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.

Keywords: Allosteric Regulation; Androgen; CYP17A1; Cytochrome P450; Cytochrome b5; Mass Spectrometry (MS); Protein Cross-linking; Steroidogenesis.

FIGURE 1.

Absorbance spectra of the purified recombinant human b₅ mutations. Characteristic spectra with maximum absorption wavelength for oxidized (412 nm, native) and reduced (424 nm, after adding solid sodium dithionite) forms are shown. Spectra were recorded with 4–8.5 μm b₅ in 1 ml of 10 mm potassium phosphate buffer (pH 7.4); ordinates are 0.5–1.5 absorbance units (AU) full scale.

FIGURE 2.

Stimulation of CYP17A1 17,20-lyase activity by b₅ and effect of mutations. A, effect of b₅ and b₅ mutations on catalytic activities of yeast microsomes containing CYP17A1 and POR. Incubations contained 10 μm 17-hydroxypregnenolone, and the results are shown as the percentage activity compared with wild-type b₅ values (=100%) from triplicate determinations, means ± S.D. B, effect of b₅ and b₅ mutations on catalytic activities of reconstituted, purified CYP17A1 and POR. Purified CYP17A1 (10 pmol) was reconstituted with POR, various b₅ mutations, and control yeast microsomes as phospholipid for reconstitution with molar ratio of P450/POR/b₅ at 1:4:2. Results are shown as the percentage activity compared with wild-type b₅ values (=100%) from triplicate determinations, means ± S.D. The 17-hydroxylase (C) and 17,20-lyase activities (D) of CYP17A1 mutations R347K and R358K are not different from the activities of wild-type CYP17A1. Incubations were carried out with 10 μm progesterone and a 1:4 molar ratio of P450/POR (C) or 10 μm 17-hydroxypregnenolone and a 1:4:1 molar ratio of P450/POR/b₅ (D) as described under “Experimental Procedures.” Values are the averages of duplicate determinations. The rate of dehydroepiandrosterone (DHEA) formation without b₅ added is 7–9% the rates in the presence of b₅.

FIGURE 3.

Immunoblots of CYP17A1 and b₅ or b₅ mutation E48G/E49G cross-linked with EDC and effect of steroid substrates on complex formation. Reactions were performed in 50 mm potassium phosphate buffer (pH 7.0), containing 2.5 μm CYP17A1, 37.5 μm b₅, 250 μm phospholipid, and 2 mm EDC in total volume of 20 μl. A 1-μl aliquot of the reaction mixture was loaded in each lane and subjected to SDS-PAGE and immunoblot with anti-polyhistidine antibody, which detects both CYP17A1 and b₅. A, wild-type b₅ (17 kDa) and CYP17A1 (55 kDa), wild-type or mutations R347K and R358K as indicated, were incubated with PE + PS (1:1) in the absence of presence of EDC. B, b₅ mutation E48G/E49G and CYP17A1, wild-type or mutations R347K and R358K as indicated, were incubated with PE + PS (1:1) in the absence or presence of EDC. C, CYP17A1 and b₅ were incubated with PE + PS (1:1) and EDC in the absence or presence of steroid substrates. Prog, progesterone; Preg, pregnenolone; 17Preg, 17-hydroxypregnenolone. The immunoreactive bands corresponding to CYP17A1 and the 1:1 CYP17A1-b₅ complex (complex) are indicated.

FIGURE 4.

Mass spectrometry of peptide pair derived from CYP17A1 cross-linked with b₅. A, EDC cross-linking of wild-type CYP17A1 and b₅ followed by SDS-PAGE analysis with Denville blue staining. Reactions were performed as described in Fig. 3, and the entire reaction mixture was loaded on the gel. B, representative high resolution fragmentation spectrum of cross-linked peptide between CYP17A1 and b₅ identified by StavroX: CYP17A1-WT (⁸⁴EVLIKK⁸⁹) and b₅ (⁵³EQAGGDATENFEDVGHSTDAR⁷³). The numbers in parentheses indicate the positions of the cross-linked peptide sequences with the cross-linked residues underlined. Signals of y-type ions are shown in blue, and signals of b-type ions are shown in red. The measurement error of the ion is 0.3 ppm. C, EDC cross-linking of R358K and b₅ followed by SDS-PAGE and Denville blue staining. D, representative high resolution fragmentation spectrum of cross-linked peptide pair between CYP17A1-R358K and b₅: CYP17A1-R358K (⁸⁴EVLIKK⁸⁹) and b₅ (⁵³EQAGGDATENFEDVGHSTDAR⁷³). Residue numbering and spectrum annotation are the same as in A and B. The measurement error of the ion is 1.5 ppm.

FIGURE 5.

Mass spectrometry of peptide pair derived from CYP17A1 mutation R347K cross-linked with b₅. A, EDC cross-linking of CYP17A1 mutation R347K and b₅ followed by SDS-PAGE analysis with Denville blue staining. Reactions were performed as described in Fig. 3, and the entire reaction mixture was loaded on the gel. B, representative high resolution fragmentation spectrum of cross-linked peptide between CYP17A1-R347K and b₅ identified by StavroX: R347K (³⁴¹TPTISDKNR³⁴⁹) and b₅ (⁴⁰FLEEHPGGEEVLR⁵²). The numbers in parentheses indicate the positions of the cross-linked peptide sequences with the cross-linked residues underlined. Signals of y-type ions are shown in blue, and signals of b-type ions are shown in red. The measurement error of the ion is 1.6 ppm.

FIGURE 6.

Docking model of CYP17A1 interaction with b₅, based on crystal structures of CYP17A1 (PDB code 3RUK) and b₅ (PDB code 2I96). The docking was performed using the programs HADDOCK with the distance constraints of two intermolecular cross-links, Lys-88(CYP17A1)-Glu-61(b₅) and Arg-347(CYP17A1)-Glu-42(b₅), as well as Glu-48 and Glu-49 of b₅. A, interaction of amino acid Lys-88 (red) in CYP17A1 and Glu-61 (blue) of b₅ are shown on a surface model. Glu-49 of b₅ in the center of complex is shown in blue. B, schematic model of CYP17A1 interacting with b₅. The heme groups in CYP17A1 and b₅ are shown in red and orange sticks, respectively, and the abiraterone molecule in the crystal structure is shown in green. The central I-helix is displayed in purple, and amino acids Lys-88, Arg-347, and Arg-358 of CYP17A1, which interact with b₅, are shown in magenta. The side chain of glutamic acids 42, 48, and 49 of b₅ are shown in blue. C, close-up view of the CYP17A1-b₅ interaction interface is shown, where the glutamic acid residues in b₅ interacting with CYP17A1 are visible. D, CYP17A1-b₅ interactions were detected using the PISA server and presented as a space-filled model. Interface residues at less than 3.9 Å from interaction sites are shown in red (including Arg-347, Arg-358, and Arg-449) for CYP17A1 and green (including Glu-42, Glu-48, Glu-49, and Arg-52) for b₅, respectively. The buried residues were colored in gray, and the rest of the residues are shown in blue for CYP17A1 and cyan for b₅.