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• W2144999739 abstract "The human cytochrome P450 17A1 (CYP17A1) enzyme operates at a key juncture of human steroidogenesis, controlling the levels of mineralocorticoids influencing blood pressure, glucocorticoids involved in immune and stress responses, and androgens and estrogens involved in development and homeostasis of reproductive tissues. Understanding CYP17A1 multifunctional biochemistry is thus integral to treating prostate and breast cancer, subfertility, blood pressure, and other diseases. CYP17A1 structures with all four physiologically relevant steroid substrates suggest answers to four fundamental aspects of CYP17A1 function. First, all substrates bind in a similar overall orientation, rising ∼60° with respect to the heme. Second, both hydroxylase substrates pregnenolone and progesterone hydrogen bond to Asn202 in orientations consistent with production of 17α-hydroxy major metabolites, but functional and structural evidence for an A105L mutation suggests that a minor conformation may yield the minor 16α-hydroxyprogesterone metabolite. Third, substrate specificity of the subsequent 17,20-lyase reaction may be explained by variation in substrate height above the heme. Although 17α-hydroxyprogesterone is only observed farther from the catalytic iron, 17α-hydroxypregnenolone is also observed closer to the heme. In conjunction with spectroscopic evidence, this suggests that only 17α-hydroxypregnenolone approaches and interacts with the proximal oxygen of the catalytic iron-peroxy intermediate, yielding efficient production of dehydroepiandrosterone as the key intermediate in human testosterone and estrogen synthesis. Fourth, differential positioning of 17α-hydroxypregnenolone offers a mechanism whereby allosteric binding of cytochrome b5 might selectively enhance the lyase reaction. In aggregate, these structures provide a structural basis for understanding multiple key reactions at the heart of human steroidogenesis. The human cytochrome P450 17A1 (CYP17A1) enzyme operates at a key juncture of human steroidogenesis, controlling the levels of mineralocorticoids influencing blood pressure, glucocorticoids involved in immune and stress responses, and androgens and estrogens involved in development and homeostasis of reproductive tissues. Understanding CYP17A1 multifunctional biochemistry is thus integral to treating prostate and breast cancer, subfertility, blood pressure, and other diseases. CYP17A1 structures with all four physiologically relevant steroid substrates suggest answers to four fundamental aspects of CYP17A1 function. First, all substrates bind in a similar overall orientation, rising ∼60° with respect to the heme. Second, both hydroxylase substrates pregnenolone and progesterone hydrogen bond to Asn202 in orientations consistent with production of 17α-hydroxy major metabolites, but functional and structural evidence for an A105L mutation suggests that a minor conformation may yield the minor 16α-hydroxyprogesterone metabolite. Third, substrate specificity of the subsequent 17,20-lyase reaction may be explained by variation in substrate height above the heme. Although 17α-hydroxyprogesterone is only observed farther from the catalytic iron, 17α-hydroxypregnenolone is also observed closer to the heme. In conjunction with spectroscopic evidence, this suggests that only 17α-hydroxypregnenolone approaches and interacts with the proximal oxygen of the catalytic iron-peroxy intermediate, yielding efficient production of dehydroepiandrosterone as the key intermediate in human testosterone and estrogen synthesis. Fourth, differential positioning of 17α-hydroxypregnenolone offers a mechanism whereby allosteric binding of cytochrome b5 might selectively enhance the lyase reaction. In aggregate, these structures provide a structural basis for understanding multiple key reactions at the heart of human steroidogenesis. The cytochrome P450 superfamily of heme monooxygenases performs diverse physiological functions, ranging from drug and xenobiotic metabolism to hormone and vitamin biosynthesis. The human cytochrome P450 (P450) 3The abbreviations used are: P450cytochrome P450b5cytochrome b5RMSDroot mean square deviation. enzyme 17A1 (CYP17A1) functions specifically at a critical juncture in human steroidogenesis (1Miller W.L. Auchus R.J. The molecular biology, biochemistry, and physiology of human steroidogenesis and its disorders.Endocr. Rev. 2011; 32: 81-151Crossref PubMed Scopus (1369) Google Scholar). Its initial substrates are also substrates for mineralocorticoid biosynthesis by other enzymes. CYP17A1 catalysis leads to either steroid precursors of glucocorticoids like cortisol that regulate immune response or androgens like testosterone that drive the development and maintenance of male characteristics or are converted to estrogens in females (2Gilep A.A. Sushko T.A. Usanov S.A. At the crossroads of steroid hormone biosynthesis: the role, substrate specificity and evolutionary development of CYP17.Biochim. Biophys. Acta. 2011; 1814: 200-209Crossref PubMed Scopus (88) Google Scholar). In later life, however, androgens drive the development of prostate cancer, the cancer of highest incidence and the second leading cause of cancer deaths in American men, whereas estrogens are a long recognized driver of hormone-responsive breast cancer (3Edwards B.K. Noone A.M. Mariotto A.B. Simard E.P. Boscoe F.P. Henley S.J. Jemal A. Cho H. Anderson R.N. Kohler B.A. Eheman C.R. Ward E.M. Annual report to the nation on the status of cancer, 1975–2010, featuring prevalence of comorbidity and impact on survival among persons with lung, colorectal, breast, or prostate cancer.Cancer. 2014; 120: 1290-1314Crossref PubMed Scopus (842) Google Scholar). Thus this enzyme has garnered substantial interest as a relatively new drug target, validated by successful use of the CYP17A1 inhibitor abiraterone in men with castration-resistant prostate cancer (4Ferraldeschi R. de Bono J. Agents that target androgen synthesis in castration-resistant prostate cancer.Cancer J. 2013; 19: 34-42Crossref PubMed Scopus (19) Google Scholar, 5de Bono J.S. Logothetis C.J. Molina A. Fizazi K. North S. Chu L. Chi K.N. Jones R.J. Goodman Jr., O.B. Saad F. Staffurth J.N. Mainwaring P. Harland S. Flaig T.W. Hutson T.E. Cheng T. Patterson H. Hainsworth J.D. Ryan C.J. Sternberg C.N. Ellard S.L. Fléchon A. Saleh M. Scholz M. Efstathiou E. Zivi A. Bianchini D. Loriot Y. Chieffo N. Kheoh T. Haqq C.M. Scher H.I. Abiraterone and increased survival in metastatic prostate cancer.New Engl. J. Med. 2011; 364: 1995-2005Crossref PubMed Scopus (3391) Google Scholar, 6Auchus M.L. Auchus R.J. Human steroid biosynthesis for the oncologist.J. Investig. Med. 2012; 60: 495-503Crossref PubMed Scopus (46) Google Scholar) and its current evaluation in breast cancer patients. cytochrome P450 cytochrome b5 root mean square deviation. Abiraterone acetate, the Food and Drug Administration-approved prodrug form of this CYP17A1 inhibitor, improves overall survival in men with metastatic castration-resistant prostate cancer, including patients for whom the disease has progressed following chemotherapy, with compounds such as docetaxel and the androgen receptor blocker enzalutamide (5de Bono J.S. Logothetis C.J. Molina A. Fizazi K. North S. Chu L. Chi K.N. Jones R.J. Goodman Jr., O.B. Saad F. Staffurth J.N. Mainwaring P. Harland S. Flaig T.W. Hutson T.E. Cheng T. Patterson H. Hainsworth J.D. Ryan C.J. Sternberg C.N. Ellard S.L. Fléchon A. Saleh M. Scholz M. Efstathiou E. Zivi A. Bianchini D. Loriot Y. Chieffo N. Kheoh T. Haqq C.M. Scher H.I. Abiraterone and increased survival in metastatic prostate cancer.New Engl. J. Med. 2011; 364: 1995-2005Crossref PubMed Scopus (3391) Google Scholar, 7Loriot Y. Bianchini D. Ileana E. Sandhu S. Patrikidou A. Pezaro C. Albiges L. Attard G. Fizazi K. De Bono J.S. Massard C. Antitumour activity of abiraterone acetate against metastatic castration-resistant prostate cancer progressing after docetaxel and enzalutamide (MDV3100).Ann. Oncol. 2013; 24: 1807-1812Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar). Abiraterone binds with high affinity to the CYP17A1 active site heme iron (8DeVore N.M. Scott E.E. Structures of cytochrome P450 17A1 with prostate cancer drugs abiraterone and TOK-001.Nature. 2012; 482: 116-119Crossref PubMed Scopus (252) Google Scholar) that is essential for catalysis, which effectively and systemically prevents androgen production. However, by doing so, this inhibitor also increases the pool of precursors for mineralocorticoid production and halts CYP17A1-mediated production of glucocorticoids, which occurs in the same active site and also requires the heme iron. The resulting steroid imbalances in patients treated with abiraterone can frequently lead to hypertension, hypokalemia, and adrenocortical insufficiency, which must then be monitored and treated with additional drugs (9Pia A. Vignani F. Attard G. Tucci M. Bironzo P. Scagliotti G. Arlt W. Terzolo M. Berruti A. Strategies for managing ACTH dependent mineralocorticoid excess induced by abiraterone.Cancer Treat. Rev. 2013; 39: 966-973Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Furthermore, there is some evidence that the increase in mineralocorticoids associated with complete inhibition of CYP17A1 may facilitate the flow of androgen precursors through a “backdoor” androgen biosynthesis pathway (10Attard G. Reid A.H. Auchus R.J. Hughes B.A. Cassidy A.M. Thompson E. Oommen N.B. Folkerd E. Dowsett M. Arlt W. de Bono J.S. Clinical and biochemical consequences of CYP17A1 inhibition with abiraterone given with and without exogenous glucocorticoids in castrate men with advanced prostate cancer.J. Clin. Endocrinol. Metab. 2012; 97: 507-516Crossref PubMed Scopus (202) Google Scholar), proposed to provide a possible escape route that could permit cancer progression. Selective inhibition of the CYP17A1-mediated androgen biosynthesis proven to increase overall survival, whereas sparing CYP17A1-mediated glucocorticoid biosynthesis to prevent corticosteroid imbalances would ameliorate both of these clinically relevant issues for prostate cancer patients. CYP17A1 impairment has also been associated with Cushing's syndrome (11Ogo A. Haji M. Ohashi M. Nawata H. Markedly increased expression of cytochrome P-450 17α-hydroxylase (P-450c17) mRNA in adrenocortical adenomas from patients with Cushing's syndrome.Mol. Cell Endocrinol. 1991; 80: 83-89Crossref PubMed Scopus (22) Google Scholar), some forms of congenital adrenal hyperplasia (12Maitra A. Shirwalkar H. Congenital adrenal hyperplasia: biochemical and molecular perspectives.Indian J. Exp. Biol. 2003; 41: 701-709PubMed Google Scholar), and polycystic ovary syndrome (13Qin K.N. Rosenfield R.L. Role of cytochrome P450c17 in polycystic ovary syndrome.Mol. Cell Endocrinol. 1998; 145: 111-121Crossref PubMed Scopus (68) Google Scholar, 14Arlt W. Martens J.W. Song M. Wang J.T. Auchus R.J. Miller W.L. Molecular evolution of adrenarche: Structural and functional analysis of p450c17 from four primate species.Endocrinology. 2002; 143: 4665-4672Crossref PubMed Scopus (86) Google Scholar, 15Strauss 3rd., J.F. Some new thoughts on the pathophysiology and genetics of polycystic ovary syndrome.Ann. N.Y. Acad. Sci. 2003; 997: 42-48Crossref PubMed Scopus (72) Google Scholar). Substantial potential for improving prostate cancer treatment and therapies for these other diseases thus lies in an improved understanding of the mechanisms whereby CYP17A1 performs catalysis. In general, CYP17A1 hydroxylates the mineralocorticoid precursor steroids pregnenolone and progesterone to yield 17α-hydroxypregnenolone and 17α-hydroxyprogesterone, respectively (see Fig. 1). These resulting C17-hydroxylated steroids can serve as substrates for glucocorticoid biosynthesis or for the subsequent CYP17A1-mediated 17,20-lyase reaction to yield the androgens dehydroepiandrosterone or androstenedione (see Fig. 1). Functional variations on this general pathway for different substrates provide clues to key protein/small molecule interactions that direct catalysis. First, although human CYP17A1 hydroxylates pregnenolone (a Δ5,3-ol steroid) only at carbon 17, progesterone (the corresponding Δ4,3-keto steroid) is hydroxylated at C17 as the major product, but also at C16 to generate an additional minor product (see Fig. 1) (14Arlt W. Martens J.W. Song M. Wang J.T. Auchus R.J. Miller W.L. Molecular evolution of adrenarche: Structural and functional analysis of p450c17 from four primate species.Endocrinology. 2002; 143: 4665-4672Crossref PubMed Scopus (86) Google Scholar, 16Swart P. Swart A.C. Waterman M.R. Estabrook R.W. Mason J.I. Progesterone 16α-hydroxylase activity is catalyzed by human cytochrome P450 17α-hydroxylase.J. Clin. Endocrinol. Metab. 1993; 77: 98-102PubMed Google Scholar). It is known that in other species CYP17A1 with Leu at position 105 generates much less 16α-hydroxyprogesterone (14Arlt W. Martens J.W. Song M. Wang J.T. Auchus R.J. Miller W.L. Molecular evolution of adrenarche: Structural and functional analysis of p450c17 from four primate species.Endocrinology. 2002; 143: 4665-4672Crossref PubMed Scopus (86) Google Scholar) and that an A105L mutation in the human CYP17A1 enzyme decreases progesterone 16α-hydroxylation and increases its 17α-hydroxylation (17Swart A.C. Storbeck K.H. Swart P. A single amino acid residue, Ala 105, confers 16α-hydroxylase activity to human cytochrome P450 17α-hydroxylase/17,20-lyase.J. Steroid Biochem. Mol. Biol. 2010; 119: 112-120Crossref PubMed Scopus (42) Google Scholar). Suggested to somehow alter substrate position in the CYP17A1 active site or alter protein flexibility, the structural basis for the effects of the A105L mutation on substrate and regioselectivity of hydroxylation have not been elucidated experimentally. Second, CYP17A1 also performs a carbon-carbon bond cleavage, which is unusual for cytochrome P450 enzymes (18Sohl C.D. Guengerich F.P. Kinetic analysis of the three-step steroid aromatase reaction of human cytochrome P450 19A1.J. Biol. Chem. 2010; 285: 17734-17743Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). For human CYP17A1, this 17,20-lyase reaction proceeds far less efficiently for the Δ4,3-keto 17α-hydroxyprogesterone substrate than for its counterpart, the Δ5,3-ol 17α-hydroxypregnenolone (see Fig. 1). As a result, the Δ5,3-ol 17α-hydroxypregnenolone 17,20-lyase product dehydroepiandrosterone (see Fig. 1) is the physiologically relevant intermediate in the formation of all human androgens and estrogens (19Flück C.E. Miller W.L. Auchus R.J. The 17,20-lyase activity of cytochrome p450c17 from human fetal testis favors the delta5 steroidogenic pathway.J. Clin. Endocrinol. Metab. 2003; 88: 3762-3766Crossref PubMed Scopus (124) Google Scholar). Although hydroxylation is well established to occur by the Groves hydrogen abstraction/oxygen rebound mechanism mediated by an Fe(IV)-oxo catalytic intermediate called Compound I (20Guengerich F.P. Mechanisms of cytochrome P450 substrate oxidation: MiniReview.J. Biochem. Mol. Toxicol. 2007; 21: 163-168Crossref PubMed Scopus (169) Google Scholar, 21Groves J.T. McClusky G.A. White R.E. Coon M.J. Aliphatic hydroxylation by highly purified liver microsomal cytochrome P-450. Evidence for a carbon radical intermediate.Biochem. Biophys. Res. Commun. 1978; 81: 154-160Crossref PubMed Scopus (479) Google Scholar) (see Fig. 1), a mechanism for cleavage of the bond between carbons 17 and 20 has been an ongoing debate in the literature. Recent data, however, favors a ferric peroxy anion intermediate (see Fig. 1) (22Akhtar M. Corina D. Miller S. Shyadehi A.Z. Wright J.N. Mechanism of the acyl-carbon cleavage and related reactions catalyzed by multifunctional P-450s: Studies on cytochrome P-450(17)α.Biochemistry. 1994; 33: 4410-4418Crossref PubMed Scopus (105) Google Scholar, 23Gregory M.C. Denisov I.G. Grinkova Y.V. Khatri Y. Sligar S.G. Kinetic solvent isotope effect in human P450 CYP17A1-mediated androgen formation: evidence for a reactive peroxoanion intermediate.J. Am. Chem. Soc. 2013; 135: 16245-16247Crossref PubMed Scopus (50) Google Scholar), and spectroscopic evidence has suggested that the two 17α-hydroxy steroids might interact differentially with this peroxy intermediate (24Gregory M. Mak P.J. Sligar S.G. Kincaid J.R. Differential hydrogen bonding in human CYP17 dictates hydroxylation versus lyase chemistry.Angew. Chem. Int. Ed. Engl. 2013; 52: 5342-5345Crossref PubMed Scopus (49) Google Scholar). Third, although the presence of cytochrome b5 has relatively little effect on the hydroxylation reactions, the presence of this small heme protein (25Auchus R.J. Lee T.C. Miller W.L. Cytochrome b5 augments the 17,20-lyase activity of human P450c17 without direct electron transfer.J. Biol. Chem. 1998; 273: 3158-3165Abstract Full Text Full Text PDF PubMed Scopus (475) Google Scholar, 26Katagiri M. Kagawa N. Waterman M.R. The role of cytochrome b5 in the biosynthesis of androgens by human P450c17.Arch. Biochem. Biophys. 1995; 317: 343-347Crossref PubMed Scopus (208) Google Scholar, 27Lee-Robichaud P. Wright J.N. Akhtar M.E. Akhtar M. Modulation of the activity of human 17α-hydroxylase-17,20-lyase (CYP17) by cytochrome b5: endocrinological and mechanistic implications.Biochem. J. 1995; 308: 901-908Crossref PubMed Scopus (133) Google Scholar, 28Onoda M. Hall P.F. Cytochrome b5 stimulates purified testicular microsomal cytochrome P-450 (C21 side-chain cleavage).Biochem. Biophys. Res. Commun. 1982; 108: 454-460Crossref PubMed Scopus (118) Google Scholar) substantially and selectively facilitates the 17,20-lyase reaction. Compartmentalization of b5 and developmental changes in b5 levels control the tissue specificity and timing of androgen production in humans. Individuals with nonfunctional b5 are unable to perform the lyase reaction and produce sex steroids, although the hydroxylase reaction required for glucocorticoid synthesis is operational (29Idkowiak J. Randell T. Dhir V. Patel P. Shackleton C.H. Taylor N.F. Krone N. Arlt W. A missense mutation in the human cytochrome b5 gene causes 46, XY disorder of sex development due to true isolated 17,20-lyase deficiency.J. Clin. Endocrinol. Metab. 2012; 97: E465-E475Crossref PubMed Scopus (74) Google Scholar, 30Kok R.C. Timmerman M.A. Wolffenbuttel K.P. Drop S.L. de Jong F.H. Isolated 17,20-lyase deficiency due to the cytochrome b5 mutation W27X.J. Clin. Endocrinol. Metab. 2010; 95: 994-999Crossref PubMed Scopus (84) Google Scholar). Facilitation of the lyase reaction by b5 occurs without electron delivery (25Auchus R.J. Lee T.C. Miller W.L. Cytochrome b5 augments the 17,20-lyase activity of human P450c17 without direct electron transfer.J. Biol. Chem. 1998; 273: 3158-3165Abstract Full Text Full Text PDF PubMed Scopus (475) Google Scholar). Thus it has been suggested that b5 might selectively stabilize the intermediate in the lyase reaction or cause substrates to assume orientations in the CYP17A1 active site more favorable for the lyase chemistry, but the mechanism remains unresolved (31Naffin-Olivos J.L. Auchus R.J. Human cytochrome b5 requires residues E48 and E49 to stimulate the 17,20-lyase activity of cytochrome P450c17.Biochemistry. 2006; 45: 755-762Crossref PubMed Scopus (61) Google Scholar). The structural basis for each of these effects—hydroxylase substrate regioselectivity, lyase reaction selectivity for the Δ4,3-keto versus Δ5,3-ol 17-hydroxylated substrate, and cytochrome b5 facilitation of the lyase versus hydroxylase reaction—is unknown. Homology models and docking studies have suggested that substrates were likely to orient essentially parallel to the plane of the heme (32Auchus R.J. Miller W.L. Molecular modeling of human P450c17 (17α-hydroxylase/17,20-lyase): insights into reaction mechanisms and effects of mutations.Mol. Endocrinol. 1999; 13: 1169-1182Crossref PubMed Google Scholar) and proposed a “bi-lobed” active site to carry out the separate hydroxylase and lyase reactions (33Schappach A. Höltje H.D. Molecular modelling of 17α-hydroxylase-17,20-lyase.Pharmazie. 2001; 56: 435-442PubMed Google Scholar, 34Haider S.M. Patel J.S. Poojari C.S. Neidle S. Molecular modeling on inhibitor complexes and active-site dynamics of cytochrome P450 C17, a target for prostate cancer therapy.J. Mol. Biol. 2010; 400: 1078-1098Crossref PubMed Scopus (26) Google Scholar, 35Lin D. Zhang L.H. Chiao E. Miller W.L. Modeling and mutagenesis of the active site of human P450c17.Mol. Endocrinol. 1994; 8: 392-402Crossref PubMed Scopus (0) Google Scholar, 36Laughton C.A. Neidle S. Zvelebil M.J. Sternberg M.J. A molecular model for the enzyme cytochrome P450(17α), a major target for the chemotherapy of prostatic cancer.Biochem. Biophys. Res. Commun. 1990; 171: 1160-1167Crossref PubMed Scopus (79) Google Scholar). The only known structures of CYP17A1, in the presence of the steroidal inhibitors abiraterone or TOK-001, were published recently (8DeVore N.M. Scott E.E. Structures of cytochrome P450 17A1 with prostate cancer drugs abiraterone and TOK-001.Nature. 2012; 482: 116-119Crossref PubMed Scopus (252) Google Scholar). These steroidal inhibitors both orient more nearly perpendicular to the heme, evoking the prediction of a similar binding mode for substrates (8DeVore N.M. Scott E.E. Structures of cytochrome P450 17A1 with prostate cancer drugs abiraterone and TOK-001.Nature. 2012; 482: 116-119Crossref PubMed Scopus (252) Google Scholar), but the actual binding of CYP17A1 substrates is unknown. To probe the binding orientations of the physiologically relevant substrates and the structural basis of CYP17A1 function, a series of experimental x-ray structures were generated for CYP17A1 in complexes with both hydroxylase and both lyase substrates with the mutation A105L. Comparisons among these structures identify steric and hydrogen bonding interactions between CYP17A1 and distal portions of the substrate that play key roles in modulating spatial relationships between sites of metabolism on the opposite end of substrates and the catalytic heme iron. A steric rationale is provided for hydroxylase regioselectivity, whereas differences observed for hydrogen bonding and substrate positioning may form the basis for substrate selectivity of the lyase reaction. This new information informs a working hypothesis for how cytochrome b5 might selectively facilitate the lyase reaction. The CYP17A1 gene in the pCWori+ vector has an N-terminal truncation of residues 1–19, a slight modification of the new N terminus, and a C-terminal 4× histidine tag, as described (8DeVore N.M. Scott E.E. Structures of cytochrome P450 17A1 with prostate cancer drugs abiraterone and TOK-001.Nature. 2012; 482: 116-119Crossref PubMed Scopus (252) Google Scholar). The A105L mutation was incorporated using the QuikChange Lightning site-directed mutagenesis kit (Agilent). The resulting pCW17A1Δ19H plasmid containing either the wild type or A105L sequence was transformed into Escherichia coli JM109 cells by heat shock at 42 °C, spread on lysogeny broth agarose plates containing 50 μg/ml ampicillin and incubated overnight at 37 °C. To select for the pCWori+ vector, all expression media were supplemented with 50 μg/ml ampicillin. Lysogeny broth (5 ml) was inoculated with a single colony from the aforementioned plate and incubated at 37 °C with shaking at 250 rpm for 6 h. Overnight cultures (200 ml of lysogeny broth) were inoculated with 50 μl of the initial culture and incubated at 37 °C for 18 h with 250 rpm shaking. Terrific Broth (1 liter/2.8-liter Fernbach flask) was inoculated with 10 ml of the overnight culture. This culture was grown at 37 °C with shaking at 250 rpm until reaching an optical density of 0.5 at 600 nm. Overexpression was then induced by the addition of isopropyl β-d-1-thiogalactopyranoside (0.5 mm). The heme precursor δ-aminolevulinic acid was added to 0.61 mm. Cultures were grown at 28 °C with shaking at 140 rpm for an additional 72 h. The cells were then collected by centrifugation at 6300 × g for 10 min. Cells were resuspended in 50 mm Tris-HCl, pH 7.4, 20% (v/v) glycerol, and 300 mm NaCl and stored at −80 °C until purification. Cells were thawed and lysed by sonication (six times for 30 s at 1-min intervals on ice). The resulting lysate was centrifuged at 9900 × g for 15 min. Membrane proteins were extracted by stirring this lysate in the presence 2% (v/v) Emulgen 913 (Desert Biologicals) for 90 min, followed by ultracentrifugation (100,000 × g for 1 h). The resulting supernatant was loaded on nickel-nitrilotriacetic acid-agarose resin (Qiagen) pre-equilibrated with Ni buffer (50 mm Tris-HCl, pH 7.4, 20% (v/v) glycerol, 300 mm NaCl, and 0.2% (v/v) Emulgen 913). The resin was subsequently washed with 2 column volumes of Ni buffer, 6 column volumes of Ni buffer supplemented with 100 mm glycine, and eluted using 4 column volumes of Ni buffer supplemented with 100 mm glycine and 80 mm histidine. Elution fractions were pooled based on absorbance of the heme Soret peak, diluted 4.2-fold in CM buffer (50 mm Tris-HCl, pH 7.4, 20% (v/v) glycerol, and 100 mm glycine), supplemented with 0.2% (v/v) Emulgen 913, and loaded onto a 5-ml carboxymethyl-Sepharose fast-flow column (GE Healthcare) previously equilibrated with CM buffer. The column was washed with 10 column volumes of CM buffer and eluted with CM buffer supplemented with 500 mm NaCl. Fractions were pooled based on the heme Soret absorbance and concentrated to ∼1 ml. Concentrated protein was injected onto a Superdex 200 gel filtration column (GE Healthcare) pre-equilibrated with buffer containing 50 mm Tris-HCl, pH 7.4, 20% (v/v) glycerol, 100 mm glycine, and 500 mm NaCl. The major peak with absorbance for the heme was collected and concentrated. All purification was performed at 4 °C. CYP17A1 A105L protein generated for crystallization studies was purified with 50 μm of one of the ligands present in all purification buffers. CYP17A1 A105L was crystallized by hanging drop vapor diffusion. Purified protein (∼29 mg/ml) containing either 50 μm of one of the substrates or 10 μm abiraterone and 0.5% (v/v) Emulgen 913 was mixed 1:1 to form 2-μl drops that were then equilibrated against precipitant solution at 20 °C. The precipitant solution used to crystallize CYP17A1 A105L with progesterone, pregnenolone, 17α-hydroxypregnenolone, and abiraterone consisted of 100 mm Tris-HCl, pH 8.5, 25% (v/v) PEG 4000 (Hampton Research), 150 mm magnesium chloride hexahydrate, and 4–6% (v/v) glycerol. The precipitant solution used to crystallize CYP17A1 A105L with 17α-hydroxyprogesterone contained 175 mm Tris-HCl, pH 8.5, 30% (v/v) PEG 3350 (Hampton Research), 250 mm lithium sulfate, and 3% (v/v) glycerol. Crystals were cryoprotected in a 7:3 mixture of mother liquor and 80% (v/v) glycerol and flash cooled in liquid nitrogen. Diffraction data were collected on Beamlines 14-1 and 12-2 of the Stanford Synchrotron Radiation Lightsource and processed using XDS (37Kabsch W. XDS.Acta Crystallogr. D Biol. Crystallogr. 2010; 66: 125-132Crossref PubMed Scopus (11232) Google Scholar). Data collection and refinement statistics and deposition codes in the Protein Data Bank are given in Table 1. Structures were solved by molecular replacement using Phaser (38McCoy A.J. Grosse-Kunstleve R.W. Adams P.D. Winn M.D. Storoni L.C. Read R.J. Phaser crystallographic software.J. Appl. Crystallogr. 2007; 40: 658-674Crossref PubMed Scopus (14444) Google Scholar) and the structure of wild type CYP17A1 (Protein Data Bank codes 3RUK or 3SWZ (8DeVore N.M. Scott E.E. Structures of cytochrome P450 17A1 with prostate cancer drugs abiraterone and TOK-001.Nature. 2012; 482: 116-119Crossref PubMed Scopus (252) Google Scholar)) as a search model using data to the respective resolution cutoff for the different structures (2.5–3.0 Å), yielding log likelihoods of 9,094–13,090. Model building and refinement were performed iteratively with Coot (39Emsley P. Lohkamp B. Scott W.G. Cowtan K. Features and development of Coot.Acta Crystallogr. D Biol. Crystallogr. 2010; 66: 486-501Crossref PubMed Scopus (17085) Google Scholar) and PHENIX (40Adams P.D. Afonine P.V. Bunkóczi G. Chen V.B. Davis I.W. Echols N. Headd J.J. Hung L.W. Kapral G.J. Grosse-Kunstleve R.W. McCoy A.J. Moriarty N.W. Oeffner R. Read R.J. Richardson D.C. Richardson J.S. Terwilliger T.C. Zwart P.H. PHENIX: a comprehensive Python-based system for macromolecular structure solution.Acta Crystallogr. D Biol. Crystallogr. 2010; 66: 213-221Crossref PubMed Scopus (16439) Google Scholar), respectively, using data to a cutoff of <I/σ(I)> of 1.5 or higher in the outer shell. Hydrogens were modeled in the riding positions for protein and substrates. Reference model restraints were incorporated only for the 3.0 Å 17α-hydroxypregnenolone data set, to avoid overfitting. After the protein structures were essentially completed, ligands were added. The ligand omit 2Fo − Fc maps shown were calculated in PHENIX. Ligand models were obtained from the Hetero-compound Information Center at Uppsala (HIC-Up) or constructed using the PRODRG2 server. Superpositions between CYP17A1 molecules were generated using the secondary structure matching algorithm in COOT (39Emsley P. Lohkamp B. Scott W.G. Cowtan K. Features and development of Coot.Acta Crystallogr. D Biol. Crystallogr. 2010; 66: 486-501Crossref PubMed Scopus (17085) Google Scholar). Superposition between P450cam and CYP17A1 was generated by the same process, then optimized using least squares fit for the hemes, also in COOT (39Emsley P. Lohkamp B. Scott W.G. Cowtan K. Features and development of Coot.Acta Crystallogr. D Biol. Crystallogr. 2010; 66: 486-501Crossref PubMed Scopus (17085) Google Scholar). Probe-occupied active site volumes were generated using VOIDOO (41Kleywegt G.J. Jones T.A. Detection, delineation, measurement and display of cavities in m" @default.
• W2144999739 created "2016-06-24" @default.
• W2144999739 creator A5005290325 @default.
• W2144999739 creator A5012368572 @default.
• W2144999739 creator A5046694826 @default.
• W2144999739 creator A5087917496 @default.
• W2144999739 date "2014-11-01" @default.
• W2144999739 modified "2023-10-17" @default.
• W2144999739 title "Structures of Human Steroidogenic Cytochrome P450 17A1 with Substrates" @default.
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