title: Molecular Recognition in Mitochondrial Cytochromes P450 That Catalyze the Terminal Steps of Corticosteroid Biosynthesis.
abstract: The mitochondrial cytochromes P450 11B1 and P450 11B2 are responsible for the final stages of cortisol and aldosterone synthesis, respectively. Dysregulation of both enzymes has been implicated in secondary forms of hypertension. Molecular recognition of the cytochromes P450 with their corresponding redox partner is a key step in the catalytic cycle, yet the precise nature of the interaction of P450 11B1 or P450 11B2 with their proximal partner, adrenodoxin (Adx), is still unknown. Here, we obtained P450 11B1·Adx2and P450 11B2·Adx2complexes using the zero-length cross-linker ethyl-3-[3-(dimethylamino)propyl]carbodiimide, which formed best under low-ionic strength conditions. R-to-K mutations were introduced into the P450s at residues predicted to form salt bridges with Adx and allow cross-linking with the carbodiimide reagent. Mass spectrometric analysis of the chymotrypsin-digested ternary complexes identified seven cross-linked peptide pairs. Consistent with the electrostatic interaction of K370 in P450 11B1-WT and K366 in P450 11B2-R366K with D79 of Adx, Adx mutation L80K abolished complex formation. Using these sites of interaction as constraints, protein docking calculations based on the crystal structures of the two proteins yielded a structural model of the P450 11B1·Adx2complex. The appositional surfaces include R/K366, K370, and K357 of P450 11B1, which interact with D79, D76, and D113 (second molecule) of Adx, respectively. Similar to P450 11B1, P450 11B2 also forms a complex with the Adx dimer via three lysine residues. We describe similarities and differences in our models of the P450 11B1·Adx2and P450 11B2·Adx2complexes with the structure of the P450 11A1-Adx fusion protein.
authors.affiliation: Division of Metabolism, Endocrinology, and Diabetes, Department of Internal Medicine, and Department of Pharmacology, University of Michigan Health System , Ann Arbor, Michigan 48109, United States.
authors.forename: Hwei-Ming
authors.initials: HM
authors.lastname: Peng
authors.affiliation: Division of Metabolism, Endocrinology, and Diabetes, Department of Internal Medicine, and Department of Pharmacology, University of Michigan Health System , Ann Arbor, Michigan 48109, United States.
authors.forename: Richard J
authors.identifier: 0000-0001-6815-6181
authors.initials: RJ
authors.lastname: Auchus
mesh_terms: D000305:Adrenal Cortex Hormones / Q000096:biosynthesis; D000325:Adrenodoxin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019943:Amino Acid Substitution; D001665:Binding Sites; D055162:Biocatalysis / Q000187:drug effects; D003432:Cross-Linking Reagents / Q000494:pharmacology; D019405:Cytochrome P-450 CYP11B2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005287:Ferredoxin-NADP Reductase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005288:Ferredoxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008928:Mitochondria / Q000201:enzymology / Q000378:metabolism; D008958:Models, Molecular; D062105:Molecular Docking Simulation; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D055672:Static Electricity; D013252:Steroid 11-beta-Hydroxylase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D000305:Adrenal Cortex Hormones; D003432:Cross-Linking Reagents; D005288:Ferredoxins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D000325:Adrenodoxin; D019405:Cytochrome P-450 CYP11B2; D013252:Steroid 11-beta-Hydroxylase; D005287:Ferredoxin-NADP Reductase