Structural characterization of human cholesterol 7α-hydroxylase

Hepatic conversion to bile acids is a major elimination route for cholesterol in mammals. CYP7A1 catalyzes the first and rate-limiting step in classic bile acid biosynthesis, converting cholesterol to 7α-hydroxycholesterol. To identify the structural determinants that govern the stereospecific hydroxylation of cholesterol, we solved the crystal structure of CYP7A1 in the ligand-free state. The structure-based mutation T104L in the B′ helix, corresponding to the nonpolar residue of CYP7B1, was used to obtain crystals of complexes with cholest-4-en-3-one and with cholesterol oxidation product 7-ketocholesterol (7KCh). The structures reveal a motif of residues that promote cholest-4-en-3-one binding parallel to the heme, thus positioning the C7 atom for hydroxylation. Additional regions of the binding cavity (most distant from the access channel) are involved to accommodate the elongated conformation of the aliphatic side chain. Structural complex with 7KCh shows an active site rigidity and provides an explanation for its inhibitory effect. Based on our previously published data, we proposed a model of cholesterol abstraction from the membrane by CYP7A1 for metabolism. CYP7A1 structural data provide a molecular basis for understanding of the diversity of 7α-hydroxylases, on the one hand, and cholesterol-metabolizing enzymes adapted for their specific activity, on the other hand. Hepatic conversion to bile acids is a major elimination route for cholesterol in mammals. CYP7A1 catalyzes the first and rate-limiting step in classic bile acid biosynthesis, converting cholesterol to 7α-hydroxycholesterol. To identify the structural determinants that govern the stereospecific hydroxylation of cholesterol, we solved the crystal structure of CYP7A1 in the ligand-free state. The structure-based mutation T104L in the B′ helix, corresponding to the nonpolar residue of CYP7B1, was used to obtain crystals of complexes with cholest-4-en-3-one and with cholesterol oxidation product 7-ketocholesterol (7KCh). The structures reveal a motif of residues that promote cholest-4-en-3-one binding parallel to the heme, thus positioning the C7 atom for hydroxylation. Additional regions of the binding cavity (most distant from the access channel) are involved to accommodate the elongated conformation of the aliphatic side chain. Structural complex with 7KCh shows an active site rigidity and provides an explanation for its inhibitory effect. Based on our previously published data, we proposed a model of cholesterol abstraction from the membrane by CYP7A1 for metabolism. CYP7A1 structural data provide a molecular basis for understanding of the diversity of 7α-hydroxylases, on the one hand, and cholesterol-metabolizing enzymes adapted for their specific activity, on the other hand. The formation of bile salts in the liver is the quantitatively most important pathway of cholesterol elimination from the body (1Chiang J.Y. Regulation of bile acid synthesis.Front. Biosci. 1998; 3: d176-d193Crossref PubMed Scopus (260) Google Scholar, 2Vlahcevic Z.R. Pandak W.M. Stravitz R.T. Regulation of bile acid biosynthesis.Gastroenterol. Clin. North Am. 1999; 28: 1-25Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). Other pathways of cholesterol metabolism include the conversion of cholesterol into steroid hormones and vitamin D3. The maintenance of cholesterol homeostasis in various tissues and cells requires complex interactions of a number of physiological factors (3Chiang J.Y. Bile acids: regulation of synthesis.J. Lipid Res. 2009; 50: 1955-1966Abstract Full Text Full Text PDF PubMed Scopus (1095) Google Scholar, 4Russell D.W. Fifty years of advances in bile acid synthesis and metabolism.J. Lipid Res. 2009; 50: S120-S125Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar), including several cholesterol-metabolizing enzymes of the cytochrome P450 family (CYP). Cytochrome P450 proteins utilize heme cofactor to perform oxidation chemistry with a vast diversity of drugs and endogenous molecules, including steroids, vitamin D, and eicosanoids. Membrane-bound CYPs localized either in the endoplasmic reticulum or mitochondria, where they use different redox partners to shuttle electrons from NADPH to molecular oxygen, resulting in the insertion of one atom of oxygen into the substrate while the other oxygen atom is reduced to water (5Poulos T.L. Johnson E.F. In Structures of cytochrome P450 enzymes.in: Ortiz de Montellano P.R. Cytochrome P450: Structure, Mechanism and Biochemistry. Kluwer Academic/Plenum, New York2005: 87-114Crossref Scopus (111) Google Scholar). The active site is buried in the core of the protein and presents a preformed cavity above the heme connected to the surface through a channel. Despite a unique but rather conserved P450-fold, the molecular mechanisms of CYP substrate specificity and selectivity remain elusive. Cholesterol oxidation products, especially 7-ketocholesterol (7KCh), are highly toxic and associated with chronic diseases including atherosclerotic and neurodegenerative processes (6Lyons M.A. Brown A.J. 7-Ketocholesterol.Int. J. Biochem. Cell Biol. 1999; 31: 369-375Crossref PubMed Scopus (80) Google Scholar, 7Brown A.J. Jessup W. Oxysterols and atherosclerosis.Atherosclerosis. 1999; 142: 1-28Abstract Full Text Full Text PDF PubMed Scopus (762) Google Scholar). 7KCh has potent pharmacological properties to induce inflammation and apoptosis (8Vejux A. Lizard G. Cytotoxic effects of oxysterols associated with human diseases: induction of cell death (apoptosis and/or oncosis), oxidative and inflammatory activities, and phospholipidosis.Mol. Aspects Med. 2009; 30: 153-170Crossref PubMed Scopus (220) Google Scholar). As a photooxidation product of nonenzymatic and possibly enzymatic pathways in the retina, 7KCh might be a pathogenetic factor in age-related macular degeneration (9Rodríguez I.R. Larrayoz I.M. Cholesterol oxidation in the retina: implications of 7KCh formation in chronic inflammation and age-related macular degeneration.J. Lipid Res. 2010; 51: 2847-2862Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). The major routes for 7KCh metabolism include a) conversion into less toxic 27-hydroxylated 7KCh by CYP27A1/CYP46A1 (10Brown A.J. Watts G.F. Burnett J.R. Dean R.T. Jessup W. Sterol 27-hydroxylase acts on 7-ketocholesterol in human atherosclerotic lesions and macrophages in culture.J. Biol. Chem. 2000; 275: 27627-27633Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 11Jessup W. Brown A.J. Novel routes for metabolism of 7-ketocholesterol.Rejuvenation Res. 2005; 8: 9-12Crossref PubMed Scopus (33) Google Scholar, 12Heo G.Y. Bederman I. Mast N. Liao W.L. Turko I.V. Pikuleva I.A. Conversion of 7-ketocholesterol to oxysterol metabolites by recombinant CYP27A1 and retinal pigment epithelial cells.J. Lipid Res. 2011; 52: 1117-1127Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 13Lyons M.A. Brown A.J. Metabolism of an oxysterol, 7-ketocholesterol, by sterol 27-hydroxylase in HepG2 cells.Lipids. 2001; 36: 701-711Crossref PubMed Scopus (45) Google Scholar) and further to more water-soluble metabolites, thus protecting mitochondria from reactive oxygen species; b) esterification including sulfonation (14Fuda H. Javitt N.B. Mitamura K. Ikegawa S. Strott C.A. Oxysterols are substrates for cholesterol sulfotransferase.J. Lipid Res. 2007; 48: 1343-1352Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar); c) lipoprotein-mediated elimina­tion (15Brown A.J. Jessup W. Oxysterols: sources, cellular storage and metabolism, and new insights into their roles in cholesterol homeostasis.Mol. Aspects Med. 2009; 30: 111-122Crossref PubMed Scopus (239) Google Scholar); and d) interconversion to 7β-hydroxycholesterol by 11β-hydroxysteroid dehydrogenase type 1 (16Schweizer R.A. Zurcher M. Balazs Z. Dick B. Odermatt A. Rapid hepatic metabolism of 7-ketocholesterol by 11beta-hydroxysteroid dehydrogenase type 1: species-specific differences between the rat, human, and hamster enzyme.J. Biol. Chem. 2004; 279: 18415-18424Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 17Larsson H. Bottiger Y. Iuliano L. Diczfalusy U. In vivo interconversion of 7beta-hydroxycholesterol and 7-ketocholesterol, potential surrogate markers for oxidative stress.Free Radic. Biol. Med. 2007; 43: 695-701Crossref PubMed Scopus (70) Google Scholar). However, the mechanisms of 7KCh formation and elimination are still not fully understood. Conversion of cholesterol into 7α-hydroxycholesterol by CYP7A1 represents the first and rate-limiting step in the classic pathway of bile salts biosynthesis. Hydroxylation of the ring system of cholesterol in a regio- and stereospecific manner with further oxidation and shortening of the side chain produces water-soluble bile acids with powerful detergent properties to emulsify dietary lipids (4Russell D.W. Fifty years of advances in bile acid synthesis and metabolism.J. Lipid Res. 2009; 50: S120-S125Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar). Bile acids also serve as signaling molecules that bind to G-protein-coupled receptors (GPCRs) and nuclear hormone receptors that regulate lipid, glucose, and energy metabolism (18Chiang J.Y. Bile acid metabolism and signaling.Compr. Physiol. 2013; 3: 1191-1212Crossref PubMed Scopus (746) Google Scholar). A modulation of both oxysterol and bile acid signaling pathways has recently emerged as a source of promising novel drug targets to treat common metabolic and hepatic diseases (19Thomas C. Pellicciari R. Pruzanski M. Auwerx J. Schoonjans K. Targeting bile-acid signalling for metabolic diseases.Nat. Rev. Drug Discov. 2008; 7: 678-693Crossref PubMed Scopus (919) Google Scholar). Bile acid synthesis is tightly regulated through the transcriptional regulation of CYP7A1 (1Chiang J.Y. Regulation of bile acid synthesis.Front. Biosci. 1998; 3: d176-d193Crossref PubMed Scopus (260) Google Scholar) and possibly by the availability of substrate to the enzyme, which is located in the cholesterol-poor endoplasmic reticulum (20Straka M.S. Junker L.H. Zacarro L. Zogg D.L. Dueland S. Everson G.T. Davis R.A. Substrate stimulation of 7 alpha-hydroxylase, an enzyme located in the cholesterol-poor endoplasmic reticulum.J. Biol. Chem. 1990; 265: 7145-7149Abstract Full Text PDF PubMed Google Scholar). In humans, three cytochrome P450 enzymes perform the 7α-hydroxylation reaction: CYP7A1 is specific for cholesterol, CYP7B1 for oxysterols and steroids, and CYP39A1 for 24(S)-hydroxycholesterol. None of these 7α-hydroxylases are structurally characterized, hindering understanding of the molecular mechanisms of their substrate selectivity. Here we present crystal structures of CYP7A1, both unliganded and bound to either the substrate cholest-4-en-3-one or the inhibitor 7KCh. Ligands are bound deep in the active site cavity, isolated from the bulk solvent, in a previously unobserved orientation complemented by unusual structural features of CYP7A1. An asparagine (Asn) residue in place of the highly conserved threonine (Thr) in I helix does not directly interact with cholest-4-en-3-one. Instead, the Asn residue appears to interact with the sixth ligand of the heme iron in the ligand-free and 7KCh structures. Maintaining the network involving the 7-keto group and interaction with the protein's active site residues along with closed conformation of the access channel explain a competitive inhibition by 7KCh. Combining these new findings with previous data prompted us to suggest a working model for cholesterol binding by CYP7A1 from the membrane. The CYP7A1 cDNA was purchased from Origene [accession code TC123882 (NM_000780)] and subcloned into a modified pCW-LIC vector. The N-terminal transmembrane anchor domain (codons 1–24) was replaced with an optimized sequence, MAKKTSS. The C-terminally His5-tagged protein was coexpressed with GroEL/ES (pGro12, Takara Bio Inc.) in Escherichia coli JM109. Cells were lysed by passing through a Microfluidizer (Microfluidics Corp.) at 18,000 psi. Sodium cholate was added to the lysate at a final concentration of 23 mM, and the lysate was incubated at 4°C for 1 h. After centrifugation at 60,000 g for 1 h, protein was purified using metal affinity chromatography on a HiTrap chelating column charged with Ni2+ (Amersham Biosciences) and cation-exchange chromatography using a Source 30S column (Amersham Biosciences). The protein storage buffer was and The molecular of purified protein by was The of purified used in was A P450 protein concentration was from reduced R. The pigment of liver I. for its Biol. Chem. Full Text PDF PubMed Google Scholar). CYP7A1 was in the of cholesterol using the at 1 of the protein with 1 of the and were with as in To obtain a complex with a T104L was and the crystals were in and at and with for in the T104L crystals were in and and are in data were at at The ligand-free structure was solved by molecular with A.J. 2007; PubMed Scopus Google Scholar) and a model from L.H. structure of the human Biol. PubMed Scopus Google Scholar). from and structure and PubMed Scopus Google Scholar) of the with an model and A. 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The of CYP7A1. was in the system at in buffer and The of recombinant proteins were and for at The final of CYP7A1 and P450 were and in was added to the at a final concentration of After of at the was by NADPH to a final concentration of were from the at were with of After and were by centrifugation at for The was and was added to the resulting and were on an with a or an system system with The in the of 7KCh was in the which of 7KCh as CYP7A1 a cytochrome with the conserved structural core of D, and the heme group The most structures in the using the were code and code is a P450 requires molecular oxygen NADPH to perform its as to the However, of the as as CYP7A1 and CYP7B1, is involved in bile acid and requires a cytochrome P450 as an Despite the chemistry of their and different CYP7A1 common structural features with L.H. structure of the human Biol. PubMed Scopus Google Scholar). the helix was to the heme a I helix the helix at to a an Asn residue of the conserved which is involved in oxygen in most and a on the a site for the interaction with the redox CYP7A1 was structurally less to the with the of sterol in the cholesterol CYP7A1 in the of cholesterol a closed conformation with access is the and closed active site was a or the in vivo of the enzyme in the of the substrate and/or physiological in the substrate binding but was to of the substrate in the Additional to and CYP7A1 with a substrate to for the cholesterol An of the closed conformation shows the and orientation of residues the active site cavity and residues that are to interact with the of 7α-hydroxylases and of helix, as potential substrate The helix is to substrate specificity and in and structural the P450 family J. Johnson E.F. cytochrome P450 structural for membrane binding and 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). 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In the group of 7KCh with and the group of A water is the heme with the of the ligand binding which from of the the water a to the 7-keto group of the inhibitor and also to the and the side chain of network might the to the with as the in the of the 7-keto the structural data are with 7KCh competitive inhibition in and that the and interactions are as for the CYP7A1 structures suggest that the steroid the active site with the side chain binding orientation might be the to access a cholesterol from the membrane. that CYP7A1 is into the of the membrane at a of of the In the N-terminal transmembrane helix, the and the helix binding the cholesterol from the of the membrane with cholesterol side chain the of the access channel in or interactions with other proteins induce the that of the substrate access into which cholesterol with its side chain positioning in the active site that the of cholesterol hydroxylation the heme with a model for cholesterol substrate directly from the binding model is by J. 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An positioning of the is in in complex with A. of a membrane cytochrome P450 that the domain to a PubMed Scopus Google Scholar) and the a) a of that its for the and with the helix, the and b) that interact with other protein which substrate of the interaction by the might also be a of proteins to and might be further adapted for chemistry In CYP7A1 structures identify residues involved in cholest-4-en-3-one binding and specific interactions with the inhibitor 7KCh. structural features that to the cholesterol ring system are not unique to CYP7A1 and different chemistry in other the for cholesterol-metabolizing cytochrome to a substrate from the we suggest that orientation of cholesterol in the different of the be for further binding by CYP7A1 or in complex with protein The and structure factors of the CYP7A1 structures in the for Structural the complex with and complex with The K. for the model of the cholest-4-en-3-one complex and for 7-ketocholesterol cytochrome P450

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