title: Catalytically relevant electrostatic interactions of cytochrome P450c17 (CYP17A1) and cytochrome b5.
abstract: Two acidic residues, Glu-48 and Glu-49, of cytochrome b5 (b5) are essential for stimulating the 17,20-lyase activity of cytochrome P450c17 (CYP17A1). Substitution of Ala, Gly, Cys, or Gln for these two glutamic acid residues abrogated all capacity to stimulate 17,20-lyase activity. Mutations E49D and E48D/E49D retained 23 and 38% of wild-type activity, respectively. Using the zero-length cross-linker ethyl-3-(3-dimethylaminopropyl)carbodiimide, we obtained cross-linked heterodimers of b5 and CYP17A1, wild-type, or mutations R347K and R358K. In sharp contrast, the b5 double mutation E48G/E49G did not form cross-linked complexes with wild-type CYP17A1. Mass spectrometric analysis of the CYP17A1-b5 complexes identified two cross-linked peptide pairs as follows: CYP17A1-WT: (84)EVLIKK(89)-b5: (53)EQAGGDATENFEDVGHSTDAR(73) and CYP17A1-R347K: (341)TPTISDKNR(349)-b5: (40)FLEEHPGGEEVLR(52). Using these two sites of interaction and Glu-48/Glu-49 in b5 as constraints, protein docking calculations based on the crystal structures of the two proteins yielded a structural model of the CYP17A1-b5 complex. The appositional surfaces include Lys-88, Arg-347, and Arg-358/Arg-449 of CYP17A1, which interact with Glu-61, Glu-42, and Glu-48/Glu-49 of b5, respectively. Our data reveal the structural basis of the electrostatic interactions between these two proteins, which is critical for 17,20-lyase activity and androgen biosynthesis.
authors.affiliation: From the Division of Metabolism, Endocrinology, and Diabetes, Department of Internal Medicine.
authors.forename: Hwei-Ming
authors.initials: HM
authors.lastname: Peng
authors.affiliation: From the Division of Metabolism, Endocrinology, and Diabetes, Department of Internal Medicine.
authors.forename: Jiayan
authors.initials: J
authors.lastname: Liu
authors.affiliation: From the Division of Metabolism, Endocrinology, and Diabetes, Department of Internal Medicine.
authors.forename: Sarah E
authors.initials: SE
authors.lastname: Forsberg
authors.affiliation: Life Sciences Institute, and.
authors.forename: Hong T
authors.initials: HT
authors.lastname: Tran
authors.affiliation: From the Division of Metabolism, Endocrinology, and Diabetes, Department of Internal Medicine.
authors.forename: Sean M
authors.initials: SM
authors.lastname: Anderson
authors.affiliation: From the Division of Metabolism, Endocrinology, and Diabetes, Department of Internal Medicine, the Department of Pharmacology, University of Michigan, Ann Arbor, Michigan 48109 rauchus@med.umich.edu.
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Auchus
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry* / Q000378:metabolism; D020134:Catalytic Domain; D003432:Cross-Linking Reagents / Q000737:chemistry; D018360:Crystallography, X-Ray; D015786:Cytochromes b5 / Q000737:chemistry* / Q000145:classification / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000737:chemistry; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000145:classification / Q000235:genetics / Q000378:metabolism; D055672:Static Electricity; D013254:Steroid 17-alpha-Hydroxylase / Q000737:chemistry* / Q000145:classification / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics
chemical_list: D000596:Amino Acids; D003432:Cross-Linking Reagents; D011994:Recombinant Proteins; D015786:Cytochromes b5; C501892:CYP17A1 protein, human; D013254:Steroid 17-alpha-Hydroxylase; D005022:Ethyldimethylaminopropyl Carbodiimide
keywords: Allosteric Regulation; Androgen; CYP17A1; Cytochrome P450; Cytochrome b5; Mass Spectrometry (MS); Protein Cross-linking; Steroidogenesis