Human cytochrome P450 21A2, the major steroid 21-hydroxylase: Structure of the enzyme?progesterone substrate complex and rate-limiting C-H bond cleavage

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 290, NO. 21, pp. 13128–13143, May 22, 2015

Human Cytochrome P450 21A2, the Major Steroid

21-Hydroxylase

STRUCTURE OF THE ENZYME⅐PROGESTERONE SUBSTRATE COMPLEX AND RATE-LIMITING

C–H BOND CLEAVAGE^*

Received for publication, February 19, 2015, and in revised form, April 6, 2015 Published, JBC Papers in Press, April 8, 2015, DOI 10.1074/jbc.M115.646307

Pradeep S. Pallan^‡1, Chunxue Wang^‡1, Li Lei^‡, Francis K. Yoshimoto^‡, Richard J. Auchus^§, Michael R. Waterman^‡,

F. Peter Guengerich^‡2, and Martin Egli^‡3

From the ^‡Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146 and the

^§Division of Metabolism, Endocrinology, and Diabetes, Department of Internal Medicine, University of Michigan Medical School,

Ann Arbor, Michigan 48109

Background: P450 21A2 catalyzes 21-hydroxylation of both progesterone and 17␣-hydroxyprogesterone, an important step

in adrenal steroidogenesis.

Results: A crystal structure of human P450 21A2 with progesterone can explain many functional variants.

Conclusion: High kinetic deuterium isotope effects show the importance of a closely spaced site for C21 hydrogen abstraction.

Significance: The structure provides insight into enzyme deficiencies in congenital adrenal hyperplasia.

Cytochrome P450 (P450) 21A2 is the major steroid 21-hy- gesting that the rate of substrate binding may also be partially

droxylase, and deficiency of this enzyme is involved in ϳ95% of rate-limiting. The structure of the human P450 21A2-substrate

cases of human congenital adrenal hyperplasia, a disorder of complex provides direct insight into mechanistic effects of

adrenal steroidogenesis. A structure of the bovine enzyme that genetic variants.

we published previously (Zhao, B., Lei, L., Kagawa, N., Sundar-

amoorthy, M., Banerjee, S., Nagy, L. D., Guengerich, F. P., and

Cytochrome P450 (P450)⁴21A2 is the major steroid 21-hy-

droxylase, which catalyzes the 21-hydroxylation of progester-

one and 17␣-hydroxyprogesterone (17␣-OH-progesterone) to

form 11-deoxycorticosterone and 11-deoxycortisol, respec-

tively (Fig. 1), primarily in the adrenal cortex (1–3). Although

P450s from some other gene families (e.g. 2C and 2D) can cat-

alyze this hydroxylation (4, 5), P450 21A2 is clearly the most

important in humans, and Ͼ100 different amino acid variants

have been identified in clinical settings (2, 6, 7). There are two

CYP21 genes in the human genome, CYP21A1 (a pseudogene)

and CYP21A2 (the functional gene) (8, 9). Both are located on

chromosome 6 in the major histocompatibility locus. These

two CYP21 genes have 98% exonic sequence identity. Some of

the clinical variants result from recombination of the two genes,

and others result from other single amino acid mutations. Defi-

ciencies in the activity lead to a type of congenital adrenal

hyperplasia, which has three clinical forms: a severe form with

concurrent defects in both cortisol and aldosterone biosynthe-

sis (“salt-wasting” type), a form with adequate aldosterone bio-

synthesis (“simple virilizing” type), and a mild, non-classic form

that can be asymptomatic or associated with signs of postpu-

bertal androgen excess without cortisol deficiency (2, 6). The

worldwide incidence of congenital adrenal hyperplasia is

1:15,000 for the classical form and at least 1:1,000 for the non-

classical form (7, 10), due primarily to gene conversion events

with the adjacent CYP21A1P pseudogene; thus, congenital

adrenal hyperplasia is one of the most common inborn meta-

Waterman, M. R. (2012) Three-dimensional structure of steroid

21-hydroxylase (cytochrome P450 21A2) with two substrates

reveals locations of disease-associated variants. J. Biol. Chem.

287, 10613–10622), containing two molecules of the substrate

17␣-hydroxyprogesterone, has been used as a template for

understanding genetic deficiencies. We have now obtained a

crystal structure of human P450 21A2 in complex with proges-

terone, a substrate in adrenal 21-hydroxylation. Substrate bind-

ing and release were fast for human P450 21A2 with both sub-

strates, and pre-steady-state kinetics showed a partial burst but

only with progesterone as substrate and not 17␣-hydroxypro-

gesterone. High intermolecular non-competitive kinetic deute-

rium isotope effects on both k_catand k_cat/K_m, from 5 to 11, were

observed with both substrates, indicative of rate-limiting C–H

bond cleavage and suggesting that the juxtaposition of the C21

carbon in the active site is critical for efficient oxidation. The

estimated rate of binding of the substrate progesterone (k_on

؊1

2.4

؋

10⁷

M

s

^؊1) is only ϳ2-fold greater than the catalytic

efficiency (k_cat/K_m

؍

1.3

؋

10^{7 ؊1}s^؊1) with this substrate, sug-

M

* Thisworkwassupported, inwholeorinpart, byNationalInstitutesofHealth

Grants R01 GM103937 (to M. E. and F. P. G.), T32 ES007028 (to F. K. Y.), and

R01 GM086596 (to R. J. A.).

The atomic coordinates and structure factors (code 4Y8W) have been deposited

in the Protein Data Bank (http://wwpdb.org/).

¹Both authors contributed equally to this work.

²To whom correspondence may be addressed: Dept. of Biochemistry, Van-

derbilt University School of Medicine, 638 Robinson Research Bldg., 2200

Pierce Ave., Nashville, TN 37232-0146. Tel.: 615-322-2261; Fax: 615-343-

0704; E-mail: f.guengerich@vanderbilt.edu.

³To whom correspondence may be addressed: Dept. of Biochemistry, Van-

derbilt University School of Medicine, 868A Robinson Research Bldg., 2200 ⁴The abbreviations used are: P450, cytochrome P450; KIE, kinetic isotope

Pierce Ave., Nashville, TN 37232-0146. Tel.: 615-343-8070; Fax: 615-322-

7122; E-mail: martin.egli@vanderbilt.edu.

effect; r.m.s.d., root mean square deviation; UPLC, ultraperformance liquid

chromatography.

13128 JOURNAL OF BIOLOGICAL CHEMISTRY

VOLUME 290•NUMBER 21•MAY 22, 2015

Human P450 21A2 Structure and Kinetics

FIGURE 1. Reactions catalyzed by P450 21A2.

bolic errors known in humans. In addition, P450 21A2 is the

major autoantigen in autoimmune Addison disease (11).

Because of the relevance of P450 21A2 to human disease,

there is considerable interest in identifying the molecular basis

for loss of function in P450 21A2 variants (6, 7). Major aspects

of structure-activity relationships among the deficient variants

include changes in the structure, the nature of the rate-limiting

steps in catalysis, and the substrate selectivity (Fig. 1). In partic-

ular, P450 21A2 is unusual in that its major 21-hydroxylase

activities generate electron-deficient primary carbon radicals

FIGURE 2. P450 catalytic cycle.

21,21,21-d₃-progesterone and 21,21,21-d₃-17␣-OH-progester-

one were synthesized as described previously (13).

Expression of Human P450 21A2—In the human CYP21A2

open reading frame, the region encoding the N-terminal trans-

membrane helix (residues 1–29) was replaced by nucleotides

coding for the peptide MAKKTSSKGK (P450 2C3 N-terminal

region), and the 3Ј-end was extended by 18 nucleotides encod-

ing six histidines. The entire modified cDNA was synthesized

by GenScript (Piscataway, NJ) and inserted into a pET17b

expression vector (EMD Millipore, Billerica, MA). The pET-

17b plasmid containing the gene encoding human P450 21A2

was co-transformed with a chaperone pGro12 plasmid into

E. coli BL21-Gold DE(3)-competent cells. The cultures were

used for inoculation in Terrific Broth medium with 100 ␮g/ml

ampicillin and 50 ␮g/ml kanamycin at 37 °C and 250 rpm.

When an OD₆₀₀of 1.0 was achieved (ϳ3.5 h), the shaker speed

was lowered to 100 rpm, and ␦-aminolevulinic acid and arabi-

nose were added to final concentrations of 1.0 and 27 m^M,

respectively. After 60 min, the temperature was lowered to

26 °C, and 1.0 m^Misopropyl 1-thio-␤-D-galactopyranoside was

added to induce the expression of human P450 21A2. The cells

were cultured for another 40 h at 26 °C (shaking at 100 rpm) and

harvested by centrifugation (2.5 ϫ 10³g, 20 min). The cells were

subsequently suspended in 50 m^MTris-HCl buffer (pH 7.4)

containing 250 m^Msucrose and 0.5 mM EDTA (200 ml/liter of

cell culture) containing 1 mg/ml lysozyme. After incubation at

4 °C for 30 min, the cells were pelleted and stored at Ϫ80 °C.

Purification of Human P450 21A2—Human P450 21A2 was

purified from the E. coli cells as described previously for the

recombinant bovine enzyme, with minor modifications (6).

Briefly, the cell pellets were thawed and sonicated in 200 ml of

sonication buffer (50 m^Mpotassium phosphate buffer (pH 7.4)

containing 20% glycerol (v/v), 0.5 ^MNaCl, 1.0% sodium cholate

(w/v) (added to extract the protein from the membranes), 0.1

m^Mdithiothreitol (DTT), 0.1 mM EDTA, 0.1 mM phenylmeth-

ylsulfonyl fluoride (PMSF), and a protease inhibitor mixture

⅐

(-CH₃3 -CH₂) in the course of the reaction (12, 13).

Despite the clinical interest in P450 21A2, only limited work

has been done directly with the human enzyme. Bovine P450

21A2 was purified from adrenal cortex (14). Arase et al. (15)

successfully expressed the bovine enzyme in Escherichia coli in

2006, and in 2012, we reported the x-ray crystal structure of

modified bovine P450 21A2 (6). That structure has been used to

model some of the possible effects of variants observed in clin-

ics (6, 7). Human P450 21A2 has been heterologously expressed

in yeast microsomes and utilized in mutagenesis and basic

kinetic isotope effect (KIE) studies (13) and also expressed in

baculovirus (11) and bacterial systems (16).

In order to address several catalytic and structural issues

related to the function of P450 21A2, we expressed the human

enzyme in E. coli and purified the enzyme. This protein was

used to obtain an x-ray crystal structure in the presence of the

substrate progesterone (6) bound at the active site, with a sec-

ond substrate molecule of low occupancy residing inside the

access channel (in the bovine complex, the corresponding sites

are fully occupied by 17␣-OH-progesterone (6)). Kinetic anal-

yses indicate that human P450 21A2 is more catalytically effi-

cient than the bovine enzyme and may be the most catalytically

efficient mammalian P450 reported (i.e. has the highest k_cat/K_m

value) (3). In contrast to previous reports on the bovine enzyme

(17), we find that the enzyme is not limited by rates of product

dissociation but, at least in large part, by the rate of C–H bond

breaking (Fig. 2, step 7), as revealed by high non-competitive

intermolecular KIEs.

Experimental Procedures

Chemicals—Progesterone was purchased from Sigma-Al- (Roche Complete Tablets, EDTA-free, EASYpack)). Unbroken

drich, and 17␣-OH-progesterone was purchased from Ster- cells and cell debris were removed by centrifugation for 60 min

aloids (Newport, RI). All radiolabeled steroids were purchased at 10⁵ϫ g. The reddish brown supernatant was applied to an

from American Radiolabeled Chemicals (St. Louis, MO). Ni^2ϩ-nitrilotriacetic acid-agarose column, followed by 10 col-

MAY 22, 2015•VOLUME 290•NUMBER 21

JOURNAL OF BIOLOGICAL CHEMISTRY 13129

Human P450 21A2 Structure and Kinetics

umn volumes of wash buffer (50 mM potassium phosphate (pH

Catalytic Activity Assays—In general, steady-state enzyme

7.4) containing 20% glycerol (v/v), 0.5 ^MNaCl, 0.25% sodium assays were done in 500-␮l reaction volumes at 37 °C in 100 mM

cholate (w/v), 20 m^Mimidazole, 0.1 mM DTT, and 0.1 mM potassium phosphate buffer (pH 7.4). The reconstituted

EDTA). The bound P450 21A2 was subsequently eluted with enzyme system contained 2 n^MP450, 0.6 ␮M recombinant rat

elution buffer (wash buffer containing 250 m^Mimidazole) and NADPH-P450 reductase (19), 15 ␮M L-␣-1,2-dilauroyl-sn-

collected in 2-ml fractions. The most concentrated fractions glycero-3-phosphocholine, and varying concentrations of pro-

were combined and dialyzed against 100 m^Mpotassium phos- gesterone or 17␣-OH-progesterone (0–40 ␮M). The reactions

phate buffer (pH 7.4) containing 20% glycerol (v/v), 0.3 ^MNaCl, were initiated by the addition of an NADPH-regenerating sys-

ϩ

and 0.1 m^MEDTA, which removed the sodium cholate.

tem containing 10 m^Mglucose 6-phosphate, 0.5 mM NADP ,

The protein for crystallization experiments was purified with and 1 IU/ml glucose-6-phosphate dehydrogenase (20). Reac-

a method similar to that used for purifying bovine P450 21A2, tions were quenched by the addition of 2 ml of CH₂Cl₂, and the

except that no detergent was used for chromatography with an steroid products were extracted into the organic solvent (lower

SP Fast-Flow Sepharose column (GE Healthcare). The protein phase) by mixing with a vortex device. After centrifugation to

was eluted with 50 m^Mpotassium phosphate buffer (pH 7.2) separate the layers (3 ϫ 10³g, 10 min), an aliquot of the organic

containing 20% glycerol (v/v), 0.1 m^MDTT, 0.1 mM EDTA, and phase was transferred and dried under an N₂stream. The

400 m^MNaCl.

extracted products were dissolved in a CH₃OH/H₂O mixture

Spectral Analyses of P450 21A2—Steady-state binding exper- (70%, v/v) and injected onto an Acquity BEH octadecylsilane

iments were carried out using an Aminco DW2/OLIS spectro- (C₁₈) UPLC column (2.1 mm ϫ 50 mm, 1.7 ␮m) in a Waters

photometer (On-Line Instruments, Bogart, GA) at 23 °C. In Acquity UPLC system connected to a UV detector set to a

general, P450 21A2 (1.0 ␮M, in 0.2 M potassium phosphate (pH wavelength of 243 nm. The UPLC column was used at 40 °C.

7.4) buffer) was titrated with increasing concentrations of The product was identified and quantified using external stan-

ligands (in C₂H₅OH) in the sample cuvette, and the same dards. With progesterone as the substrate, the following

amount of C₂H₅OH was added to the reference cuvette. The CH₃OH/H₂O linear gradient was used, at a flow rate of 0.1

UV-visible spectra from 350 to 500 nm were recorded. The ml/min: 0–2 min, hold at 70% CH₃OH (v/v); 2–6 min, linear

spectral change ⌬(A₃₉₀Ϫ A₄₁₈), as a function of ligand concen- increase from 70 to 95% CH₃OH (v/v); 6–6.5 min, hold at 95%

tration, was fit to a quadratic equation of the form, y ϭ B ϩ CH₃OH (v/v); 6.5–7 min, linear decrease from 95 to 70%

(A/2)*(1/E)*((K_dϩ E ϩ X) Ϫ sqrt((K_dϩ E ϩ X)^2 Ϫ (4*E*X)), CH₃OH (v/v); 7–10 min, hold at 70% CH₃OH (v/v). With 17␣-

using Prism software (GraphPad, Inc.).

OH-progesterone as the substrate, the following CH₃OH/H₂O

In order to obtain more accurate K_dvalues for the ligands linear gradient was used, at a flow rate of 0.1 ml/min: 0–2 min,

with initial K_dvalues of Ͻ1 ␮M, titration experiments were hold at 60% CH₃OH (v/v); 2–6 min, linear increase from 60 to

repeated in a Cary 14/OLIS spectrophotometer using a 10-cm 85% CH₃OH (v/v); 6–6.5 min, hold at 85% CH₃OH (v/v); 6.5–7

path length cylindrical cuvette (Starna Cells (Atascadero, CA), min, linear decrease from 85 to 60% CH₃OH (v/v); 7–10 min,

catalogue no. 34-Q-100, volume 25 ml). P450 21A2 (0.10 ␮M, in hold at 60% CH₃OH (v/v).

0.2 ^Mpotassium phosphate (pH 7.4) buffer) was titrated with

Pre-steady-state Kinetics of Product Formation—Pre-steady-

increasing concentrations of ligands (dissolved in C₂H₅OH). state 21-hydroxylation reactions with P450 21A2 were car-

The spectral change ⌬(A₃₉₀Ϫ A₄₁₈), as a function of ligand ried out in a KinTek instrument (model RQF-3, KinTek

concentration, was fit to a quadratic equation (see above) using Corp., State College, PA). All reactions were performed in 100

GraphPad Prism software.

m^Mpotassium phosphate buffer (pH 7.4) at 37 °C. Human P450

An OLIS RSM-1000 instrument (1.24-mm slits, 20-mm path 21A2 (2 ␮M) was reconstituted with 4 ␮M recombinant rat

length) was used to study pre-steady-state kinetics of ligand NADPH-P450 reductase (19), 15 ␮M L-␣-1,2-dilauroyl-sn-

binding to P450 21A2. Experiments were performed in the glycero-3-phosphocholine, and either 20 ␮M progesterone ([4-

rapid scanning mode at 23 °C in 0.2 ^Mpotassium phosphate ¹⁴C]progesterone) or 17␣-OH-progesterone (17␣-OH-[1,2,6,

buffer (pH 7.4) containing 60 ␮M L-␣-1,2-dilauroyl-sn-glycero- 7-³H]progesterone). The reactions were initiated by rapidly

3-phosphocholine. Briefly, 2 or 4 ␮M P450 was mixed with 4 ␮M mixing with an NADPH-regenerating system (20 m^Mglucose

ligand, and spectral data were collected for 1 s. The traces at 390 6-phosphate, 2 m^MNADPH, and 2 IU/ml glucose-6-phosphate

nm were fit to equations using the manufacturer’s software dehydrogenase (20)) and quenched with 1 ^MHCl as a function

(GlobalWorks). The basis of the experimental design is of time from 10 ms to 1 s. A 50-␮l aliquot of each reaction

described by Daniels and Alberty (18). A second-order reaction mixture was spotted onto the loading zone of a channeled silica

2A 3 A₂is kinetically equivalent to A ϩ B 3 AB, if similar gel G TLC plate (Analtech, Newark, DE), and the plates were

concentrations of A and B are mixed; the analysis is straightfor- developed with ethyl acetate/CH₂Cl₂(1:4 (v/v) for progester-

ward, especially if the back-reaction is slow (AB 3 A ϩ B), one and 1:3 (v/v) for 17␣-OH-progesterone). With progester-

which was the case here. This approach was used instead of one as the substrate, the plate was exposed to a phosphor

trying to achieve pseudo-first-order kinetics with excess sub- imager screen (Imaging Screen K, Bio-Rad) for 7 days and ana-

strate, because of the low K_dvalues (i.e. K_dϽ 1–2 ␮M, which is lyzed with a molecular imager system (Pharos FX Plus Molec-

a minimum for detection of changes in the kinetic binding ular Imager, Bio-Rad) using the manufacturer’s Quantity One

experiments). Experiments with higher concentrations of sub- software. With 17␣-OH-progesterone as the substrate, the

strate did not result in faster rates, in that the enzyme was plate was scanned with an AR-2000 Radio-TLC imaging scan-

already saturated with substrate.

ner (Bioscan, Poway, CA) and analyzed with the manufacturer’s

13130 JOURNAL OF BIOLOGICAL CHEMISTRY

VOLUME 290•NUMBER 21•MAY 22, 2015

Human P450 21A2 Structure and Kinetics

B

A

250 kDa

75 kDa

0.04

0.02

50 kDa

37 kDa

0.00

-0.02

25 kDa

15 kDa

-0.04

400

420

440

460

480

500

Wavelength, nm

FIGURE 3. Purification of human P450 21A2. A, SDS-PAGE. The molecular weight maker (left) and purified P450 21A2 (right) are indicated. B, Fe^2ϩ⅐CO versus

Fe^2ϩdifference spectrum (0.38 ␮M P450) (28). (Safranin T (1 ␮M) was added as an electron mediator (29).)

Winscan software. The data points were fit to a burst equation, mined by molecular replacement with the program MOLREP

y ϭ A(1 Ϫ e^Ϫkpt) ϩ k_sst, where A is the burst amplitude, k_pis the (22) (23), using the crystallographic coordinates of bovine P450

first-order rate of the pre-steady-state burst, and k_ssis the 21A2 as the search model (6) (Protein Data Bank code 3QZ1).

steady-state rate, using GraphPad Prism software.

Refinement was carried out with the program Refmac5 (23, 24),

Crystallization—To obtain the human P450 21A2-progester- and manual rebuilding was performed with Coot (25). The

one complex, substrate (dissolved in C₂H₅OH) was added in a r.m.s.d. values from the superimposition were calculated with

1:5 (P450/progesterone) molar ratio, followed by mixing and the Superpose routine in CCP4 (23). All structural figures were

concentrating to 25 mg/ml in 50 m^Mpotassium phosphate generated with the program UCSF Chimera (26).

buffer (pH 7.2) containing 5% glycerol (v/v), 0.1 m^MDTT, 0.1

Results

m^MEDTA, and 100 mM NaCl. Aliquots of the complex solution

were frozen in liquid N₂and then stored at Ϫ80 °C. Human

Purification of Human P450 21A2 and Catalytic Properties—

P450 21A2-progesterone complex crystals were grown using To facilitate the expression of human P450 21A2 in E. coli, the

the sitting drop vapor diffusion technique by mixing equal vol- N terminus was replaced with the sequence MAKKTSSKGK to

umes (200 nl) of complex solution and mother liquor (0.10 ^Mincrease the solubility of this enzyme; this sequence has been

sodium HEPES (pH 7.5) containing 0.20 M ammonium sulfate utilized to improve heterologous expression of several P450s

and 25% (w/v) polyethylene glycol 3350) and equilibrating (27). P450 21A2 was stabilized in high ionic strength buffers;

droplets against 60 ␮l of reservoir solution at 20 °C. Microcrys- thus, at least 0.3 ^MNaCl was added in the buffers during puri-

tals were obtained within 2 weeks, and in order to increase their fication. The protein was efficiently purified to Ͼ90% electro-

size, one round of microseeding was performed. Wells contain- phoretic purity after one step of Ni^2ϩ-nitrilotriacetic acid affin-

ing microcrystals were washed with 10 ␮l of reservoir buffer ity chromatography. The Fe^2ϩ⅐CO versus Fe^2ϩ-reduced

and transferred to a microcentrifuge tube with 40 ␮l of mother difference spectra of purified human P450 21A2 displayed a

liquor. After vortex mixing the seeds for 2 min and diluting typical P450 spectrum with a Soret peak at 450 nm (Fig. 3B).

1:1000 with mother liquor, 200 nl of the seed solution was Only a small 420 nm peak was observed in the assays (when 1

mixed with 200 nl of protein for each drop. Larger crystals of the ␮M safranin T was added as a mediator to increase the rate of

complex grew from microseeded droplets in 5 days. Crystals reduction (29)), indicating that the purified human P450 21A2

were mounted in nylon loops and swiped through a droplet of is very low in the presence of the inactive form (cytochrome

25% glycerol (v/v) in mother liquor for flash freezing in liquid P420) (Ͻ5%).

nitrogen.

Steady-state Kinetics of Hydroxylation Reactions Catalyzed

X-ray Data Collection, Structure Determination, and Refine- by Human and Bovine P450 21A2—Steady-state kinetic param-

ment—Diffraction data were collected on the insertion device eters for both progesterone and 17␣-OH-progesterone 21-hy-

beam line (21-ID-G) of the Life Sciences Collaborative Access droxylation by purified human and bovine P450 21A2 were

Team, located at Sector 21 of the Advanced Photon Source, determined (Fig. 4 and Table 1). The bovine P450 21A2

Argonne National Laboratory (Argonne, IL) at a wavelength of C3B21RA mutant, designed for crystallization of bovine P450

0.9782 Å using a Mar300 CCD detector (at 100 K). Processing 21A2, has a similar functional activity as the wild-type protein

was done with the program HKL2000 (21), and selected crystal and was studied for direct comparison. Bovine P450 21A2 had

Ϫ1

data and refinement statistics are summarized in Table 2. The comparable catalytic efficiency (ϳ90 min ␮M^Ϫ1) for the sub-

structure of the P450 21A2-progesterone complex was deter- strates progesterone and 17␣-OH-progesterone, with similar

MAY 22, 2015•VOLUME 290•NUMBER 21

JOURNAL OF BIOLOGICAL CHEMISTRY 13131

Human P450 21A2 Structure and Kinetics

300

200

100

0

200

B

A

150

100

50

0

10

20

30

40

50

0

10

20

30

40

50

⁵⁰D

60

C

40

20

0

30

20

10

0

10

20

30

40

50

0

10

20

30

40

50

α-

[17 OH Progesterone], μM

[Progesterone], μM

FIGURE 4. Steady-state kinetics of 21-hydroxylation of progesterone and 17␣-hydroxyprogesterone by human and bovine P450 21A2. The curves

represent the best non-linear fits to the Michaelis-Menten equation. A, human P450 21A2 with the substrate progesterone; B, human P450 21A2 with the

substrate 17␣-OH-progesterone. C, bovine P450 21A2 with the substrate progesterone; D, bovine P450 21A2 with the substrate 17␣-OH-progesterone.

TABLE 1

due to the fact that the purified enzyme was utilized in the

Steady-state kinetics parameters for 21-hydroxylation catalyzed by

recombinant human P450 21A2

assays rather than whole cells or cell lysates containing

expressed human P450 21A2.

Substrate

k_cat

K_m

k

_cat/K_m

^DV

^D(V/K)

Binding of Substrates to Human P450 21A2—The binding of

the substrates progesterone and 17␣-OH-progesterone to P450

21A2 produces a classic Type I binding spectrum (Fig. 5), indi-

cating the effects of substrate binding on the heme iron spin

state equilibrium. High affinities of P450 21A2 for the steroid

substrates progesterone and 17␣-OH-progesterone were

observed, with K_dvalues of ϳ10 Ϯ 3 and 30 Ϯ 3 nM, respec-

tively. In order to estimate the rate constants (k_on) for substrate

binding, rates of spectral changes at 390 nm were monitored

using stopped-flow spectroscopy, and the k_onvalues were

determined by fitting. The estimated k_onvalues were 2.4 ϫ 10⁷

Ϫ1

min

␮M

␮M^Ϫ1min

d₀-Progesterone

168 Ϯ 4 0.21 Ϯ 0.03 800 Ϯ 120

6.5 Ϯ 0.3 11 Ϯ 2

8.0 Ϯ 1.0 5.3 Ϯ 0.6

21,21, 21-d₃- Progesterone 26 Ϯ 1 0.37 Ϯ 0.01 70 Ϯ 2

d₀-17␣-OH progesterone 240 Ϯ 5 1.5 Ϯ 0.1

160 Ϯ 11

30 Ϯ 3

21,21, 21-d₃-17␣-OH

30 Ϯ 1 1.0 Ϯ 0.1

progesterone

TABLE 2

Steady-state kinetics parameters for 21-hydroxylation catalyzed by

recombinant bovine P450 21A2

Substrate

k_cat

min

K_m

k

_cat/K_m

Ϫ1

␮M

␮M^Ϫ1min

91 Ϯ 14

94 Ϯ 13

Progesterone

17␣-OH progesterone

47 Ϯ 1

40 Ϯ 1

0.50 Ϯ 0.07

0.44 Ϯ 0.07

and 2.2 ϫ 10⁷^Ϫ1s^Ϫ1for progesterone and 17␣-OH-proges-

M

terone, respectively. The dissociation rate constants (k_off) were

_cat(ϳ40 min^Ϫ1) and K_m(ϳ0.5 ␮M) values (Table 2). The cat-

estimated using the relationship K_dϭ k_off/k_onand were 0.24

k

Ϫ1

and 0.66 s

for progesterone and 17␣-OH-progesterone,

alytic activities determined for this mutant were significantly

higher than those reported previously (6), probably due to the

lack of residual detergent (Cymal 5) used in that work. In par-

ticular, for progesterone, the k_catvalue is about 3-fold higher,

and the K_mvalue is about 5-fold lower, yielding a 15-fold

increase in the catalytic efficiency compared with the previous

kinetic data reported for the purified bovine P450 21A2

C3B21RA mutant (6). Human P450 21A2 is a more efficient

enzyme than bovine P450 21A2, with catalytic efficiencies of

respectively. These studies indicated that substrate binding and

release are relatively fast for P450 21A2 with both substrates.

The monophasic nature of the binding of both substrates differs

from the apparent biphasic binding of 17␣-OH-progesterone

to bovine P450 21A2 (6).

Binding of Products to Human P450 21A2—Titration of P450

21A2 with the 21-OH products deoxycorticosterone and

11-deoxycortisol also yielded Type I binding spectra (Fig. 6).

The K_dvalues were 1.2 and 2.9 ␮M for deoxycorticosterone and

11-deoxycortisol, respectively, which are 2 orders of magnitude

higher than those for the substrates. Pre-steady-state binding

studies using stopped-flow spectroscopy yielded k_onvalues of

800 and 160 ␮M^Ϫ1min^Ϫ1for progesterone and 17␣-OH-pro-

gesterone, respectively (i.e. 1.3 ϫ 10⁷and 2.7 ϫ 10⁶

M

s

,

Ϫ1 Ϫ1

respectively). Although the k_catvalues were similar with both

substrates, the 7-fold lower K_mvalue with the substrate proges-

terone resulted in much higher catalytic efficiency than with

the substrate 17␣-OH-progesterone. Compared with other

previous studies (11, 30), the significantly higher enzymatic

2.2 ϫ 10⁷and 1.9 ϫ 10⁷^Ϫ1s^Ϫ1for deoxycorticosterone and

M

11-deoxycortisol, respectively, which are comparable with the

rate constants for binding of substrates. The calculated k_off

activities of human P450 21A2 reported herein are probably rates were 26 s^Ϫ1for deoxycorticosterone and 55 s^Ϫ1for 11-de-

13132 JOURNAL OF BIOLOGICAL CHEMISTRY

VOLUME 290•NUMBER 21•MAY 22, 2015

Human P450 21A2 Structure and Kinetics

FIGURE 5. Binding of steroid substrates to human P450 21A2. Steady-state titrations of P450 21A2 (1.0 ␮M) were conducted with varying concentrations of

steroids in 0.2 M potassium phosphate buffer (pH 7.4) (shown in A (progesterone) and B (17␣-OH-progesterone)). The plots in C (progesterone) and D

(17␣-OH-progesterone) show quadratic fits of spectral changes (⌬(A₃₉₀Ϫ A₄₁₈)) when the titrations were repeated with 0.10 ␮M P450 21A2 (progesterone

K_dϭ 0.010 Ϯ 0.003 ␮M; 17␣-OH-progesterone K_dϭ 0.030 Ϯ 0.003 ␮M). Stopped-flow absorbance changes (⌬A₃₉₀) for binding of different substrates (2 ␮M) to

P450 21A2 (1 ␮M) are shown in E and F. The absorbance changes at 390 nm were fit using the manufacturer’s software (OLIS GlobalWorks): binding of

progesterone to human P450 21A2, k_onϭ 2.4 ϫ 10⁷

analysis is shown in the insets in E and F.

M M

^Ϫ1s^Ϫ1; binding of 17␣-OH-progesterone to human P450 21A2, k_onϭ 2.2 ϫ 10^{7 Ϫ1}s^Ϫ1). The residuals

oxycortisol. The lower affinities of products for P450 21A2 are parameters were determined with the deuterium-labeled sub-

probably due to fast product dissociation from P450 21A2, as is strates 21,21,21-d₃-progesterone and 17␣-OH-21,21,21-d₃-

the case with most proteins (31).

progesterone. Comparison of the k_catand K_mvalues between

Analysis of Burst Kinetics—In order to determine whether non-deuterated (d₀) and deuterated (d₃) substrates yielded

steps following product formation are rate-limiting for 21-hy- non-competitive intermolecular KIEs, designated ^DV and

droxylation by P450 21A2, pre-steady-state kinetic analysis was ^D(V/K) following the convention of Northrop (32). ^DV and

performed (Fig. 7). Product formation was monitored over the ^D(V/K) values determined for progesterone were 6.5 and 11,

range of 10 ms to 1 s using a rapid quench flow instrument. A respectively. ^DV and ^D(V/K) values determined for 17␣-OH-

small kinetic burst within the initial 80 ms (20%) was followed progesterone were 8.0 and 5.3, respectively (Fig. 8 and Table 1).

by a slower product formation with the substrate progesterone, The relatively high non-competitive intermolecular isotope

whereas an almost linear curve was observed with 17␣-OH- effects indicate that C–H bond cleavage is at least partially rate-

progesterone as the substrate.

limiting in both 21-hydroxylation reactions catalyzed by

Kinetic Isotope Effects—In order to determine the kinetic human P450 21A2.

contribution of step 7 (C–H bond breaking; Fig. 2) in the cata-

Crystal Structure of Human P450 21A2—The structure of the

lytic cycle of P450 21A2, KIE approaches were used to analyze complex between human P450 21A2 and progesterone was

the rate-limiting nature of this step. The steady-state kinetic phased by molecular replacement using the bovine P450 21A2

MAY 22, 2015•VOLUME 290•NUMBER 21

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Human P450 21A2 Structure and Kinetics

FIGURE 6. Binding of steroid products to human P450 21A2. Steady-state titrations of P450 21A2 (1 ␮M) were conducted with varying concentrations of

steroid products in 0.2 M potassium phosphate buffer (pH 7.4) (shown in A and B). The plots show quadratic fits of spectral changes (⌬A₃₉₀Ϫ A₄₁₈), and insets

show spectral changes. C, titration of human P450 21A2 with 11-deoxycorticosterone (K_dϭ 1.2 Ϯ 0.2 ␮M). D, titration of human P450 21A2 with 11-deoxycor-

tisol (K_dϭ 2.9 Ϯ 0.7 ␮M). Stopped-flow absorbance changes (⌬A₃₉₀) for binding of different steroids (2 ␮M) to P450 21A2 (2 ␮M) are shown in E and F. The

absorbance changes at 390 nm were fit using the manufacturer’s software (OLIS GlobalWorks). E, kinetics of binding of 11-deoxycorticosterone to human P450

21A2 (k_onϭ 1.1 ϫ 10⁷

insets in E and F.

M

^Ϫ1s^Ϫ1), F, kinetics of binding of 11-deoxycortisol to human P450 21A2 (k_onϭ 9.5 ϫ 10^{6 Ϫ1}s^Ϫ1). The residuals analysis is shown in the

M

3

protein portion from the previously determined structure of

the complex of the latter with 17␣-OH-progesterone as the

search model (6). The monoclinic crystal of human P450 21A2

contained three molecules of the complex with progesterone

per asymmetric unit, and the structure was refined to a resolu-

tion of 2.64 Å (Table 3 and Fig. 9A). The conformations adopted

by the independent complexes exhibit close similarity (Fig. 9B),

with 440, 440, and 442 amino acids traced for complexes 1, 2,

and 3, respectively, in the electron density map. The sequence

identity between the human and bovine P450 21A2 proteins is

80% (Fig. 10A). However, the initial model of the human P450

21A2⅐progesterone complex based on the molecular replace-

ment solution obtained from the search with the coordinates of

the 3 Å resolution structure of the bovine P450 21A2⅐17␣-OH-

progesterone complex required extensive rebuilding to arrive

1.0

A

B

2

1

0

0.5

0.0

0. 0

0.5

1.0

0.0

0.5

1.0

Time (s)

FIGURE 7. Pre-steady-state-kinetics for 21-hydroxylation catalyzed by

human P450 21A2. The final P450 concentration was 1.0 ␮M. The curves

represent the best fits to the equation, y ϭ A(1 Ϫ e^Ϫkpt) ϩ k_sst, where A

represents the burst amplitude, k_prepresents the first-order rate of the

pre-steady-state burst, and k_ssrepresents steady-state rate. A, formation

of deoxycorticosterone from progesterone. A ϭ 0.19 Ϯ 0.02 ␮M, k_pϭ 32 Ϯ

10 s^Ϫ1, and k_ssϭ 0.63 Ϯ 0.03 s^Ϫ1. B, formation of 11-deoxycortisol from

17␣-OH-progesterone. The parameters were not calculated due to the

lack of burst reaction.

13134 JOURNAL OF BIOLOGICAL CHEMISTRY

VOLUME 290•NUMBER 21•MAY 22, 2015

Human P450 21A2 Structure and Kinetics

200

150

100

50

300

200

100

0

A

B

d0

d3

d0

d3

0

10

20

30

40

50

0

10

20

30

40

50

α-

[Progesterone], μM

[17 OH Progesterone], μM

FIGURE 8. Kinetic isotope effects for 21-hydroxylation reactions catalyzed by human P450 21A2. A, progesterone; B, 17␣-OH-progesterone.

TABLE 3

Selected crystal data, data collection, and refinement parameters

P450 21A2⅐progesterone

Crystal data

Space group

C2

Unit cell constants a, b, c (Å), and ␤ (degrees)

150.38, 86.86, 108.92, and 102.1

Data collection

Wavelength (Å)

Temperature

Resolution (outer shell) (Å)

Unique reflections

Completeness (%)

I/␴(I)

0.9782

110 K

30.00-2.64 (3.42-2.64)^a

40,299 (3,979)

100 (100)

10.2 (1.4)

R_pim

0.455 (0.081)

7.5 (7.1)

Redundancy

Refinement

Phasing method

Molecular replacement (Protein Data Bank code 3QZ1 model)

R_work

0.230 (0.225)^b

0.281 (0.281)^b

1,322 (440,440,442)^c

10,537

R_free

No. of amino acids

No. of protein atoms

No. of heme atoms

129

No. of ligand atoms (3 progesterones)

No. of water molecules/sulfate ions

Average B-factor, protein atoms (Å²)

Average B-factor, ligand/heme atoms (Å²)

Average B-factor, water/ions (Å²)

Wilson B-factor (Å²)

69

81/4

43.1

32.5/26.6

30.2/35.4

44.1

r.m.s.d. bond lengths (Å)

r.m.s.d. bond angles (degrees)

Ramachandran analysis: no. of favored/allowed residues/outliers (%)

Protein Data Bank code

0.005

1.2

94.2/4.3/1.5

4Y8W

^aNumbers in parentheses refer to the outer shell.

^bRefinement outer shell 2.71–2.64 Å.

^cBelonging to three independent chains: for molecule A, residues 29–266/276–326/333–411/414–485; for molecule B, 29–267/276–324/333–484; for molecule C,

29–266/275–325/333–485.

at the final structure. The degree of deviation between the deviations between the tertiary structures of the human and

superimposed human P450 21A2⅐progesterone and bovine bovine P450 21A2 proteins.

P450 21A2⅐17␣-OH-progesterone complexes (Fig. 11A) is indi-

The overall structure adopted by human P450 21A2 exhibits

cated by an r.m.s.d. of 4.68 Å for main chain atoms (C, O, N, C_␣; the typical P450 fold that consists of at least 12 ␣-helices and a

residue range 29–485; complex A). Residue by residue r.m.s.d. ␤-sheet domain near the N terminus; it resembles a triangular

values for main chain atoms along the entire chain based on an prism (34). Human P450 21A2 features 13 ␣-helices (treating B

overall superimposition of the human and bovine P450 21A2 and BЈ as well as F and FЈ as single helices; Fig. 9C) and a total of

complex structures are depicted in Fig. 10B. The computed nine ␤-strands. The latter fold into an N-terminal ␤-sheet

average values for r.m.s.d. regarding main chain atoms and domain (␤1–␤3 (blue) and ␤4–␤7 (yellow); Fig. 9C) and a

based on separate superimpositions of individual segments of C-terminal sheet (␤8 (orange) and ␤9 (red); Fig. 9C). Thus,

the human and bovine P450 21A2 proteins are 0.85 Å (aa human and bovine P450 21A2 exhibit very similar secondary

29–100), 2.38 Å (aa 101–200), 1.50 Å (aa 201–300), 1.05 Å (aa structure arrangements (Fig. 11A), and differences in the distri-

301–400), and 3.21 Å (aa 401–485). This analysis provides a bution of ␣-helices and ␤-strands are most likely not at the

clear indication that the N-terminal portions of the two crystal origin of the considerable conformational differences indicated by

structures exhibit the most similar conformations among the theabover.m.s.d. values. Rather, therelativeorientationsofhelices

analyzed segments. On the other hand, even when just main and sheets as well as deviating conformations in the extensive loop

chain atoms are included, the comparison reveals significant regions are probably more important in this regard.

MAY 22, 2015•VOLUME 290•NUMBER 21

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Human P450 21A2 Structure and Kinetics

Inspection of the progesterone substrates at the active sites of

the three human P450 21A2 complexes shows a roughly 45°

orientation of the long axis of the substrate relative to the heme

plane. The spacing between heme and substrate is quite large in

all cases, with the average distance between the progesterone

C21 atom and Fe^3ϩin the heme prosthetic group amounting to

4.15 Å; Fig. 11B). However, if one assumes a perferryl state with

a single oxygen wedged between iron and the progesterone car-

bon atom (Fe^4ϩ-O⅐⅐⅐C21), the Fe⅐⅐⅐C distance is reduced to ϳ3.0

Å, and that between the iron and the H21(C21) atom is reduced

to ϳ2.3 Å. At the other end of the substrate molecule, the car-

bonyl group (O3) is engaged in a hydrogen bond with the gua-

nidino moiety of Arg-234 in the G helix (average distance of

2.66 Å between O3 and NH₂), which protrudes from the ceiling

of the active site (Fig. 11C). The inclination between the plane

defined by the core rings of the progesterone substrate and

the heme plane is ϳ45°. The substrate cavity wall facing the

␤-face interacts primarily with the I-helix, via the hydrophobic

side chain atoms of Val-287 and Ile-291 as well as the Gly-292

residue (Fig. 11, B and C). The ␣-face does not make any notice-

able contacts with nearby loops between helices BЈ and C, helix

K and ␤4, and between the ␤8 and ␤9 strands. The C19 methyl

group of progesterone lies close to Trp-202 from the F-helix

and probably engages in an H⅐⅐⅐␲ type interaction. Other note-

worthy residues located in the vicinity of the substrate are Asp-

107, with its carboxylate moiety residing at ϳ3.90 Å from the

C6 atom of progesterone, Asp-288 from helix I (average dis-

tances of 3.34 Å from C6 and 3.36 Å from C7), and Ser-109, with

C␥ located 3.37 Å from C7 of progesterone (Fig. 11B).

Comparison between the Structures of the Human P450

21A2⅐Progesterone and Bovine P450 21A2⅐17␣-OH-Progester-

one Complexes—The superimposition of the overall structures

of human and bovine P450 21A2 reveals conformational simi-

larities (Fig. 11A), consistent with the high sequence identity

(Fig. 10A), but also clear differences. However, there are some

notable differences as far as the substrate binding sites are con-

cerned. Thus, in the case of bovine P450 21A2, two distinct

17␣-OH-progesterone molecules are visible in all four indepen-

dent complex molecules, with the proximal one located close to

the heme and the distal one residing in the substrate access

channel, located between the FЈ-helix and the ␤4/␤7 sheet. In

the human P450 21A2 complexes, only the proximal site is fully

occupied by progesterone at first observation. However,

inspection of the electron density map at a reduced threshold in

the region of the human P450 21A2 structure that corresponds

to the distal 17␣-OH-progesterone binding site in the crystal

structure of the bovine P450 21A2 complex reveals density that

is consistent with a progesterone molecule of partial occupancy

(Fig. 11D). Further, in the structure of the bovine P450 21A2

complex, accommodation of 17␣-OH-progesterone leads to a

movement of the I-helix relative to the structure of human P450

21A2⅐progesterone complexes. This helical shift is stabilized by

an OH⅐⅐⅐␲ interaction between the Thr-177 O␥H and the Phe-

288 ring (Fig. 11C). In the 17␣-OH-progesterone complexes

with bovine P450 21A2, the 17␣-OH group makes a hydrogen

bond with the carbonyl moiety of Gly-290 (average distance

3.37 Å). However, without a structure of 17␣-OH-progesterone

in complex with human P450 21A2 available, it is difficult to

FIGURE 9. Crystal structure of the human P450 21A2⅐progesterone com-

plex. A, quality of the final 2F_oϪ F_comit electron density (1.2␴ level) around

the progesterone substrate. Helices and selected amino acids are labeled,

including Val-359 (V359), which limits 16␣-hydroxylation activity (12). B,

superimposition of the three independent complex molecules per asymmet-

ricunit. ComplexesA, B, andCarecoloredinbeige, cyan, andlilac, respectively.

C, tertiary structural fold of complex A. The residue ranges for ␣-helices A–M

and ␤-strands 1–9 are as follows: ␤1, 32–36; A, 45–56; ␤2, 59–64; ␤3, 67–72; B,

75–84; BЈ, 86–89; C, 115–131; D, 136–153; E, 161–178; F, 188–202; FЈ, 205–

212;G, 224–246;H, 257–264;I, 279–310;J, 312–322;K, 344–356; ␤4, 364–368;

␤5, 372–374; ␤6, 377–379; ␤7, 384–387; L, 389–394; M, 432–447; ␤8, 450–

454; ␤9, 479–483.

13136 JOURNAL OF BIOLOGICAL CHEMISTRY

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Human P450 21A2 Structure and Kinetics

FIGURE10.PrimaryandtertiarystructurecomparisonofhumanandbovineP45021A2proteins.A,sequencealignmentforhuman(h,topline)andbovine

(b, bottom line) P450 21A2. The sequence alignment was generated with CLUSTAL Omega (33). For the nucleotide sequence and the protein sequence and

numbering of human P450 21A2 (normal 21-hydroxylase B gene), see Ref. 42. B, r.m.s.d. values (in Å) for backbone atoms in the three-dimensional structures

of human and bovine P450 21A2 along the entire chain. The r.m.s.d. values from the superimposition were calculated with the Superpose routine (43) in CCP4

(23).

assess exactly how the human enzyme accommodates 17␣- majority of residues are conserved, and only a few are different:

OH-progesterone. An overlay of the active sites in the human Gly-468 (bovine) versus Val-470 (human); Ser-97 (bovine) ver-

P450 21A2⅐progesterone and bovine P450 21A2⅐17␣-OH-pro- sus Thr-97 (human); and Met-197 (bovine) versus Leu-199

gesterone complex is depicted in Fig. 11B. As can be seen, the (human) (Fig. 10A). Interestingly, all residues interacting with

MAY 22, 2015•VOLUME 290•NUMBER 21

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Human P450 21A2 Structure and Kinetics

FIGURE 11. Comparison between the crystal structures of the human P450 21A2⅐progesterone and bovine P450 21A2⅐17␣-OH-progesterone com-

plexes. A, overall view of the superimposed complexes (human P450 21A2 and bovine P450 21A2 are colored in green and pink, respectively). The overlay was

generated using the Matchmaker option in UCSF Chimera (26). B, close-up view of the active sites in the superimposed complexes. Selected amino acid side

chains in the two structures are depicted and labeled, with carbon, oxygen, nitrogen, and iron atoms colored red, blue, and beige, respectively (in green for

human P450 21A2 and pink for bovine P450 21A2). Selected hydrogen bonds are indicated with dashed lines and distances in Å. C, significant deviations in the

conformation of the central region of helix I in the human and bovine P450 21A2 structures. D, differences in the conformation of the loop region connecting

helices FЈ and G in the human and bovine P450 21A2 structures and evidence for the binding of a second progesterone molecule at a distal site in the structure

of the human protein complex. Fourier 2F_oϪ F_csum electron density at the 0.7␴ level (green meshwork) in the human P450 21A2 structure is consistent with

the presence of a partially occupied progesterone molecule at virtually the same location where 17␣-OH-progesterone (pink wire diagram) resided in the

previously determined structure of the bovine P450 21A2 complex (6).

the porphyrin moiety are identical except for one residue (Ile- structure of bovine P450 21A2 with its substrate 17␣-OH-pro-

108 (bovine) versus Leu-108 (human)). Another residue close to

the C11 atom of the substrate C ring is Met-197 (bovine) (aver-

age distance 4.04 Å); Leu-199(human) features an average dis-

tance of 3.78 Å.

gesterone (6), but this is apparently the first structure of a P450

21A2 enzyme with the substrate progesterone. The human

P450 21A2-progesterone structure has single ligand occupancy

and differs from the bovine structure, which had two molecules

of the substrate 17␣-OH-progesterone bound (6). We also

report kinetic characterization of the human P450 21A2

enzyme and conclude that the rate-limiting step(s) differs from

that reported for bovine P450 21A2 by another group (17).

The catalytic activities of our purified human P450 21A2

enzyme were higher than reported for the bovine enzyme,

which had been used in some previous kinetic studies (14, 15,

17). Some of the differences may be due to the enzyme recon-

stitution conditions, in that previous studies included relatively

high concentrations of detergents and glycerol, and our own

studies with the bovine enzyme yielded higher rates (see above;

Table 2), but not as high as for the human enzyme. The k_cat

values for progesterone and 17␣-OH-progesterone 21-hy-

droxylation were similar (Table 1), but due to a lower K_m, the

reaction was more efficient with progesterone. The catalytic

efficiency (for progesterone 21-hydroxylation) appears to be

A further difference seen in the structures of the complexes

of human and bovine P450 21A2 concerns the acetyl group of

progesterone and 17␣-OH-progesterone. In the human P450

21A2⅐progesterone complex, the acetyl carbon is pointing

toward the heme moiety, whereas in the bovine P450

21A2⅐17␣-OH-progesterone complex, the carbonyl group is

pointing away from the heme (Fig. 11B). Acetyl groups in ste-

roid molecules can rotate more or less freely, and both orienta-

tions are possible in principle. Another factor contributing to

the different orientations of the acetyl groups could be consti-

tuted by the interaction between the progesterone O20 atom

and a methyl group from Val-470 (average distance 3.14 Å).

Discussion

Congenital adrenal hyperplasia is one of the most common

heritable metabolic diseases in humans, and Ͼ95% of cases are

attributed to mutations in P450 21A2 (1, 2). Much of the liter-

ature involving P450 21A2 biochemistry used enzymes from

other species, and this appears to be the first reported purifica-

the highest reported for any mammalian P450 enzyme

(ϳ1.3 ϫ 10⁷

M

s

at 37 °C), which compares favorably

Ϫ1 Ϫ1

tion of human P450 21A2. We previously published a crystal with the apparent k_onrate estimated for the reaction (at

13138 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290•NUMBER 21•MAY 22, 2015

Human P450 21A2 Structure and Kinetics

23 °C) (Fig. 6). Of the other mammalian P450s examined in substrate complex (the “16␣-OH” form) in equilibrium. The

Ϫ1

detail, only human P450 7A1 (2 ϫ 10⁶

M

s

^Ϫ1) (35) and equilibrium could be estimated by kinetic modeling (38) but the

human P450 27C1 (8 ϫ 10⁵^Ϫ1s^Ϫ1)⁵have been found to be equilibrium may not be reflected in the comparison of the ratio

M

close to being this efficient (3).

of the 21- and 16␣-hydroxylation ratios.

Because of the high catalytic efficiency of human P450 21A2,

The three-dimensional overlay of the human and bovine

we were interested in what the rate-limiting step(s) in catalysis P450 21A2 complex structures revealed that the two proteins

is. Substrate binding was relatively fast, calculated to be 2.4 ϫ exhibit significant deviations even in their main chain confor-

10⁷^Ϫ1s^Ϫ1(at 23 °C) (Fig. 5, C and D). If we consider the K_dmations despite the high sequence identity (Figs. 10 and 11).

M

values of the substrates and products (Fig. 5, A and B) and Some of these are accrued within secondary structure elements,

assume that the k_onrates are similar for the products, the k_offas established for a section of helix I (Fig. 11C). It should not

values for the products 11-deoxycorticosterone and 11-deoxy- come as a surprise, however, that the most noticeable confor-

cortisol are 40–60 s^Ϫ1(at 23 °C), which is not very rate-limiting mational differences occur in loop regions. A good case in point

for steady-state reactions with rates of 3 s^Ϫ1(Fig. 4).

is offered by the loop connecting the FЈ and G helices that maps

The KIE studies provide strong evidence that C–H bond to the surface of the protein (Fig. 11D). The FЈ helix lies adjacent

breaking (step 7 in Fig. 2) is rate-limiting. A non-competitive to the distal binding site occupied by 17␣-OH-progesterone in

intermolecular KIE provides the most direct evaluation of the the structure of bovine P450 21A2, and together with the ␤4/␤7

extent of rate limitation of the C–H bond-breaking step in an sheet, the loop connecting ␤2 and ␤3 as well as a portion of the

enzyme reaction (32, 36). The ^DV and ^D(V/K) values were high loop between helices BЈ and C, forms the outer section of the

for the P450 21A2 reactions (5–11) (Fig. 8 and Table 1), strongly substrate access channel. The loop leading from helix FЈ to helix

indicating that C–H bond breaking is at least partially rate- G features a short curl and makes a tighter turn between resi-

limiting. Yoshimoto et al. (13) also reported significant KIE dues Val-212 and Arg-217 in the structure of human P450 21A2

values for human P450 21A2 using other KIE experimental compared with the bovine protein (Fig. 12A). The tighter turn is

designs, and the results are consistent with ours.

caused in part by a sulfate ion that stitches together the side

The high KIEs and rapid product k_offrates are not consonant chains of Arg-217, Arg-103, and Lys-99. The orientations of

with the conclusions of Kominami et al. (17) with bovine P450 side chains from the corresponding residues Arg-215, Gln-103,

21A2, who concluded that product dissociation was rate-limit- and Lys-99, respectively, in the structure of the bovine protein

ing in the case of progesterone 21-hydroxylation but not 17␣- are substantially different, and it is unlikely that these could

OH-progesterone. In contrast, we found high KIEs for both shape the loop conformation in bovine P450 21A2 to resemble

steroid hydroxylations with human P450 21A2. The conclusion the one in human P450 21A2, even if a sulfate ion were present

that product (11-deoxycorticosterone) dissociation is rate-lim- and could be trapped between Arg-215 and Lys-99. Although

iting in the progesterone 21-hydroxylation reaction was based the r.m.s.d. values for main chain atoms discussed above offer a

largely on the appearance of a burst in the pre-steady-state measure for the distinct geometries adopted by the human and

reaction (17) (the reaction with 17␣-OH-progesterone did not bovine P450 21A2 proteins, the differences are even more obvi-

show a burst). We also observed a partial burst in the proges- ous at the level of amino acid side chains, as demonstrated by

terone reaction (Fig. 7A). The partial (20%) burst (cf. 40% for the loop region between the FЈ and G helices. Differences

bovine P450 21A2 in the report of Kominami et al. (17)) has between the conformations of the loop region connecting heli-

several possible explanations. One is that only part of the P450 ces FЈ and G in the two structures (Fig. 12A) are not the result of

21A2 is catalytically active, although this scenario would not deviating crystal packing interactions. Both space groups and

explain the complete absence of a burst with the 17␣-OH-pro- packing interactions differ between the human and bovine

gesterone reaction (17) (Fig. 7B). The possibility can be consid- P450 21A2 crystal structures. Bovine P450 21A2 crystals are of

ered that the product release is only partially rate-limiting, space group P2₁with four molecules per asymmetric unit. Hel-

although modeling with the program DynaFit (37) did not yield ices F, G, and J are involved in crystal packing interactions. In

partial bursts when k_offwas systematically lowered (results not the case of human P450 21A2 crystals, the space group is C2

presented). Another possibility is one that we established for with three molecules per asymmetric unit. Crystal packing

DNA polymerases (38). The appearance of a quantitative burst interactions are primarily mediated by helices B, BЈ, C, J, and M.

is evidence that the rate-limiting step occurs after product for- The loop region between helices FЈ and G is not involved in

mation, but a reversible equilibrium of an enzyme-substrate packing in either the crystal of human P450 21A2 or that of the

complex between an active and an alternate form can explain a bovine protein. Instead, residues from loops participating in

partial burst (38, 39), and this proved to be the case in DynaFit lattice interactions in the former include 35–36, 66–67, 213–

kinetic modeling (results not presented). Such an explanation is 223,247–252,380–382,412–414,andterminalresiduesofheli-

possible here, in that it is known that the enzyme also forms an ces F (position 189) and H (position 264).

alternate product from progesterone, 16␣-OH-progesterone

With the human P450 21A2 crystal structure now in hand, it

(this reaction (i.e. 16␣-hydroxylation) does not occur with the is interesting to evaluate the structural consequences of some

substrate 17␣-OH-progesterone (13)). Thus, one can explain selected missense mutations in human P450 21A2 that cause

the partial burst with an alternate conformation of the enzyme- complete loss of function in some cases and congenital adrenal

hyperplasia, resulting in salt-wasting disease, or steep reduc-

tions in enzyme activity and simple virilizing disease. An exam-

⁵L. D. Nagy and F. P. Guengerich, unpublished results.

ple of the former is constituted by the E352K mutation located

MAY 22, 2015•VOLUME 290•NUMBER 21

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Human P450 21A2 Structure and Kinetics

in helix K (7). Glu-352 is part of the so-called ERR triad together

with Arg-355 and Arg-409, the latter mapping to the meander

region leading up to helix M (Fig. 12B). Using a model of human

P450 21A2 based on the crystal structure of the bovine protein,

it was predicted that the E352K mutation would disrupt the

triad as a result of repulsions between the three basic side

chains (7). The crystal structure of human P450 21A2 indeed

confirms this view in that the ERR triad seen in the bovine

protein is maintained and that replacement of the Glu-352 by a

lysine would disrupt the salt bridge to Arg-409 (Fig. 12B).

Another salt-wasting disease-causing mutation, R409H (7), can

also be expected to disrupt the ERR triad, because the shorter

histidine side chain might not be able to reach the carboxylate

moiety of Glu-352 (Fig. 12B).

Although humanized models of P450 21A2 (6, 7) based on

the crystal structure of the bovine protein (6) may be helpful in

some cases for interpreting changes at the structural level as a

result of disease-causing mutations in patients (e.g. those affect-

ing the above ERR triad), clear differences between the human

and bovine 21A2 crystal structures set limitations for the use-

fulness of a computational model. Thus, the V305D mutation in

helix I, which is implicated in S-channel gating (simple viriliz-

ing phenotype), was interpreted to result in a salt bridge to

Lys-473 based on the modeled structure of human 21A2 (7).

However, Lys-473 in the crystal structure of human P450 21A2

and the corresponding Lys-472 in the structure of bovine P450

21A2 adopt very different orientations relative to Val-305 (Glu-

305 in the mutant) and Val-303, respectively (Fig. 12C). Lys-473

in human P450 21A2 engages in a salt bridge to Glu-295 located

further up in helix I. The same glutamate also forms a hydrogen

bond to Asn-299 (Fig. 12C). The amino nitrogen of Lys-473 lies

at a distance of 13.9 Å from the C atom of the side chain of

Val-305, a gap that obviously canno^␤t be bridged even if valine is

mutated to glutamate. Conversely, in the structure of bovine

P450 21A2, the corresponding distance between Lys-472 and

Val-303 is 8.6 Å, a gap that could conceivably be bridged if

valine were replaced by glutamate and the lysine side chain

unraveled to assume a more extended conformation. In the

crystal structure of bovine P450 21A2, methylene groups of the

lysine side chain stack onto Trp-303 (Fig. 12C). In the structure

of human P450 21A2, it is the side chain of Met-474 that snakes

along the plane of Trp-303 (Fig. 12C).

Two additional salt-wasting mutations in the N-terminal

region, G65E (40) and G91V (41), that reduce P450 21A2 activ-

ity to Ͻ1%,⁶can now be readily interpreted in light of the

human P450 21A2 crystal structure. Gly-65 maps to a hydro-

phobic patch, and mutation to a glutamate would disrupt

hydrophobic interactions and result in a direct clash with the

side chain of Val-212 (Fig. 13A). The mutation can therefore be

expected to destabilize the protein and alter the local confor-

mation considerably. The opposite scenario, namely disturbing

a largely negatively polarized region by introducing a bulky

hydrophobic moiety, would play out in the case of the G91V

mutation (Fig. 13B). Thus, in the mutant, the valine side chain

would be surrounded by main chain keto groups as well as

FIGURE12. Distinctsidechaininteractionsinthestructuresofhumanand

bovine P450 21A2 complexes and insights into the structural conse-

quences of mutations. A, a sulfate ion is bound by amino acids from loop

regions connecting helices F and G (top left, Arg-217) and helices B and C

(center, Lys-99 and Arg-103), respectively, in the structure of human P450

21A2. The corresponding residues in bovine P450 21A2 are Arg-215, Lys-99,

and Gln-103, respectively, which exhibit completely different relative orien-

tations compared with the situation in the structure of the human protein. B,

view of a portion of an extended loop (meander) region connecting helices L

and M, with Arg-409 in human P450 21A2 engaging in interactions with main

chain oxygen atoms of amino acids Glu-400 and Arg-401 as well as the car-

boxylate moiety of the Glu-352 side chain from the K helix. A further amino

acid in close vicinity of Arg-409 is Arg-355. The R409H and R352K mutations

(7) as well as the R409C and R409L mutations (6, 44–47) could disrupt the ERR

triad. C, view of interactions between I helix residue Glu-295 and the side

chains of the adjacent Asn-299 and Lys-473 located in the ␤8-␤9 loop. The

V305D mutation was predicted to result in a salt bridge between Asp-305 and

Lys-473 (7), but the structure of the human P450 21A2 complex reveals that

the two residues are spaced too far apart.

⁶C. Wang and F. P. Guengerich, unpublished data.

13140 JOURNAL OF BIOLOGICAL CHEMISTRY

VOLUME 290•NUMBER 21•MAY 22, 2015

Human P450 21A2 Structure and Kinetics

the latter structure revealed a second progesterone of lower

occupancy that is located at the distal site in the substrate access

tunnel (Fig. 11D), where the second 17␣-OH-progesterone

molecule is lodged in the structure of the bovine P450 21A2

complex. Thus, the differences in the binding modes of proges-

terone and 17␣-OH-progesterone in the structures of the

human and bovine P450 21A2 protein complexes are more

likely reflective of deviating binding affinities for the substrates

and different crystallization conditions. Although the human

and bovine P450 21A2 enzymes share 80% sequence identity,

the conformational differences in the main chains and orienta-

tions of side chains in the two structures are most likely a re-

flection of intrinsic differences between the two proteins rather

than a consequence of the crystallizations with different sub-

strates. This conclusion is also supported by very similar bind-

ing modes of progesterone and 17␣-OH-progesterone at the

proximal sites in the human and bovine complexes (Fig. 11, B

and C).

The crystal structure of human P450 21A2 now enables

insight into the structural underpinnings of the phenotypes

exhibited by the numerous mutations identified for the P450

21A2 enzyme and associated in many cases with congenital

adrenal hyperplasia. By analyzing just a few of these mutants in

the context of the structure of human P450 21A2, we have dem-

onstrated that a computational model of the three-dimensional

structure of human P450 21A2 derived by others (7) on the

basis of our previously determined structure of the bovine

enzyme (6) does not provide a meaningful understanding of

the consequences for structure and stability by mutation in

some cases. A more extensive correlative analysis of the struc-

ture and phenotypes of the many P450 21A2 mutants identified

to date goes beyond the focus of this paper and will be presented

elsewhere.

FIGURE 13. Structural consequences of two salt-wasting mutations in the

N-terminal region of human P450 21A2. A, mutation of Gly-65 located in

the ␤2-␤3 loop to glutamate (40) (side chain carbon atoms highlighted in

gray) introduces a charged residue into a hydrophobic patch and is expected

to result in a clash with Val-212, located in the loop region linking helices F

and G. B, mutation of Gly-91 located in the loop between the B and BЈ helices

to valine (41) (side chain carbon atoms highlighted in gray) introduces a

hydrophobic residue into a highly polar region near the surface of the protein

and is expected to result in a clash with Asp-112, located in the loop region

linking helices BЈ and C.

In summary, the human P450 21A2 structure, complexed

with progesterone, provides a better template for a biochemical

understanding of the disease variants. The juxtaposition of the

substrate is considered to be very important in explaining the

very efficient catalysis and the rate-limiting nature of the C–H

bond cleavage in the reaction. Both the C–H bond-breaking

hydrophilic or negatively charged side chains from threonine or and substrate-binding steps appear to contribute in the kinetic

aspartate, respectively. Moreover, the carboxylate moiety of the analysis of the overall hydroxylation reaction.

latter residue, Asp-112, would clash with the Val-91 side chain.

Acknowledgments—We thank K. Trisler for assistance in the prepa-

ration of the manuscript. Vanderbilt University is a member institu-

tion of the Life Sciences Collaborative Access Team at Sector 21 of the

Advanced Photon Source (Argonne, IL). Use of the Advanced Photon

Source at Argonne National Laboratory was supported by the United

States Department of Energy, Office of Science, Office of Basic Energy

Sciences, under Contract DE-AC02-06CH11357.

Our study provides a complete kinetic framework for human

P450 21A2 and the first crystal structure of the human enzyme

with bound substrate. The kinetic data demonstrate that

human P450 21A2 catalyzes 21-hydroxylation of both proges-

terone and 17␣-OH-progesterone with higher rates than the

bovine enzyme and that human P450 21A2 is among the fastest

mammalian P450 enzymes if not the fastest (3). The crystal

structures of human P450 21A2 in complex with progesterone

and the bovine enzyme in complex with 17␣-OH-progesterone

exhibit significant differences. Importantly, these are not lim-

ited to loop regions and areas closer to the surface but are also

found within secondary structure elements conserved in P450

enzymes. Could some of these conformational differences be

the result of two 17␣-OH-progesterone molecules being bound

to bovine P450 21A2 but only one progesterone to human P450

21A2? The electron density computed at a lower threshold for

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