Catalytically Relevant Electrostatic Interactions of Cytochrome P450c17 (CYP17A1) and Cytochrome b5

Richard J Auchus,Sean M Anderson,Sarah E Forsberg,Hong T Tran,Hwei-Ming Peng,Jiayan Liu

doi:10.1074/jbc.m114.608919

Richard J Auchus, Sean M Anderson + Show 4 more

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https://doi.org/10.1074/jbc.m114.608919

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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.

Highlights

Cytochrome b5 (b5) stimulates the 17,20-lyase activity of cytochrome P450c17 (CYP17A1)
When yeast microsomes containing CYP17A1 and P450 oxidoreductase (POR) were incubated with b5 and 17-hydroxypregnenolone, the presence of wild-type b5 increased the rate of product formation 11-fold
The GD and DD mutations partially restored the capacity to stimulate 17,20-lyase activity at 23 and 38% that of wild-type b5, respectively. These results were replicated in a reconstituted system containing purified CYP17A1, POR, b5, and control yeast microsomes as lipid source (Fig. 2B)

Summary

Introduction

Cytochrome b5 (b5) stimulates the 17,20-lyase activity of cytochrome P450c17 (CYP17A1). Glu-48 and Glu-49, of cytochrome b5 (b5) are essential for stimulating the 17,20-lyase activity of cytochrome P450c17 (CYP17A1). 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. 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: 84EVLIKK89-b5: 53EQAGGDATENFEDVGHSTDAR73 and CYP17A1-R347K: 341TPTISDKNR349-b5: 40FLEEHPGGEEVLR52. 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. 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

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