Substrate-modulated Cytochrome P450 17A1 and Cytochrome b75 Interactions Revealed by NMR

被引:78
作者
Estrada, D. Fernando [1 ]
Laurence, Jennifer S. [2 ]
Scott, Emily E. [1 ]
机构
[1] Univ Kansas, Dept Med Chem, Lawrence, KS 66045 USA
[2] Univ Kansas, Dept Pharmaceut Chem, Lawrence, KS 66045 USA
关键词
17,20-LYASE ACTIVITY; P450; OXIDOREDUCTASE; ABIRATERONE ACETATE; STRUCTURAL-ANALYSIS; MASS-SPECTROMETRY; CATIONIC CHARGES; BINDING-SITE; HUMAN CYP17; B(5); REDUCTASE;
D O I
10.1074/jbc.M113.468926
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The membrane heme protein cytochrome b(5) (b(5)) can enhance, inhibit, or have no effect on cytochrome P450 (P450) catalysis, depending on the specific P450, substrate, and reaction conditions, but the structural basis remains unclear. Here the interactions between the soluble domain of microsomal b(5) and the catalytic domain of the bifunctional steroidogenic cytochrome P450 17A1 (CYP17A1) were investigated. CYP17A1 performs both steroid hydroxylation, which is unaffected by b5, and an androgen-forming lyase reaction that is facilitated 10-fold by b5. NMR chemical shift mapping of b5 titrations with CYP17A1 indicates that the interaction occurs in an intermediate exchange regime and identifies charged surface residues involved in the protein/protein interface. The role of these residues is confirmed by disruption of the complex upon mutagenesis of either the anionic b(5) residues (Glu-48 or Glu-49) or the corresponding cationic CYP17A1 residues (Arg-347, Arg-358, or Arg-449). Cytochrome b(5) binding to CYP17A1 is also mutually exclusive with binding of NADPH-cytochrome P450 reductase. To probe the differential effects of b(5) on the two CYP17A1mediated reactions and, thus, communication between the superficial b(5) binding site and the buried CYP17A1 active site, CYP17A1/b(5) complex formation was characterized with either hydroxylase or lyase substrates bound to CYP17A1. Significantly, the CYP17A1/b(5) interaction is stronger when the hydroxylase substrate pregnenolone is present in the CYP17A1 active site than when the lyase substrate 17 alpha-hydroxypregnenolone is in the active site. These findings form the basis for a clearer understanding of this important interaction by directly measuring the reversible binding of the two proteins, providing evidence of communication between the CYP17A1 active site and the superficial proximal b(5) binding site.
引用
收藏
页码:17008 / 17018
页数:11
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