Substrate-modulated cytochrome P450 17A1 and cytochrome b5 interactions revealed by NMR

Abstract

The membrane heme protein cytochrome b5 (b5) 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 b5 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 b5 residues (Glu-48 or Glu-49) or the corresponding cationic CYP17A1 residues (Arg-347, Arg-358, or Arg-449). Cytochrome b5 binding to CYP17A1 is also mutually exclusive with binding of NADPH-cytochrome P450 reductase. To probe the differential effects of b5 on the two CYP17A1-mediated reactions and, thus, communication between the superficial b5 binding site and the buried CYP17A1 active site, CYP17A1/b5 complex formation was characterized with either hydroxylase or lyase substrates bound to CYP17A1. Significantly, the CYP17A1/b5 interaction is stronger when the hydroxylase substrate pregnenolone is present in the CYP17A1 active site than when the lyase substrate 17α-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 b5 binding site.

Keywords: Cytochrome P450 17A1; Cytochrome b5; Enzyme Mechanisms; NMR; Protein Conformation; Protein-Protein Interactions; Steroidogenesis.

FIGURE 1.

Chemical shift mapping of cytochrome b₅/CYP17A1 interaction shows binding on an intermediate exchange time scale. A, HSQC spectra of ¹⁵N-b₅ titrated with unlabeled CYP17A1 show a combination of chemical shift perturbations and line broadening. Residues indicated by solid arrows showed substantial line broadening so that a signal was no longer detectible or very weak at a half-molar equivalent of CYP17A1, whereas small shifts were observed in other residues (dashed arrows). B, peak intensity for two titration points (b₅:CYP17A1 1:0.25 in open bars and 1:1 in black bars) plotted as a fraction of the free b₅ intensity (b₅:CYP17A1 1:0 molar ratio) revealed consistent and disproportionate line broadening for residues ⁴⁷GEEV⁵⁰ throughout the titration. C, these broadened residues (red) occur in the b₅ α2 helix and its preceding loop. Other residues with small shifts (yellow) include His-44 and His-68 on both sides of the b₅ heme, suggesting a change in the heme position. Residues shown in gray have little or no perturbation, whereas those shown in white were not evaluated because of either spectral overlap or the presence of a proline.

FIGURE 2.

Conserved anionic (b₅) and cationic (CYP17A1) residues are required to form the b₅-CYP17A1 complex. Shown are 2D ¹⁵N-HSQC spectra of b₅ (1:0, black) overlaid with the same samples containing an equal molar equivalent (1:1, red) of CYP17A1. A, effects of b₅ mutation. Broadening of Glu-48 or Glu-49 was not observed in the b₅ E48Q and E49Q mutants (top and center panels, respectively), suggesting that the b5/CYP17A1 complex was not formed. However, the b₅ V50S mutant did show broadening of both resonances (bottom panel), suggesting b5-CYP17A1 complex formation similar to wild-type b₅. B, effects of CYP17A1 mutation. None of the CYP17A1 R347H, R358Q, and R449L mutant proteins induced line broadening in either Glu-48 or Glu-49, suggesting that the b5-CYP17A1 complex was not formed.

FIGURE 3.

Addition of P450 reductase disrupts the b₅-CYP17A1 complex. Representative segments of the overlaid 2D ¹⁵N-HSQC spectra demonstrated that b5/CYP17A1 binding (shown in the absence of CPR in A in red) can be disrupted by adding an equal molar amount of full-length rat CPR, as indicated by the return of the liberated b₅ spectrum (B, blue).

FIGURE 4.

Alterations in b₅-CYP17A1 complex formation in the presence of CYP17A1 ligands including the 17α-hydroxylase substrate pregnenolone, the 17,20-lyase substrate 17α-hydroxypregnenolone, and the heme-coordinating inhibitor abiraterone. A, representative HSQC spectra of ¹⁵N-b₅ alone (1:0) and in the presence of an equimolar concentration of CYP17A1 (b5/CYP17A1 1:1) were collected in the presence of each substrate, showing that in addition to broadening of ⁴⁷GEEV⁵⁰ observed during b₅ binding to unliganded CYP17A1 (solid arrows), enhanced line broadening also occurred for specific residues near the binding interface (dashed arrows). B, the steroidal inhibitor abiraterone coordinates the CYP17A1 heme iron via its pyridine ring, resulting in a low-spin system that is less strongly paramagnetic, allowing distinction of paramagnetic effects on residues like His-44, Lys-39, and Glu-49 (dashed arrows) from intermediate exchange effects on Glu-48 and Val-50 because of complex formation (solid arrows). C, plots of the relative intensities for all b₅ resonances in the ligand bound versus free state illustrate substrate-induced reductions in intensity because of line broadening in regions adjoining the ⁴⁷GEEV⁵⁰ sequence for substrates but not for the inhibitor abiraterone. Residues marked with an asterisk are located near the binding site but were either not assigned or consist of a proline. For each liganded condition, residues line-broadened below 15% of the original signal are mapped onto the structure in red in the right panel.

FIGURE 5.

CYP17A1 substrate differentially affects CYP17A1/b₅ interaction. A, representative spectra of b₅ in the presence of a half-molar equivalent of CYP17A1:substrate. At a reduced substrate-bound CYP17A1 concentration, b₅ line broadening is less severe. As a result, minor differences can be observed in the binding to unliganded (black), pregnenolone bound (red), and 17α-hydroxypregnenolone bound (green) CYP17A1. In particular, Lys-39, Asp-65, and His-68 are perturbed differently for each liganded state (black arrows), whereas His-44 and Glu-49 (dashed arrows) are both perturbed as well as line-broadened to a different extent. B, the differentially affected residues (orange) map onto both sides of b₅, suggestive of a substrate-influenced change in the b₅ heme.

FIGURE 6.

CYP17A1 substrate differentially affects the ability of CPR to disrupt binding to b₅. Samples containing ¹⁵N-b₅ and CYP17A1 in 1:1 molar ratios were titrated with CPR, and HSQC spectra were reacquired. A, as demonstrated, in the absence of CYP17A1 substrate, an equal molar amount of CPR (1:1:1) was sufficient to observe the complete return of resonances for Glu-48 and Glu-49, indicating the liberation of free b₅. B, when pregnenolone is bound to CYP17A1, a greater molar equivalent of CPR was required to liberate free b₅, as indicated by a weak Glu-48 and Glu-49 signal even with 1.5 molar equivalents of CPR. C, when 17α-hydroxypregnenolone is the CYP17A1 ligand, more CPR is required to liberate b₅ than in the substrate-free situation, as indicated by weak signals in the 1:1:1 condition, but the free b₅ signal fully reappears upon increasing CPR to the 1.5 molar equivalent (1:1:1.5).

FIGURE 7.

Model describing the second-order effects of 17α-hydroxylase and 17,20-lyase substrates on modulation of the b₅-CYP17A1 and CPR-CYP17A1 complexes. A, the current data support a scheme in which, when in the presence of pregnenolone, the b5-CYP17A1 electrostatically driven association is enhanced relative to the same interaction in the absence of substrate, thus resulting in decreased access to electron transfer via CPR. B, the scales of this balance tip in the presence of 17α-hydroxypregnenolone, in which a weaker b5:CYP17A1 association (relative to pregnenolone) results in enhanced electron transfer via CPR.