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Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee, USADepartment of Biological Sciences, Konkuk University, Seoul, Republic of Korea
It has been recognized for >50 years that cytochrome b5 (b5) stimulates some cytochrome P450 (P450)–catalyzed oxidations, but the basis of this function is still not understood well. The strongest stimulation of catalytic activity by b5 is in the P450 17A1 lyase reaction, an essential step in androgen synthesis from 21-carbon (C21) steroids, making this an excellent model system to interrogate b5 function. One of the issues in studying b5–P450 interactions has been the limited solution assay methods. We constructed a fluorescently labeled variant of human b5 that can be used in titrations. The labeled b5 bound to WT P450 17A1 with a Kd of 2.5 nM and rapid kinetics, on the order of 1 s−1. Only weak binding was observed with the clinical P450 17A1 variants E305G, R347H, and R358Q; these mutants are deficient in lyase activity, which has been hypothesized to be due to attenuated b5 binding. Kd values were not affected by the presence of P450 17A1 substrates. A peptide containing the P450 17A1 Arg-347/Arg-358 region attenuated Alexa 488-T70C-b5 fluorescence at higher concentrations. The addition of NADPH–P450 reductase (POR) to an Alexa 488-T70C-b5:P450 17A1 complex resulted in a concentration-dependent partial restoration of b5 fluorescence, indicative of a ternary P450:b5:POR complex, which was also supported by gel filtration experiments. Overall, these results are interpreted in the context of a dynamic and tight P450 17A1:b5 complex that also binds POR to form a catalytically competent ternary complex, and variants that disrupt this interaction have low catalytic activity.
). In addition to playing major roles in areas as diverse as drug metabolism and the biosynthesis of natural products, P450s are the major catalysts involved in steroid metabolism and catalyze most of the oxidations beginning with the side-chain cleavage of cholesterol to form pregnenolone (
). P450 17A1 plays a critical role in the synthesis of androgens as well as in the generation of 17α-hydroxy (17α-OH) steroids and in the synthesis of mineralocorticoids and glucocorticoids (Fig. 1). More than 125 clinically deficient variants of P450 17A1 have been identified (
). Some of the variants show normal 17α-hydroxylation activity but are deficient in the second, so-called “lyase,” reaction (Fig. 1), attenuating androgen production and subsequently estrogen levels. Conversely, prostate cancers are dependent on androgens (
Antitumour activity of abiraterone acetate against metastatic castration-resistant prostate cancer progressing after docetaxel and enzalutamide (MDV3100).
Orteronel plus prednisone in patients with chemotherapy-naive metastatic castration-resistant prostate cancer (ELM-PC 4): A double-blind, multicentre, phase 3, randomised, placebo-controlled trial.
Figure 1Reactions catalyzed by P450 17A1. The first reaction in each sequence is a 17α-hydroxylation, and the second reaction is a 17α, 20-cleavage reaction of the 21-carbon steroid (lyase reaction). DHEA, dehydroepiandrosterone.
Aside from its clinical relevance, P450 17A1 is of inherent biochemical interest in the context of the two reactions it catalyzes. The first reaction (Fig. 1) is a relatively straightforward P450 hydroxylation involving compound I (FeO3+) chemistry, but the mechanism of the second, the lyase step, is not without controversy (
). Although a number of mammalian P450 enzymes are stimulated by another hemeprotein, cytochrome b5 (CYB5A, b5), the P450 17A1 lyase reaction is the one stimulated the most (
). This effect has been shown to be important biologically (i.e., the interaction can be demonstrated using fluorescence resonance energy transfer in cells (
The cationic charges on Arg347, Arg358 and Arg449 of human cytochrome P450c17 (CYP17) are essential for the enzyme's cytochrome b5-dependent acyl-carbon cleavage activities.
). Zebrafish P450 17A1 lyase activity is only slightly enhanced by b5, and the related zebrafish P450 17A2 enzyme catalyzes only 17α-hydroxylation, not lyase activity, with or without b5 (
One of the experimental deficiencies in this research field has been useful assays for b5 binding. We considered several approaches with fluorescence, in order to utilize sensitive and solution-based methods, and were able to use labeling of a previously described mutant with a dye (
Putidaredoxin competitively inhibits cytochrome b5-cytochrome P-450cam association: A proposed molecular model for a cytochrome P-450cam electron-transfer complex.
Determination of cytochrome b5 association reactions: Characterization of metmyoglobin and cytochrome P-450cam binding to genetically engineered cytochrome b5.
). Fluorescence attenuation assays allowed for analysis with submicromolar concentrations of proteins, with estimates of binding constants and rates of association and dissociation. In contrast to previous proposals about this system, we provide evidence that P450–b5 interactions are not tightly linked to substrate identity and occupancy and that a ternary P450 17A1–b5–NADPH–P450 reductase (POR) complex is preferred to a model in which b5 and POR shuttle at a single site.
Results
Expression and purification of P450 17A1 variant enzymes
Plasmids for WT P450 17A1 and five clinically observed variants were constructed in a pCW expression vector and expressed in Escherichia coli JM109 cells (Figs. 2 and S1). WT P450 17A1 showed an expression level of ∼450 nmol per liter culture, and the expression levels of the E305G, R347H, and R358Q variants were 400, 100, and 200 nmol per liter, respectively (Fig. S1). However, no P450 holoenzyme spectra were detected for the R347C and P428L variants (Fig. S1, C and F). Purified WT P450 17A1 and three variant proteins (E305G, R347H, and R358Q) were prepared by Ni2+–nitrilotriacetate (NTA) affinity column chromatography. The spectra of these purified variant proteins showed very little of peaks corresponding to inactive cytochrome P420 (Fig. 2, B–D). All subsequent assays were based on the amount of spectrally detectable P450 heme for WT and variant P450 17A1 enzymes.
Figure 2Fe2+–CO versus Fe2+binding spectra of purified WT P450 17A1 and variant enzymes.A, WT; B, E305G; C, R347H; and D, R358Q. The P450 concentrations in these particular assays were 3.1, 0.41, 0.82, and 0.34 μM, respectively. These spectra are presented to demonstrate the lack of (inactive) cytochrome P420 and do not reflect the ratio of bound heme in the individual proteins.
Steady-state kinetic parameters for all (four) hydroxylation and lyase reactions (Fig. 1) of WT P450 17A1 and the three purified variants (E305G, R347H, and R358Q) were measured. For the 17α-hydroxylation of progesterone, all three variants formed the 17α-OH product, but their activities (specificity constants, kcat/Km) were attenuated to 3 to 11% of that of WT P450 17A1 (Fig. 3A and Table 1). The 17α-OH progesterone lyase activities of the R347H and R358Q variants were reduced more dramatically (to <1%) (Fig. 3B and Table 1). However, the E305G variant differed in that progesterone 17α-hydroxylation activity was decreased (to 8%) more than the lyase reaction activity (28%) (Fig. 3A and Table 1).
Figure 3Steady-state kinetics of enzyme reactions catalyzed by WT P450 17A1 and variants.A, WT (blue); B, E305G (green); C, R347H (orange); and D, R358Q (purple). Each point is a mean of duplicate assays, shown as a mean ± range. The kinetic parameters (kcat, Km, and kcat/Km) are indicated in Table 1.
The R347H and R358Q variants showed similar levels of pregnenolone 17α-hydroxylation (slight reduction) but only very low levels of 17α-OH pregnenolone lyase activity (Fig. 3, C and D and Table 1). The E305G variant displayed highly reduced pregnenolone 17α-hydroxylation and 17α-OH pregnenolone lyase activities (Fig. 3, C and D and Table 1). One previous study reported a similar result (
). These results indicated that the 17,20-lyase activity in all three variants was impaired even when excess b5 was present.
Substrate-binding affinities of P450 17A1 mutants
Substrate binding of purified P450 17A1 (WT and three variants) was analyzed in titrations with all four major substrates. WT P450 17A1 and all variant enzymes showed a typical “type І” substrate-binding spectral titration change (increase at 388 nm, decrease at 423 nm, corresponding to a shift in the iron spin state from low to high (
)) (Figs. S2 and S3). The calculated Kd value of WT P450 17A1 with progesterone was ∼0.065 μM, although even with quadratic analysis, there is considerable error in such a low value (Table 2). The Kd values of the three variants were somewhat increased, for example, from 0.17 to 0.26 μM (Table 2). The Kd values of variants for 17α-OH progesterone, pregnenolone, and 17α-OH pregnenolone were similar to or slightly higher than measured with WT P450 17A1 (Table 2). These results are interpreted to mean that changes in the substrate-binding affinities of the three variants are not primarily responsible for the decreased enzymatic activities, which were observed even at high substrate concentrations (Fig. 3).
). b5 did not significantly stimulate the progesterone and pregnenolone 17α-hydroxylation reactions by WT P450 17A1 or the three variants (Fig. 4). WT P450 17A1 lyase activities were stimulated by b5 in a concentration-dependent manner (Fig. 4). However, the R347H and R358Q variants did not display b5 concentration-dependent stimulation in either of the lyase reactions. The E305G variant displayed weak b5 stimulation of the 17α-OH progesterone lyase reaction but not the 17α-OH pregnenolone lyase reaction (Fig. 4, C and D).
Figure 4Stimulation of reactions catalyzed by WT P450 17A1 and variants by b5.A, WT (blue); B, E305G (magenta); C, R347H (orange); and D, R358Q (purple). Each point is a mean of duplicate assays, shown as a mean ± range.
Attenuation of Alexa 488-T70C-b5 fluorescence by WT P450 17A1 and variants
WT b5 has no cysteine residues, and the T70C substitution in b5 was chosen based on previous studies of fluorescent labeling with an acrylodan derivative and interactions of that modified protein with bacterial and other hemeproteins (
Putidaredoxin competitively inhibits cytochrome b5-cytochrome P-450cam association: A proposed molecular model for a cytochrome P-450cam electron-transfer complex.
Determination of cytochrome b5 association reactions: Characterization of metmyoglobin and cytochrome P-450cam binding to genetically engineered cytochrome b5.
). The proprietary “Red Reagent” was unsatisfactory for fluorescence titrations because of low sensitivity. The “Blue Reagent” provided much more sensitivity, and the resulting Blue-T70C-b5 protein stimulated the lyase reactions of (WT) P450 17A1 with both 17-OH progesterone and 17-OH pregnenolone as substrates, in a concentration-dependent manner (Fig. S4).
The spectral properties of the Blue derivative suggested that the dye is Alexa 488 (or a closely related molecule), which is commercially available as a maleimide derivative for attachment to cysteine residues, and the protein modified with Alexa 488 (Alexa 488-T70C-b5) also stimulated the lyase reactions (results not shown). The fluorescence spectrum of Alexa 488-T70C-b5 showed an emission maximum at 513 nm (excitation wavelength of 480 nm) (Fig. 5), and the fluorescence emission displayed linearity with respect to the concentration of Alexa 488-T70C-b5 (Fig. S5). Titration of Alexa 488-T70C-b5 with WT P450 17A1 caused a loss of fluorescence intensity, indicating the perturbation of fluorescence by binding between Alexa 488-T70C-b5 and P450 17A1 (Fig. 5). Fitting of the loss of fluorescence with increased concentrations of P450 17A1 (using a quadratic equation) provided Kd values for the affinity of Alexa 488-T70C-b5 and P450 17A1 (Fig. 6 and Table 3). The apparent Kd value for WT P450 17A1 was ∼2.5 nM (Fig. 6 and Table 3), indicating very tight binding.
Figure 5Fluorescence titration of Alexa 488-T70C-b5with WT P450 17A1 and variants. Excitation of Alexa 488-T70C-b5 (50 nM) was at 480 nm. The emission spectra (λmax of 513 nm) decreased with increasing concentrations of P450 17A1. A, WT; B, E305G; C, R347H; and D, R358Q. See Figure 6 for plots.
Figure 6Fitting of binding titrations of Alexa 488-T70C-b5with P450 17A1 WT and variants. The data points at the emission maximum (513 nm) in fluorescence titrations (Fig. 5) were fitted with a quadratic equation and nonlinear regression analysis in GraphPad Prism software: WT (blue, •); E305G (magenta, ▪); R347H (orange, ⋄); and R358Q (purple, ▵). The estimated Kd value for WT P450 17A1 was 2.5 ± 0.6 nM (Table 3).
With the three clinical variants, very little change in fluorescence spectra was observed in the binding titrations of Alexa 488-T70C-b5, indicating weak or no binding interaction between the b5 protein and the variant enzymes (Fig. 6). The effects of P450 17A1 substrates on the binding affinity of b5 and P450 17A1 were examined (Fig. S6 and Table 3). Very tight binding affinities (Kd: 2.2–12 nM) were observed for WT P450 17A1/Alexa 488-T70C-b5 in the presence of each of the four substrates (present at 10 μM), not substantially altered from the binding of Alexa 488-T70C-b5 in the absence of substrate. The variants did not show binding in the presence or absence of substrates in the titrations (Fig. S6).
Competition of POR with b5 for P450 17A1
The loss of Alexa 488-T70C-b5 fluorescence induced by binding of P450 17A1 was partially reversed by titration with POR (Fig. 7). The P450 interaction of POR appeared to be competitive with Alexa 488-T70C-b5, but the original fluorescence values were never reached. The calculated Kd value of POR (for the WT P450 17A1:Alexa 488-T70C-b5 complex) was 0.11 μM, suggesting much lower affinity than b5 with P450 17A1 (Fig. 7).
Figure 7Partial recovery of Alexa 488-T70C-b5fluorescence of an Alexa 488-T70C-b5:P450 17A1 complex by titration with POR. A, the fluorescence of Alexa 488-T70C-b5 (50 nM) is shown with a red trace. Addition of P450 17A1 (50 nM) caused a drop in the fluorescence, to give the blue trace. The intensity of the fluorescence spectra increased upon adding increasing concentrations of POR (to 2.6 μM), in the direction shown with the arrow. B, recovery of fluorescence in the Alexa 488-T70C-b5:P450 17A1 complex as a function of POR concentration. The estimated Kd was 0.11 μM.
Binding rates for interaction of Alexa 488-T70C-b5 and P450 17A1
The rate of binding of Alexa 488-T70C-b5 to (WT) P450 17A1 was measured by observing attenuation of fluorescence upon mixing the two proteins in a stopped-flow fluorimeter. When concentrations of 0.50 μM Alexa 488-T70C-b5 and 0.50 μM P450 17A1 were mixed, the first-order kobs value was 0.7 s−1 (Fig. 8, A and B).
Figure 8Rates of binding and dissociation of Alexa 488-T70C-b5and P450 17A1.A, stopped-flow analysis of binding of Alexa 488-T70C-b5 to P450 17A1 (1 μM concentrations of each protein in each syringe, excitation at 488 nm, emission >530 nm, and 23 °C). The decrease in fluorescence was fit to a single exponential using the OLIS GlobalWorks program (red line, 0.7 ± 0.1 s−1). B, residuals analysis for part A. C, stopped-flow analysis of binding of dissociation of Alexa 488-T70C-b5:P450 17A1. One syringe contained 100 nM Alexa 488-T70C-b5:P450 17A1, and the other contained 30 μM b5 (excitation at 488 nm and emission >530 nm). (When Alexa 488-T70C-b5 dissociated, it was replaced by b5.) The increase in fluorescence was fit to a single exponential using the OLIS GlobalWorks program (red line, 1.9 ± 0.2 s−1).
The loss of Alexa 488-T70C-b5 fluorescence induced by binding of P450 17A1 was reversed by adding unlabeled b5 protein (data not presented), indicating that the unlabeled b5 replaces Alexa 488-T70C-b5 in interacting with P450 17A1. When the kinetics were measured, the reaction occurred at a rate of 1.9 s−1 (Fig. 8, C and D). This value is considered to be the first-order koff rate.
In principle, for binding reactions in a freely reversible system, kobs = kon + koff (
), but the apparent koff rate here was higher than the kobs (on) rate. Thus, the true kon rate cannot be calculated from these measurements. However, the fluorescence traces clearly show that the binding and dissociation events are both occurring with rates of ∼1 s−1.
Interaction of peptides with Alexa 488-T70C-b5 and P450 17A1
Peptides were obtained corresponding to the putative binding sites of P450 17A1 and b5 (see Experimental procedures section and Fig. S7). The peptide corresponding to the putative b5 binding region of P450 17A1 (residues 347–358, “P450 peptide”) attenuated the fluorescence of Alexa 488-T70C-b5, although high concentrations were required (Fig. 9). The “b5 peptide,” however, did not result in a gain of fluorescence when added to an Alexa 488-T70C-b5:P450 17A1 complex, even at a concentration of 160 μM (data not presented).
Figure 9Attenuation of the fluorescence of Alexa 488-T70C-b5by a peptide corresponding to the putative binding region of P450 17A1. Increasing concentrations of the peptide were added. The inset shows the fluorescence at 513 nm as a function of concentration. The concentration of Alexa 488-T70C-b5 was 50 nM.
When added to reconstituted P450 17A1 steroid oxidation systems (Fig. 10), the P450 17A1 peptide was more inhibitory to the lyase reaction (17α-OH pregnenolone) than the b5 peptide, which may appear to be surprising in that b5 was present in the reaction at a concentration 10-fold higher than the P450. This peptide might be expected to bind to POR and block its interaction with P450 17A1, although that should have also inhibited the 17α-hydroxylation reaction. Inhibition was only observed at the very highest concentration.
Figure 10Inhibition of P450 17A1 reactions by peptides corresponding to the putative binding regions of P450 17A1 and b5.A, progesterone 17α-hydroxylation; B, 17α-OH pregnenolone lyase activity. The “b5 peptide” corresponds to residues 43 to 52 of b5, and the “17A1 peptide” corresponds to residues 347 to 358 of P450 17A1 (see Experimental procedures section and Fig. S7).
Demonstration of a ternary P450 17A1:POR:b5 ternary complex using size-exclusion chromatography
The fluorescence titration results (Fig. 7) suggested that P450 17A1, POR, and b5 form a ternary complex. Accordingly, we tested this hypothesis further using a different approach, that is, gel filtration (Fig. 11A). In a previous work with P450 17A1 (
), we reported that about one-half of the protein migrated as a monomer, but in this case, all the P450 17A1 eluted as a single monomeric peak on a Superose 12 column, at an elution volume between those of ovalbumin (45 kDa) and bovine serum albumin (67 kDa, monomer) (Fig. S8). b5 eluted later, as might be expected, and POR eluted as a multimer near the void volume of the column (Fig. 11A). (The identity of the second peak in the POR sample is unknown and presumed to be a small molecule, in that no proteins were visualized upon SDS-gel electrophoresis and Coomassie Blue staining; Fig. 11B).
Figure 11Gel filtration analysis of complexes of P450 17A1, b5, and POR. All analyses were done using a Superose 12 10/300 FPLC column. A, absorbance at 280 nm profiles of individual proteins (POR [green] and P450 17A1 [orange]) and a binary (blue) and a ternary mixture (red) are shown. Individual fractions were collected and analyzed by SDS-polyacrylamide gel electrophoresis, and densitometry was done of the Coomassie Blue–stained bands corresponding to the individual proteins. The POR preparation contained unknown 280 nm-absorbing material eluting near the position of free b5 but not showing any protein after electrophoresis and staining. B, densitometry traces of P450 17A1 (orange) and b5 (purple) eluted in a binary equimolar mixture of the two proteins. The migration positions of the individual proteins (P450 [17A1] and b5) are indicated. C, Coomassie Blue staining of the proteins in a ternary complex, as eluted from the column in Part A. The numbers on the left indicate Mr values of markers relevant to the three proteins of interest, which have approximate Mrs of 79 kDa (POR), 57 kDa (P450 17A1), and 17 kDa (b5). D, densitometry traces of P450 17A1 (orange), POR (green), and b5 (purple) eluted in a ternary equimolar mixture of the three proteins. The migration positions of the individual proteins (POR, P450 [17A1], and b5) are indicated.
A complex of P450 17A1 and b5 yielded peaks in the monomeric P450 17A1 and b5 regions plus a larger complex eluting earlier, as verified with gel electrophoresis (Fig. 11, A and B). A mixture of POR, P450 17A1, and b5 had most of the 280 nm-absorbing material (protein) in a large peak eluting later than free POR but earlier than the P450 17A1–b5 complex, as validated by gel electrophoresis (Fig. 11, A, C, and D). The presence of all three proteins in the ternary complex peak fractions (Fig. 11, A and D) is documented in Figure 11C.
Discussion
The development of a sensitive fluorescence-based assay for b5 binding, based on a literature precedent with an unrelated system (
Putidaredoxin competitively inhibits cytochrome b5-cytochrome P-450cam association: A proposed molecular model for a cytochrome P-450cam electron-transfer complex.
Determination of cytochrome b5 association reactions: Characterization of metmyoglobin and cytochrome P-450cam binding to genetically engineered cytochrome b5.
), enabled assays that could address several issues in the field of P450 17A1. A major finding was the very tight binding of b5 to P450 17A1, with an estimated Kd of ∼2.5 nM (Fig. 6 and Table 2). Comparisons with P450 17A1 variants known to be deficient in lyase activity (
) and documented here (Table 1) showed the importance of the P450 17A1 residues Arg-347 and Arg-358, previously proposed to be involved in the interaction (
The cationic charges on Arg347, Arg358 and Arg449 of human cytochrome P450c17 (CYP17) are essential for the enzyme's cytochrome b5-dependent acyl-carbon cleavage activities.
). The binding phenomena we studied are attributed to the ionic charges in P450 17A1 and b5, in that all binding assays were done in the absence of phospholipid vesicles.
Stopped-flow measurements with P450 17A1 showed that both the binding of b5 and its dissociation are rapid processes. The rates are on the order of 1 s−1, although the kobs value for binding (Fig. 8A) is probably not accurate in that it should be greater than the (first-order) dissociation rate (Fig. 8C). The kobs value for binding is probably a reflection of a faster “on” rate followed by a conformational change to a complex in which the fluorescence is decreased (
). The kobs values for binding and dissociation are fast enough not to be rate limiting in the overall P450 17A1 oxidation reactions (Table 1). However, the results can be used to argue against a model in which POR and b5 are “switching” at the same position on P450 17A1 during catalysis, unless this would be facilitated in the phospholipid vesicles. That is, the (substrate-bound) P450 Fe2+O2 complex needs to receive an electron, thus converting it to the “compound 0” form, Fe3+–O2–. If POR donated the electron, then it would have to leave and allow the b5 to bind (k ∼ 1 s−1) (
) and induce a conformational change to permit the lyase reaction. It is unlikely that compound 0 would have enough stability for this to occur (i.e., a kobs of 1 s−1 is equivalent to a t1/2 of 0.7 s). The bulk of the evidence is that b5 does not donate the second electron in the reaction (
). Furthermore, lyase activity was stimulated in mammalian cells in which apo-b5 was expressed, demonstrating the biological relevance of these findings (
). In the event that b5 were donating the second electron, the Fe2+O2 complex would have to be stable enough for the binding to occur after POR left the P450. We conclude that our proposal of a ternary P450:b5:POR complex (Figs. 7, 11, and 12) describes the mechanism more accurately than does a shuttle system.
Figure 12Proposed scheme for interaction of P450 17A1, b5, and POR. The fluorescence of Alexa 488-T70C-b5 (F depicts the fluorophore Alexa 488) is decreased upon interaction with P450 17A1. POR binds to form a ternary complex and causes a shift of the fluorophore (Alexa 488) to a position in which the fluorescence is partially restored (Fig. 7).
The fluorescence attenuation titrations (Figs. 5 and 6) were critical in development of the conclusions about the tight binding of b5 to (WT) P450 17A1. The attenuation of fluorescence in the presence of P450 17A1 cannot be attributed to an inner filter effect, because of the low absorbance, the reversal of the decrease by b5 (Fig. 8C) or a second protein (POR), and the lack of attenuation by the three P450 17A1 variants, even at the highest P450 concentrations. The Kd values (Table 3) should be considered estimates and have some uncertainty associated with them, for several reasons. With WT P450 17A1, the value is very low, and even with a low concentration of Alexa 488-T70C-b5 (50 nM) and a quadratic equation, there is probably error in the Kd value (2.5 nM, i.e., 20-fold less than the enzyme concentration) (Table 2). With regard to the variants, they clearly did not attenuate the Alexa 488-T70C-b5 fluorescence much (Fig. 5), but any Kd calculations have the caveat that we do not know exactly what the titration endpoint should be, that is, a complex formed with Alexa 488-T70C-b5 and one of the variants may have the b5 (and the Alexa 488 dye) in a position in which it does not show as much loss of fluorescence.
A molecular description of the fluorescence of the Alexa 488 derivative of b5 is beyond the scope of this investigation. Glu-48 and Glu-49 are on the edge of b5 (Protein Data Bank: 2I96), and the heme is between these residues and Thr-70. Stayton et al. (
Putidaredoxin competitively inhibits cytochrome b5-cytochrome P-450cam association: A proposed molecular model for a cytochrome P-450cam electron-transfer complex.
Determination of cytochrome b5 association reactions: Characterization of metmyoglobin and cytochrome P-450cam binding to genetically engineered cytochrome b5.
) attached the fluorescent dye acrylodan, which has a very different structure than Alexa 488, to Cys-65, engineered to replace Thr-65 in rat b5 (corresponding to human Thr-70). The fluorescence of this derivative increased upon binding to metmyoglobin, cytochrome c, or (bacterial) P450cam, with estimates of Kd of ∼1 μM for each. Blue shifts in the emission spectrum were also observed (
Putidaredoxin competitively inhibits cytochrome b5-cytochrome P-450cam association: A proposed molecular model for a cytochrome P-450cam electron-transfer complex.
), which is known to be very sensitive to changes in dielectric constants. We have not prepared the acrylodan derivative of T70C b5 for use with P450 17A1. In other work (not shown), the fluorescence of Alexa 488-T70C-b5 was attenuated by several other human P450 enzymes (e.g., 2C9, 2E1, 3A4), but the Kd values were not as low as with P450 17A1. Apparently, the heme of P450 (17A1) is not absolutely required for fluorescence attenuation, in that the P450 17A1 peptide was able to reduce the fluorescence (Fig. 9), albeit with weaker affinity. Preliminary screening studies have also identified several small molecules (at 10 μM concentration) that can attenuate the fluorescence of Alexa 488-T70C-b5 (results not presented). Whether the heme of b5 is critical for the attenuation of fluorescence by small molecules of proteins is unknown, in that we have not studied the apo-b5 version of the conjugate. Another issue is any conformational changes that occur upon P450 17A1–b5 binding. The stepwise addition of P450 17A1 to 15N-labeled b5 induced some NMR shift perturbations, which may be indicative of conformational changes (in b5), although the Thr-70 signal did not appear to be changed (
). Exactly how a conformational change, if it occurs, would effect the fluorescence is unknown in the absence of more information.
Our fluorescence results are in agreement with earlier reports on the roles of Arg-347 and Arg-358 in b5 binding and the basis of loss of lyase activity (
The cationic charges on Arg347, Arg358 and Arg449 of human cytochrome P450c17 (CYP17) are essential for the enzyme's cytochrome b5-dependent acyl-carbon cleavage activities.
) that binding of b5 to P450 17A1 is relatively tight. However, our results lead to several different conclusions about the interaction of P450 17A1 and b5, primarily because of the sensitivity of the assays (Figs. 5 and 6), which involve the components in solution.
The Kd values for binding of substrates to P450 17A1 (Table 2) are all lower than previously reported from this laboratory (
). The main reason is that the previous results were based on assays involving endpoints of stopped-flow measurements of binding rates, and the values reported here were obtained in steady-state assays with lower P450 concentrations (Fig. S3 and Table 2). Although the Kd values for substrate binding were somewhat higher with some of the variants (Table 2), attenuated substrate binding is not the major reason for low activity, and activities were low even at high concentrations of substrate (Fig. 3). The results of the work with E305G are not consistent with some of the conclusions of Sherbet et al. (
), who used an indirect approach to characterizing substrate binding. As discussed later, the basis of loss of activity of the E305G variant is more complex than the other variants.
), who used NMR measurements at very high enzyme concentrations, we did not find a major effect of any substrate on b5 binding (Figs. S2 and S3 and Table 2). Our results are consonant with our published work indicating that the presence of b5 did not alter rates of substrate binding or dissociation (
) also show relatively low Kd values for b5 binding, but these values are an order of magnitude higher that our own (Table 3). The SPR method involves immobilization of a protein on a chip, and a major deficiency of the approach is “mass transfer,” a term used to describe the diffusion of the ligand from the solution through the matrix to reach the receptor (P450 17A1 in this case) (
) are extremely low (i.e., 360–5000 M−1 s−1) for the binding of a substrate to an enzyme, as is usually the case in SPR work, and are not in line with our own on- and off-rates (Fig. 8), even if our on-rates may not be completely accurate.
A key finding of our study is that POR only partially disrupted the Alexa 488-T70C-b5:P450 17A1 complex (Fig. 7). If all the Alexa 488-T70C-b5 had become displaced by POR and completely free, the fluorescence would have reverted to the original value. The hyperbolic nature of the binding (Fig. 7B) could be used to calculate a Kd, with multiple equilibria if there were complete displacement, but if a ternary complex is formed, then the level of residual fluorescence may not be estimated. If there is a ternary complex, then the observed Kd for binding the POR (110 nM) is valid (∼25 times the Kd for b5). Mixing POR with Alexa 488-T70C-b5 did not decrease its fluorescence, although interaction must exist between those two proteins in that POR reduces b5 (
The gel filtration results provide more evidence for the existence of a ternary P450 17A1–POR–b5 complex (Fig. 11). The elution profile of a 17A1–b5 complex was shifted relative to either monomer and the complex of a 1:1:1 M mixture of all three proteins yielded a new peak that contained all three proteins (Fig. 11, C and D). These results, along with the fluorescence results presented in Figure 7, are consistent with a ternary complex (Fig. 12). The complex is dynamic, as shown by the kinetic assays of P450 17A1–b5 binding (Fig. 8) but stable enough to persist during migration in a gel filtration column (Fig. 11). This model of a ternary complex (Fig. 12) differs from a shuttle mechanism developed on the basis of NMR measurements (
A homodimer model can resolve the conundrum as to how cytochrome P450 oxidoreductase and cytochrome b5 compete for the same binding site on cytochrome P450c17.
), in which b5 and POR bind to the far ends of a P450 17A1 dimer (in a ternary complex) but are not in contact with each other. However, it is conceivable that, in such a model, the binding of POR on one end of a P450 dimer could transduce a fluorescence increase of the b5 on the other end. A shuttle mechanism with POR and b5 alternately binding transiently to P450 has also been proposed for (rabbit) P450 2B4 (
Cytochrome b5 increases the rate of product formation by cytochrome P450 2B4 and competes with cytochrome P450 reductase for a binding site on cytochrome P450 2B4.
), but at this time, we cannot speculate on the general application of this model (Fig. 12) to other P450s that use b5.
The P450 17A1 E305G variant is complex in that, in our assays, it lost most of the catalytic activities, with the exception of only partial loss of 17α-OH progesterone conversion to androstenedione (Fig. 3B). These results contrast with a previous report using a different system (heterologous expression in yeast and use of yeast microsomes), in which the lower lyase activity was attributed to attenuated substrate binding (
). We did not observe altered substrate binding (Table 2 and Figs. S2 and S3). In addition, the E305G variant did not quench the fluorescence of Alexa 488-T70C-b5, which we interpret as lack of binding of b5, even though Glu-305 is not at the face of P450 17A1 thought to bind directly to b5. Our current hypothesis is that the E305G substitution leads to a conformational change in P450 17A1 that disrupts b5 binding (Figs. 4, B and D and 5B).
Although we have provided new information about the nature of the interaction of P450 17A1 and b5, a number of issues and questions still remain. A sensitive interaction assay is described and applied, but there may be other dyes that could provide even greater sensitivity. As mentioned previously, the E305G variant is more complex than previously thought, and the charge distant from the putative b5 binding site has an effect on binding. Also, this variant retained some lyase activity with 17α-OH progesterone but not 17α-OH pregnenolone (Figs. 3 and 4), even though b5 binding was poor (Figs. 5B and 6). Finally, if our conclusion about a ternary complex (of P450 17A1, b5, and POR) is correct, we do not know the spatial relationship of the proteins.
As mentioned earlier, a phosphorylation event postulated to be at Ser-258 of P450 17A1 has been proposed to facilitate the lyase reaction (
), and that charge (negative) is opposite to that of the arginine residues. What has not been clear from that work is whether the phosphorylation is sufficient in itself to stimulate the lyase reaction or whether the phosphorylation enhances the functional binding of b5 to produce the enhancement. Resolving the issue would require the purification of a specifically phosphorylated P450 17A1 or the analysis of activity in a system (cells?) with a b5−/− background.
In teleost fish, there are two P450 17A enzymes, 17A1 and 17A2 (
). Fish P450 17A1 enzymes resemble human P450 17A1 and catalyze both 17α-hydroxylation and lyase activities, but P450 17A2 only catalyzes the former reaction. Zebrafish P450 17A1 showed only a twofold stimulation of the lyase activity by human or zebrafish b5, and P450 17A2 shows no activity with or without b5 (
). In contrast to fish, Xenopous laevis (frog) appears to have only P450 17A1, not 17A2 (UniProt search). Although recombinant X. laevis P450 17A1 androgen biosynthesis has been suggested to be independent of b5 (
), a closer examination of that report and calculation of kcat/Km values indicates that lyase activity was stimulated 12-fold by X. laevis b5 in the case of 17α-OH pregnenolone and 1.5-fold in the case of 17α-OH progesterone. The extent of b5 stimulation is difficult to reach conclusions about in such systems where P450 17A1 and POR are present in microsomal membranes and soluble b5 is added, in that the b5 may not have the same access to the P450 as seen in a more homogeneous or even a vesicular system, although stimulations are qualitatively seen in systems with b5 added to yeast microsomes (
The results with the peptides (Fig. 10) showed weak inhibition of the lyase activity by the P450 17A1 peptide but inhibition of progesterone 17α-hydroxylation only at the very highest concentration of that peptide. In principle, it might be possible to develop drugs that block binding of b5 to P450 17A1 to discover selective inhibitors of the lyase reactions, an unmet need in treating prostate cancer (
In conclusion, we have developed a sensitive fluorescence assay that can be utilized to study b5 interactions with P450 17A1. We confirm previous conclusions that Arg-347 and Arg-358 of P450 17A1 are important in b5 binding. We also implicated loss of b5 binding in the low activity of the P450 17A1 variant E305G. In contrast to previous work, we did not observe effects of P450 17A1 on its interactions with b5, and we provide evidence for a functional P450 17A1–POR–b5 ternary complex instead of a shuttle mechanism.
Experimental procedures
Chemicals and enzymes
Progesterone, pregnenolone, 17-OH progesterone, 17-OH pregnenolone, androstenedione, dehydroepiandrosterone, protease inhibitor cocktail, cholesterol oxidase, and 1,2-dilauroyl-sn-glycero-3-phosphocholine were purchased from Sigma–Aldrich. Ni2+–NTA–agarose was purchased from Qiagen. 5-Aminolevulinic acid was purchased from Frontier Scientific. IPTG and CHAPS were purchased from Anatrace. Other chemicals were of the highest grade commercially available. Peptides were purchased from New England Peptides, with purity and identity analysis provided (Fig. S8):
with the numbering of the residues shown from the primary sequence.
Enzyme expression and purification
E. coli JM109 cells were purchased from Invitrogen. Recombinant rat POR and human b5 were expressed in E. coli and purified as described previously (
Mechanism of 17α,20-lyase and new hydroxylation reactions of human cytochrome P450 17A1: 18O labeling and oxygen surrogate evidence for a role of a perferryl oxygen.
). Briefly, E. coli JM109 cells transformed using pCW (Ori+) vectors were inoculated into terrific broth medium containing 100 μg ml−1 ampicillin, 0.5 mM 5-aminolevulinic acid, and 1.0 mM IPTG. The expression cultures (500 ml) were grown at 37 °C for 3 h and then at 30 °C under conditions of shaking at 200 rpm for 28 h in 2.8-l Fernbach flasks. Soluble fractions containing P450 17A1 enzymes were prepared after ultracentrifugation (105g, 60 min). The soluble fraction was then loaded onto a Ni2+–NTA column (Qiagen), and (after washing), the purified protein was eluted with 100 mM potassium phosphate buffer (pH 7.4) containing 0.5 M NaCl, 0.5% (w/v) CHAPS, 20% (v/v) glycerol, and 250 mM imidazole. The eluted fraction containing highly purified P450 17A1 was dialyzed at 4 °C against 100 mM potassium phosphate buffer (pH 7.4) containing 20% (v/v) glycerol and 0.1 mM EDTA to remove CHAPS and NaCl.
Fe2+–CO versus Fe2+ binding spectra were recorded using the method of Omura and Sato (
), using an extinction coefficient of 91,000 M−1 cm−1 (Δε450–490). Protein concentrations were estimated from A280 measurements using a Nanodrop spectrophotometer (Thermo Scientific) and the amino acid compositions of human P450 17A1 and b5. The estimated specific content of WT P450 17A1 was 7.3 nmol P450 (mg protein)−1, which is somewhat low but within the historical range of 5.6 to 16.8 nmol P450 (mg protein)−1 for purified P450s expressed in E. coli and purified in this laboratory (
Expression of modified human cytochrome P450 1A1 in Escherichia coli: Effects of 5′ substitution, stabilization, purification, spectral characterization, and catalytic properties.
Recombinant enzymes overexpressed in bacteria show broad catalytic specificity of human cytochrome P450 2W1 and limited activity of human cytochrome P450 2S1.
). The estimated specific contents of the purified E305G, R437H, and R358Q variants were 5.5, 2.3, and 4.2 nmol P450 (mg protein)−1, respectively. We have observed heme contents as low as 10% of the WT values for expressed variants of other human P450s, for example, P450 21A2 (
). All catalytic and titration assays comparing the WT and variant P450 17A1 enzymes were done on the basis of spectrally detected P450 heme, though (as before (
)), in order to avoid differences because of heme content.
Construction of Alexa 488-T70C-b5
The expression plasmid for T70C-b5 was constructed using an Agilent quikChange site-directed mutagenesis kit according to the manufacturer's instructions. The constructed mutant plasmid was verified with DNA nucleotide sequencing analysis and then transformed into E. coli DH5α. The purification was performed with the same procedure for WT b5 previously described (
). The calculated specific content of b5 heme in T70C-b5 was 59.9 nmol (mg protein)−1, within experimental error of the theoretical amount for a protein of 16.9 kDa.
The purified protein was labeled using a NanoTemper Monolith Protein Labeling Kit BLUE NHS second Generation (NanoTemper Technologies), which reacts with sulfhydryl groups in a protein sample to label the protein with a fluorescent dye (BLUE) (proprietary). The extent of labeling was determined by a modification of the manufacturer instructions.
Labeling of the T70C-b5 mutant was then performed using a maleimide conjugation reaction with Alexa Fluor 488 dye (Thermo Scientific). Briefly, the b5 T70C mutant (in 100 mM phosphate buffer, pH 7.4) was mixed with Alexa Fluor 488 maleimide (dissolved in dimethylsulfoxide) at a 1:10 M ratio, and the conjugation reaction was allowed to proceed at room temperature for 20 h in the dark. The labeling mixture was then passed through a Zeba spin column (Thermo Scientific) to eliminate unreacted dye. The concentration of (labeled) Alexa 488-T70C-b5 was calculated by measuring the absorbance, using ε493 = 72,000 M−1 cm−1. The extent of modification was 74%.
Substrate-binding analysis
Binding titration analysis was carried out using purified WT P450 17A1 and variant enzymes to determine the binding affinity parameters of the substrate–enzyme complexes. The purified enzymes were diluted in 100 mM potassium phosphate buffer (pH 7.4) and divided in two 1.0-ml glass cuvettes. Spectra (350–500 nm) were recorded using an OLIS-DW2 spectrophotometer (On-Line Instrument Systems) with subsequent additions of the substrate. Differences in the absorbance between the wavelength maximum and minimum were plotted versus the substrate concentration (
). P450 17A1 enzymes were reconstituted and preincubated in 500 μl of 100 mM potassium phosphate buffer (pH 7.4) containing POR, 1,2-dilauroyl-sn-glycero-3-phosphocholine, and the substrate (progesterone, 17-OH progesterone, pregnenolone, or 17-OH pregnenolone). Typical enzyme concentrations used were P450 17A1 (0.1–0.2 μM), POR (0.4–0.8 μM), and b5 (2–4 μM) for hydroxylation/lyase reactions (e.g., Figs. 3 and 4 and Table 1).
The reactions were initiated by adding an NADPH-regenerating system (final concentrations of 15 mM glucose 6-phosphate, 1.5 mM NADP+, and 1 IU ml−1 glucose 6-phosphate dehydrogenase) (
) and terminated by quenching and extracting with 2.0 ml CH2Cl2. Aliquots of the organic phases were dried and then dissolved in 100 μl of a CH3CN/H2O (1:1, v/v) mixture. The extracts from the pregnenolone and 17-OH pregnenolone reactions were dissolved in 50 μl of CH3OH, mixed with 200 μl of cholesterol oxidase (0.5 units/reaction) in 100 mM phosphate buffer (pH 7.4), and incubated at 30 °C with shaking at 150 rpm for 12 h. The extraction procedure was then repeated, and the (Δ4) reaction products were analyzed in a Waters Acquity UPLC system using a BEH C18 octadecylsilane column (2.1 mm × 100 mm, 1.7 μm). The reaction products were resolved using a mobile phase composed of solvents A (70% CH3OH, 30% H2O, v/v) and B (CH3CN), at a flow rate of 0.2 ml min−1. The mobile phase linear gradient proceeded as follows: 0 to 1 min, 5% B; 4 min, 30% B; 4.5 min, 40% B; 4.55 to 6.75 min, 95% BA; and 7 to 10 min, 5% B. The reaction products were identified by coelution with commercial standards and quantified by the A243 peak areas. Steady-state kinetic parameters were estimated using nonlinear regression of hyperbolic fits in GraphPad Prism software, with equations used for kcat and the specificity constant kcat/Km (
): Y = (ksp ∗ X)/(1 + (ksp ∗ X/kcat)) and then deriving Km by division.
P450 titrations with fluorescent b5
Steady-state fluorescence spectra were collected on an OLIS DM-45 spectrofluorimeter. For optimal fluorescence measurements, 1.24 mm slits (corresponding to a 5.0 nm bandwidth) were employed. The purified Alexa 488-T70C-b5 protein was diluted to 50 nM in 100 mM potassium phosphate buffer (pH 7.4) in a 1.0-ml cuvette. Binding titrations were carried out using increasing concentrations of WT P450 17A1 and variant enzymes. Emission spectra (500–630 nm) were recorded after subsequent additions of P450 enzymes (excitation wavelength of 480 nm). The data points at the emission maximum (513 nm) were used for analysis with a quadratic equation and nonlinear regression analysis in GraphPad Prism software, set as Y = B + (A/2) ∗ (1/E) ∗ ((Kd + E + X)-sqrt((Kd + E + X)ˆ2-(4 ∗ E ∗ X))).
Association and dissociation kinetics of Alexa 488-T70C-b5 and P450 17A1
Association kinetics of b5 and P450 17A1 were measured using a stopped-flow spectrofluorimeter (RSM-1000; On-Line Instrument Systems) equipped with a 4 × 4 mm cell (23 °C). The two drive syringes were filled with 1.0 μM Alexa 488-T70C-b5 protein and 1.0 μM P450 17A1 (in 100 mM potassium phosphate buffer, pH 7.4), respectively. After mixing, the instrument recorded the emission >530 nm (Orion long-pass filter) with an excitation wavelength of 488 nm, over a period of 5 s. To estimate the dissociation rate (koff), unlabeled b5 (30 μM) in one syringe was mixed with a preformed complex of Alexa 488-T70C-b5 (1.0 μM) and P450 17A1 (1.0 μM) in the other syringe. Rates were estimated using GlobalWorks software analysis of the experimental data, fitting to a single exponential (data from at least three replicates were averaged.)
Size-exclusion chromatography
Chromatography was done using a Superose 12 10/300 GL column (11 μm, 10 × 300 mm; GE Healthcare) with an NGC Quest 100 Plus Chromatography system (BioRad). The buffer was 50 mM potassium phosphate (pH 7.4) containing 0.15 M NaCl, and the flow rate was 1.0 ml min−1. The column was equilibrated for each run with one column volume (23.6 ml), and the injection volume was 3% of the column volume (10 nmol of each protein was injected, i.e., 100 μl of 100 μM solutions). Elution was with 1.5 column volumes of buffer. Absorbance was monitored at 280 nm. Fractions were collected (1.0 ml) and analyzed by SDS-polyacrylamide gel electrophoresis (4–15% gradient gel), with staining with Coomassie Blue and densitometry using GelAnalyzer 19.1 software (www.gelanalyzer.com; Istvan Lazar and Istvan Lazar Jr). Mr standards included ovalbumin and bovine serum albumin (Fig. S9).
The authors declare that they have no conflict of interest with the contents of this article.
Acknowledgments
We thank J. G. Chapman for purifying P450 17A1 and b5 and M.-S. Kim for the initial construction and purification of T70C b5. Thanks are also extended to K. Trisler for assistance in preparation of the article.
Author contributions
F. P. G. conceptualization; D. K. and F. P. G. data curation; D. K., V. K., K. D. M., and F. P. G. formal acquisition; D. K. and F. P. G. supervision; F. P. G. funding acquisition; D. K. and F. P. G. validation; D. K., V. K., and K. D. M. investigation; D. K., V. K., K. D. M., and F. P. G. visualization; D. K. and F. P. G. methodology; F. P. G. project administration; and D. K. and F. P. G. writing-review and editing.
Funding and additional information
This study was supported by the National Institutes of Health grant R01 GM118122 (F. P. G.). This work constitutes part of a Konkuk University research support program for its faculty on sabbatical leave in 2020 (D. K.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
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