. Here we report three crystal structures of CYP3A4: unliganded, bound to the inhibitor metyrapone, and bound to the substrate progesterone. The structures revealed a surprisingly small active site, with little conformational change associated with the binding of either compound. An unexpected peripheral binding site is identified, located above a phenylalanine cluster, which may be involved in the initial recognition of substrates or allosteric effectors.

[1]  T. Ahn,et al.  Membrane properties induced by anionic phospholipids and phosphatidylethanolamine are critical for the membrane binding and catalytic activity of human cytochrome P450 3A4. , 2003, Biochemistry.

[2]  Jean-Paul Renaud,et al.  Conformational heterogeneity of cytochrome P450 3A4 revealed by high pressure spectroscopy. , 2003, Biochemical and biophysical research communications.

[3]  T. Poulos.  Cytochrome P450 flexibility , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[4]  Jose Cosme,et al.  Crystal structure of human cytochrome P450 2C9 with bound warfarin , 2003, Nature.

[5]  Michael J. Hartshorn,et al.  AstexViewerTM †: a visualisation aid for structure-based drug design , 2002, J. Comput. Aided Mol. Des..

[6]  Yoshitsugu Shiro,et al.  Thermophilic cytochrome P450 (CYP119) from Sulfolobus solfataricus: high resolution structure and functional properties. , 2002, Journal of inorganic biochemistry.

[7]  T. Tracy,et al.  Atypical kinetic profiles in drug metabolism reactions. , 2002, Drug metabolism and disposition: the biological fate of chemicals.

[8]  J. Halpert,et al.  Midazolam oxidation by cytochrome P450 3A4 and active-site mutants: an evaluation of multiple binding sites and of the metabolic pathway that leads to enzyme inactivation. , 2002, Molecular pharmacology.

[9]  J. Houston,et al.  Multisite kinetic models for CYP3A4: simultaneous activation and inhibition of diazepam and testosterone metabolism. , 2001, Drug metabolism and disposition: the biological fate of chemicals.

[10]  M. Machius,et al.  Pivotal role of water in the mechanism of P450BM-3. , 2001, Biochemistry.

[11]  A. D. Rodrigues,et al.  Testosterone, 7-benzyloxyquinoline, and 7-benzyloxy-4-trifluoromethyl-coumarin bind to different domains within the active site of cytochrome P450 3A4. , 2001, Drug metabolism and disposition: the biological fate of chemicals.

[12]  J. Halpert,et al.  Phenylalanine and tryptophan scanning mutagenesis of CYP3A4 substrate recognition site residues and effect on substrate oxidation and cooperativity. , 2001, Biochemistry.

[13]  Eric F. Johnson,et al.  Engineering Microsomal Cytochrome P450 2C5 to Be a Soluble, Monomeric Enzyme , 2000, The Journal of Biological Chemistry.

[14]  J B Houston,et al.  CYP3A4 drug interactions: correlation of 10 in vitro probe substrates. , 1999, British journal of clinical pharmacology.

[15]  J. Halpert,et al.  Use of the steroid derivative RPR 106541 in combination with site-directed mutagenesis for enhanced cytochrome P-450 3A4 structure/function analysis. , 1999, The Journal of pharmacology and experimental therapeutics.

[16]  T. Poulos,et al.  Structure of a cytochrome P450-redox partner electron-transfer complex. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[17]  F. Guengerich,et al.  Cytochrome P-450 3A4: regulation and role in drug metabolism. , 1999, Annual review of pharmacology and toxicology.

[18]  D. Kroetz,et al.  Structure-function relationships of human liver cytochromes P450 3A: aflatoxin B1 metabolism as a probe. , 1998, Biochemistry.

[19]  J. Halpert,et al.  Analysis of human cytochrome P450 3A4 cooperativity: construction and characterization of a site-directed mutant that displays hyperbolic steroid hydroxylation kinetics. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[20]  A. Rettie,et al.  Evaluation of atypical cytochrome P450 kinetics with two-substrate models: evidence that multiple substrates can simultaneously bind to cytochrome P450 active sites. , 1998, Biochemistry.

[21]  J. Liu,et al.  Analysis of four residues within substrate recognition site 4 of human cytochrome P450 3A4: role in steroid hydroxylase activity and alpha-naphthoflavone stimulation. , 1998, Archives of biochemistry and biophysics.

[22]  P. Kollman,et al.  Graphical visualization of mean hydration from molecular dynamics simulations. , 1997, Journal of Molecular Graphics and Modelling.

[23]  Grazyna D. Szklarz,et al.  Molecular modeling of cytochrome P450 3A4 , 1997, J. Comput. Aided Mol. Des..

[24]  James R. Halpert,et al.  Alanine-scanning Mutagenesis of a Putative Substrate Recognition Site in Human Cytochrome P450 3A4 , 1997, The Journal of Biological Chemistry.

[25]  A. P. Koley,et al.  Differential Mechanisms of Cytochrome P450 Inhibition and Activation by α-Naphthoflavone* , 1997, The Journal of Biological Chemistry.

[26]  F. Guengerich,et al.  Cooperativity in oxidations catalyzed by cytochrome P450 3A4. , 1997, Biochemistry.

[27]  C. Jefcoate.  Physiological Functions of Cytochrome P450 in Relation to Structure and Regulation , 1996 .

[28]  M H Tarbit,et al.  Molecular modelling of CYP3A4 from an alignment with CYP102: identification of key interactions between putative active site residues and CYP3A-specific chemicals. , 1996, Xenobiotica; the fate of foreign compounds in biological systems.

[29]  A. P. Koley,et al.  CO Binding Kinetics of Human Cytochrome P450 3A4 , 1995, Journal of Biological Chemistry.

[30]  C. Nave.  Radiation damage in protein crystallography , 1995 .

[31]  T. Poulos,et al.  Structure of cytochrome P450eryF involved in erythromycin biosynthesis , 1995, Nature Structural Biology.

[32]  K. Korzekwa,et al.  Activation of CYP3A4: evidence for the simultaneous binding of two substrates in a cytochrome P450 active site. , 1994, Biochemistry.

[33]  E. Johnson,et al.  Modulation of rabbit and human hepatic cytochrome P-450-catalyzed steroid hydroxylations by alpha-naphthoflavone. , 1988, Molecular pharmacology.