(Received for publication, December 16, 1996, and in revised form, June 13, 1997)
From the Section of Hematology Research and the Department of Biochemistry and Molecular Biology, Mayo Clinic and Foundation, Rochester, Minnesota 55905
Calcineurin belongs to a family of
serine/threonine protein phosphatases that contain active site
dinuclear metal cofactors. Bacteriophage protein phosphatase is
also considered to be a member of this family based on sequence
comparisons (Lohse, D. L., Denu, J. M., and Dixon, J. E. (1995)
Structure 3, 987-990). Using EPR spectroscopy, we
demonstrate that
protein phosphatase accommodates a dinuclear metal
center. Calcineurin and
protein phosphatase likewise contain a
conserved histidine that is not a metal ligand but is within 5 Å of
either metal in calcineurin. In this study the conserved histidine in
calcineurin was mutated to glutamine and the mutant protein analyzed by
EPR spectroscopy and kinetic methods. Parallel studies with an
analogous
protein phosphatase mutant were also carried out. Kinetic
studies using paranitrophenyl phosphate as substrate showed a decrease
in kcat of 460- and 590-fold for the
calcineurin and
protein phosphatase mutants, respectively, compared
with the wild type enzymes. With a phosphopeptide substrate, mutagenesis of the conserved histidine resulted in a decrease in
kcat of 1,300-fold for calcineurin. With the
analogous
protein phosphatase mutant, kcat
decreased 530-fold compared with wild type
protein phosphatase
using phenyl phosphate as a substrate. EPR studies of the
iron-reconstituted enzymes indicated that although both mutant enzymes
can accommodate a dinuclear metal center, spectroscopic differences
compared with wild type proteins suggest a perturbation of the ligand
environment, possibly by disruption of a hydrogen bond between the
histidine and a metal-coordinated solvent molecule.
Calcineurin, also known as protein phosphatase 2B, consists of a 58-kDa catalytic subunit, calcineurin A, and a 19-kDa regulatory subunit, calcineurin B. It is a serine/threonine protein phosphatase whose activity is regulated by Ca2+/calmodulin. Calcineurin is the target of the immunosuppressant drugs cyclosporin A and FK506 (1, 2). These drugs bind to intracellular proteins, termed immunophilins; cyclophilin is the binding protein for cyclosporin A, and FK506 binds to the FK506-binding proteins. The complex of immunosuppressant drug and immunophilin in turn binds to and inhibits the phosphatase activity of calcineurin. Calcineurin inhibition prevents the transcriptional activation of the interleukin 2 gene in helper T cells, leading to suppression of the immune response.
Calcineurin is a member of the class of serine/threonine protein
phosphatases, whose members include protein phosphatases 1 (PP1)1 and 2A (PP2A),
phosphatases essential for a number of signal transduction pathways in
eukaryotic cells (3, 4). Another protein phosphatase from bacteriophage
,
PP, also belongs to this family (5). In addition, a number of
less characterized enzymes containing the "phosphoesterase"
consensus motif of this family,
DXH(X)nGDXXD(X)nGNHD/E,
have been identified via protein sequence comparisons (6, 7). It has
been hypothesized that this motif provides a scaffold for an active
site dinuclear metal center (7, 8), similar to the dinuclear metal
centers in PP1 (9, 10) and calcineurin (11-13). A variety of
experimental evidence indicates that this cluster in calcineurin is an
Fe3+-Zn2+ center.
Although little is known about the catalytic mechanism of the serine/threonine protein phosphatases, several pieces of experimental data indicate that the dinuclear metal center is a key component of the active site. First, x-ray crystallographic data of calcineurin and PP1 indicate that the dinuclear metal center has a ligand environment nearly identical to that of mammalian and plant purple acid phosphatases, enzymes that contain either dinuclear Fe-Fe or Fe-Zn centers that have been demonstrated to be essential for catalytic activity (14, 15). Second, these crystallographic studies indicate that the product of the reaction, phosphate, and the product analog, tungstate, directly coordinate both metal ions (10, 11, 16). Third, redox titrations of either the Fe3+-Zn2+ (12) or Fe3+-Fe2+ forms2 of calcineurin indicate a correlation between enzyme activity and the oxidation state of the bound metal ions.
In addition to the dinuclear metal center, there are several conserved
amino acids within the active site which are likely to contribute to
catalysis. One of these residues in calcineurin is histidine 151 (numbering based on the rat calcineurin A sequence (18)). His-151 is
not a ligand to either metal but is within 5 Å of both metal ions and
is conserved in other metallophosphoesterases such as PP1 (histidine
125) and
PP (histidine 76) (7). In one crystal structure, His-151
was modeled to participate in a hydrogen bond to a metal-coordinated
solvent molecule (13). The importance of this residue has been
demonstrated by site-directed mutagenesis of both
PP and PP1 which
found substantial effects on catalytic activity and/or protein
stability.
In this study the conserved histidine residue in calcineurin A,
His-151, was changed to glutamine by site-directed mutagenesis. After
reconstitution with calcineurin B, the calcineurin H151Q heterodimer
was purified to homogeneity. EPR spectroscopy was used to assess
whether mutagenesis affected the ligand environment of the dinuclear
metal center. Kinetic studies were also carried out using either pNPP
or [P]-RII peptide as substrates. The analogous PP
mutant, H76N, was characterized by EPR in a similar fashion and assayed
using either pNPP or phenyl phosphate. For both enzymes, mutagenesis
resulted in decreases in kcat of
102-103. EPR spectroscopy of
iron-reconstituted
PP confirms that this enzyme accommodates a
dinuclear metal center as predicted for members of the
metallophosphatase family. EPR also indicates that mutagenesis does not
prevent assembly of the dinuclear metal center in either calcineurin
H151Q or
PP(H76N). Nevertheless, differences observed in the EPR
spectra of wild type versus mutant enzymes indicate a
perturbation of the ligand environment, possibly by disruption of a
hydrogen bond between the histidine and a metal-coordinated water
molecule.
Competent BL21(DE3) cells were obtained from
Novagen (Madison, WI). Bovine serum albumin, DEAE-Sepharose CL-6B,
bovine pancreatic DNase I, type II, pNPP, phenyl phosphate, and
Sephacryl S300 were purchased from Sigma. Coomassie Plus Protein Assay
Reagent and 8 M guanidine hydrochloride were from Pierce
Chemical Co. [-32P]ATP (~3,000 Ci/mmol) was
purchased from Amersham Corp. Calmodulin was prepared from bovine brain
(19, 20) and coupled to Affi-Gel active ester agarose (Bio-Rad) for use
in calmodulin affinity chromatography. PM30 and YM30 membranes and
Centricon-30 concentrators were purchased from Amicon (Beverly, MA).
Tryptone, yeast extract, and Luria Bertani medium were purchased from
Difco. Sephadex NAP-25 columns were purchased from Pharmacia Biotech
Inc. The protein expression vectors used were pRCNAT77 (21) and
pRCNBT775-3 (22), encoding the genes for calcineurin A and B,
respectively, and the plasmid, pBB131 (23, 24), encoding the gene for
N-myristoyl transferase. The construction of pT7-7 plasmids
containing the genes for wild type and H76N mutant of
PP is
described elsewhere (5, 25). The Wizard Maxipreps and Wizard Minipreps
DNA purification kits and T4 polynucleotide kinase were purchased from
Promega (Madison, WI). The Geneclean II kit was purchased from BIO 101, Inc. (Vista, CA). The oligonucleotide required for site-directed mutagenesis was synthesized by the Mayo Clinic Molecular Biology Core
Facility. RII peptide (DLDVPIPGRFDRRVSVAAE) and
[31P]-RII peptide (DLDVPIPGRFDRRVS(p)VAAE)
were synthesized by the Mayo Clinic Protein Core Facility.
Protein concentrations were determined by the
Bradford assay using the Pierce Coomassie Plus Protein Assay Reagent
with bovine serum albumin as a standard (26). Alternatively,
calcineurin concentrations were determined by UV-visible
spectrophotometry (27) using 281 = 50,000 M
1 cm
1. Protein concentration
values determined using this extinction coefficient agreed within 10%
with concentrations determined by amino acid analysis.
The H151Q mutation of calcineurin
A, CN(H151Q), was created using a 5 Prime 3 Prime MORPH
site-specific plasmid DNA mutagenesis kit (Boulder, CO). The primer
5
-GCCTACATTCCTGGTTTCCAC-3
, complementary to the coding
strand of calcineurin A, was used for mutagenesis with the underlined
bases representing the codon of the mutated residue. This primer was
phosphorylated by T4 kinase and used according to the manufacturer's
instructions. Half-mutant plasmid DNA was subsequently transformed into
Escherichia coli strain BMH 71-18. Plasmid DNA isolated from
colonies was screened for the desired mutation by DNA sequencing of the
entire calcineurin A gene and yielded the expression plasmid
pCNAT77(H151Q).
The plasmid pCNAT77(H151Q) was transformed into competent BL21(DE3) cells. The growth of these cells, crude extract isolation, reconstitution with myristoylated calcineurin B, and purification of the calcineurin heterodimer were performed as described previously (21, 22).
Expression and Purification of Wild TypeExpression
of wild type PP was performed as described (5). All purification
steps were performed at 4 °C. After growth and induction with
isopropyl
-D-thiogalactopyranoside, the cells were
harvested by centrifugation at 3,400 × g for 30 min,
washed with 250 ml of 0.1 M Tris-Cl, pH 7.5, and
recentrifuged at 4,200 × g for 20 min. The cells were
resuspended in 25 mM Tris-Cl, pH 8.0, 20% glycerol, 1 mM EGTA (TGE buffer) and lysed by three passages through a
French pressure cell operating at 16,000 p.s.i. The cell lysate was
subsequently centrifuged at 39,000 × g for 3 h. The supernatant (40 ml) was batch adsorbed onto 150 ml of
DEAE-Sepharose CL-6B preequilibrated with TGE buffer. The resin was
washed in a fritted funnel with 300 ml of TGE and
PP eluted with TGE
buffer containing 0.1 M NaCl. Fractions containing
PP
were pooled and precipitated by the addition of ammonium sulfate to
50% saturation. After centrifugation at 34,800 × g,
the protein pellet was resuspended in TGE buffer + 0.5 M
NaCl and applied to a phenyl-Sepharose column (20 × 1-cm
diameter) previously equilibrated with TGE buffer + 0.5 M
NaCl. The column was washed with 200-300 ml of the same buffer and
then with 250 ml of 20 mM Tris-Cl, pH 7.5. The enzyme was
eluted with 250 ml of 50 mM Tris-Cl in 50% glycerol, pH
7.5. Fractions were assayed using pNPP as a substrate, pooled, and stored at
70 °C in 50 mM Tris-Cl and 50% glycerol, pH
7.5.
The PPT77(H76N) plasmid (25)
was transformed into BL21(DE3) cells and single colonies used to
inoculate 10 ml of Luria Bertani medium/ampicillin (0.1 mg/ml) for
overnight culture at 37 °C. Overnight cultures were then used to
inoculate 5 liters of 2 × YT/ampicillin medium (10 g/liter yeast
extract, 20 g/liter tryptone, 10 g/liter NaCl, 0.05 g/liter ampicillin)
in a New Brunswick Bioflo 3000 fermentor. Cells were grown overnight at
22 °C maintaining aeration at 30% of air saturation to a cell
density that gave an absorbance at 595 nm of ~9. Glucose was added to
a final concentration of 0.4%, and the cells were induced with 1 mM isopropyl
-D-thiogalactopyranoside. Another aliquot of glucose was added to a final concentration of 0.4%
when the cell density corresponded to an absorbance at 595 nm of 16. The cells were harvested 20 h postinduction by centrifugation at
3,400 × g for 20 min. The cell pellet was resuspended
in ~2 ml of 50 mM Tris-Cl, pH 7.5/g of cells, wet weight.
To this resuspension, 0.4 mg/ml lysozyme, 23 mM EDTA, and
0.05% Triton X-100 were added sequentially with stirring on ice for 30 min followed by a freeze/thaw process to lyse the cells. To reduce the
viscosity, MgCl2 (20 mM), DNase (0.1 unit/ml
final concentration), and 2% protamine sulfate (1/6 total volume) were
added sequentially with stirring on ice. After centrifugation at
10,000 × g for 1 h, the protein was purified as
described above for wild type
PP.
Circular dichroism spectra
were recorded at 25 °C on a Jasco J-710 circular dichroism
spectrometer. A quartz cell of 0.0202-cm path length was used for all
measurements. Mean residue ellipticities were calculated from the
relationship m =
obs/(10Crl) where
m is the mean residue ellipticity,
obs is
the observed ellipticity in millidegrees, Cr is
the mean residue molar concentration, and l is the path
length of cell in cm.
m is measured in degree
cm2 dmol
1. Samples of calcineurin (18 µM) and CN(H151Q) (24 µM) were examined in
10 mM Hepes, pH 7.5, 1 mM MgCl2,
0.1 M EGTA, 0.2 mM DTT.
Calmodulin-dependent phosphatase
activity of calcineurin and CN(H151Q) was measured using pNPP as a
substrate at 30 °C in 25 mM MOPS, pH 7.0, 1.0 mM MnCl2, 0.1 mM CaCl2,
1 µM calmodulin, and 15-23 nM wild type
calcineurin or 710 nM CN(H151Q). Wild type PP and
PP(H76N) activities were measured at 30 °C in 100 mM Tris-Cl, pH 7.8, 10 mM DTT, 1 mM
MnCl2, and 0.64 nM wild type
PP or 860 nM
PP(H76N). After incubation for 5 min at 30 °C, reactions were started by the addition of pNPP. Specific activity was
measured by following the increase in absorbance at 410 nm with time
using
410 = 7,180 M
1
cm
1 at pH 7.0 and 14,400 M
1
cm
1 at pH 7.8 based on a pKa of 7.17 and
410 = 17,800 M
1
cm
1 for the p-nitrophenolate anion. The
concentration of pNPP was varied from 2 to 100 mM, and the
kinetic parameters kcat and
Km were determined by fitting the data to the
Michaelis-Menten equation using a nonlinear least squares analysis
method.
RII
peptide was phosphorylated with [-32P]ATP to a
specific activity of 833 µCi/µmol using the catalytic subunit of
bovine cardiac cyclic-AMP dependent protein kinase and purified as
described (28). Assays were done as described (28) in 100 mM MOPS, pH 7.0, 1 mM MnCl2, 0.1 mM CaCl2, 0.5 mM DTT, 0.1-28
µM calmodulin, and 1.0 mg/ml bovine serum albumin.
[32P]-RII peptide was diluted with
[31P]-RII peptide and substrate added in
final concentrations from 9 µM to 1 mM.
Calcineurin concentrations were 10 nM for wild type and
7.7-15 µM for CN(H151Q). Data were obtained in duplicate
and fitted to the Michaelis-Menten equation by a nonlinear least
squares analysis method.
Assays were performed by
determining the amount of inorganic phosphate released during
hydrolysis of phenyl phosphate as described (29). Assays were done in
100 mM Tris-Cl, pH 7.8, containing 1 mM
MnCl2 and 10 mM DTT. Phenyl phosphate
concentrations were varied from 1 to 70 mM for PP and 1 to 90 mM for
PP(H76N). Wild type protein concentrations
ranged from 7.0 to 630 nM, whereas
PP(H76N)
concentrations ranged from 2.2 to 12 µM. Enzyme was incubated 30 °C, 5 min, and reactions started by the addition of
phenyl phosphate. At various times from 0.5 to 7.0 min, 50 µl of the
reaction was taken and added to 800 µl of a solution containing a 3:1
ratio of 0.045% malachite green hydrochloride to 4.2% ammonium
molybdate in 4 N HCl. After 1 min, 100 µl of 34% sodium
citrate was added and the absorbance at 660 nm measured. Free phosphate
was determined from a standard curve prepared using solutions of
KH2PO4. Kinetic parameters for
PP and
PP(H76N) were determined as described above.
About
0.4 mg/ml CN(H151Q) in 20 mM Tris-Cl, pH 7.5, 100 mM KCl, 1.0 mM magnesium acetate, 1.0 mM DTT, 0.1 mM EGTA, or 0.6 mg/ml PP(H76N)
in 50 mM Tris-Cl, pH 7.5, 100 mM NaCl, 10%
glycerol was added to septum-sealed vials and made anaerobic by
flushing repeatedly with oxygen-free argon. BME was added to a final
concentration of 0.715 M and
Fe(NH4)2(SO4)2·6H2O
added dropwise to a final concentration of 0.375 mM. The
solutions were flushed with argon and incubated an additional 17 h
at room temperature. Samples were concentrated using YM30 membranes in
an Amicon filtration cell to ~ 2 ml; buffer exchanged over
Sephadex NAP-25 gel filtration columns equilibrated with 50 mM MOPS, pH 7.0, 1 mM BME; and concentrated
using Centricon-30 membranes to ~250 µl. Samples were then
transferred to quartz EPR cuvettes and frozen in liquid nitrogen.
Iron-reconstituted forms of wild type calcineurin and
PP were
prepared in a similar manner except that incubation proceeded at
4 °C for 17 h, and both enzymes were desalted into 100 mM Tris-Cl, pH 7.5, 1 mM BME prior to the final
concentration step.
To the iron-reconstituted EPR sample of CN(H151Q), a solution of 0.5 M potassium phosphate, pH 7.5, was added anaerobically to a final concentration of 20 mM, incubated for 5 min at room temperature, and frozen in liquid nitrogen.
EPR AnalysisEPR spectra were recorded using a Bruker ESP300E spectrometer operating at 9 GHz (X-band) microwave frequency equipped with an Oxford Instruments ESR 900 continuous flow cryostat for temperature regulation. Background cavity resonances were subtracted from all spectra.
Metal AnalysisMetal analysis was performed by the Mayo Clinic Metals Laboratory using inductively coupled plasma emission spectrometry.
The relationship of His-151 in calcineurin relative to
the dinuclear metal center can be seen in Fig.
1. His-151 was mutated to a glutamine by
site-directed mutagenesis to investigate the effect on enzyme activity
and assembly of the dinuclear metal cofactor. The mutant calcineurin A
subunit was expressed in E. coli in a fashion identical to
that of the wild type calcineurin A subunit and reconstituted with
myristoylated calcineurin B to generate the mutant protein CN(H151Q).
The presence of the proper codon as well as the lack of inadvertently
introduced mutations in the entire calcineurin A gene were confirmed by
DNA sequence analysis. Although the yield of CN(H151Q) was less than
that obtained for the wild type reconstituted protein, enough material
could be obtained and purified to homogeneity for biochemical and
spectroscopic (EPR) analyses. A typical purification yielded
approximately 1 mg of CN(H151Q) protein/liter of cell culture.
The analogous residue in PP, identified by primary sequence
comparisons as His-76 (7), was mutated to an asparagine residue (25).
In this study, the
PP(H76N) protein was purified to homogeneity as
described under "Methods" to yield approximately 17 mg/liter of
culture.
Circular dichroism analysis of wild type calcineurin and CN(H151Q) provided evidence for a native-like conformation of CN(H151Q); CD spectra from 200 to 250 nm of both recombinant wild type calcineurin and CN(H151Q) are comparable to spectra of bovine calcineurin (30) (data not shown).
Phosphatase Activities of Wild Type Calcineurin and CN(H151Q)Kinetic parameters using pNPP and [P]-RII
peptide as substrates for wild type calcineurin and CN(H151Q) in the
presence of 1 mM MnCl2 are compared in Table
I. Using either substrate, the values of
kcat for the mutant enzyme were significantly
lower than the kcat values for wild type enzyme.
Thus, the kcat for CN(H151Q) using pNPP as a
substrate, 5.6 × 102 s
1, is 460-fold
lower than recombinant wild type calcineurin prepared in an identical
fashion. Using [P]-RII peptide, the
kcat values for wild type calcineurin and
CN(H151Q) were 1.2 × 101 s
1 and
9.0 × 10
3 s
1, respectively, a
difference of 1,300-fold. Using [P]-RII peptide, Km values for both forms of calcineurin were the
same within the error of the measurement. However, a 10-fold decrease in Km was observed for CN(H151Q) compared with wild
type calcineurin using pNPP as a substrate.
|
Kinetic parameters using pNPP and phenyl phosphate as
substrates for wild type PP and
PP(H76N) are compared in Table
II. These parameters were also determined
by inclusion of 1.0 mM MnCl2 in assay buffers.
Using pNPP as substrate, the kcat values for wild type
PP and
PP(H76N) were 3.9 × 102
s
1 and 6.6 × 10
1 s
1,
respectively. This represents a 590-fold difference, which is comparable to the decrease in kcat observed for
calcineurin for the analogous substitution. The difference in
kcat for
PP is less than the
105 fold difference found previously (25) mostly because
of a 30-fold higher activity measured for the
PP(H76N) protein
isolated in the present study. Using phenyl phosphate as substrate, the
kcat value for
PP was 1.7 × 101 s
1, compared with 3.2 × 10
2 s
1 for
PP(H76N), a difference of
530-fold. The Km values were very similar for both
mutant and wild type proteins using either substrate.
|
Although not a metal ligand, H151Q in calcineurin is
close enough to either metal ion such that mutagenesis might perturb the environment surrounding the metal cluster. To investigate the
effect of mutagenesis on the dinuclear center, wild type calcineurin and CN(H151Q) were reconstituted with iron to generate a mixed valence
Fe3+-Fe2+ cluster as a spectroscopic probe of
the active site and analyzed by EPR spectrometry. The EPR spectra of
wild type calcineurin, CN(H151Q), and CN(H151Q) in the presence of 20 mM potassium phosphate are compared in Fig.
2. Several features are evident in the
EPR spectra including a minor high spin Fe3+ species with
g values of 9.2 and 4.3 (about 700-2,000 Gauss region, not
shown for clarity), a minor radical species centered at
gav = 2.0, and a component with
gav < 2.0 representing the major paramagnetic species.
Simulations of the EPR signal in Fig. 2A to an S = 1/2 species yielded g values of 1.93, 1.77, and 1.64. This signal is identical to the signal observed previously in bovine brain calcineurin reconstituted with iron and arises from a dinuclear iron center in the mixed valence (Fe3+-Fe2+) oxidation state (12). The spin Hamiltonian He that describes the magnetic properties of the dinuclear iron center is
![]() |
(Eq. 1) |
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Fig. 2B shows the EPR spectrum of iron-reconstituted CN(H151Q). An EPR signal representative of a dinuclear iron center in the mixed valence state is evident and indicates that a dinuclear metal center can be assembled in the mutant enzyme. This signal, however, is broader than the signal observed from the mixed valence center in wild type enzyme. Metal analyses of the EPR sample found 1.5 mol each of iron and zinc/mol of protein, consistent with the formation of a dinuclear iron center but also indicating the possible presence of a mixed metal Fe-Zn center or adventitious zinc, either of which would contribute to the EPR spectrum in the gav < 2.0 region.
Further proof that the signal in Fig. 2B results from an active site metal center was demonstrated by adding 20 mM potassium phosphate to the sample. The addition of phosphate led to a noticeable sharpening of the EPR signal (Fig. 2C), whereas none of the other species was affected, suggesting that phosphate coordinates to one or both of the metal ions of the dinuclear iron center.
EPR Analysis of Iron-reconstitutedIn a
fashion similar to that for calcineurin, PP and
PP(H76N) were
reconstituted with iron to generate a spectroscopic probe of the active
site metal cluster.
PP as purified contains very little iron, zinc,
or manganese as determined by metal analysis using inductively coupled
plasma emission spectrometry (
0.05 mol of iron, 0.09 mol of zinc,
and 0.01 mol of manganese/mol of protein). Likewise,
PP(H76N) also
contained low amounts of these metals (
0.3 mol of iron, 0.09 mol of
zinc, and 0.01 mol of manganese/mol of protein). Reconstitution of wild
type
PP and
PP(H76N) with iron yielded samples that exhibited low
temperature EPR spectra with gav < 2.0 (Fig.
3, A and B). Metal
analysis of both EPR samples found 1.74 iron/mol of protein and 0.20 zinc/mol of protein for wild type
PP, and 1.7 iron/mol of protein
and 0.05 zinc/mol of protein for
PP(H76N). Thus both
PP and
PP(H76N) can accommodate dinuclear metal clusters. As in the case of
calcineurin, the EPR spectrum of the
PP(H76N) mutant is different
from the spectrum of wild type
PP.
Recent crystallographic models of calcineurin (11, 13), PP1 (9,
10), and purple acid phosphatase (16, 31) have identified a conserved
histidine in each active site which, although not coordinated to either
metal of the dinuclear metal center, is within 5 Å of both
metal ions. In this study we have mutated the corresponding residue of
calcineurin (His-151) to glutamine to explore its significance in
catalysis and effect, if any, on the active site dinuclear metal
center. An analogous histidine in the bacteriophage
protein
phosphatase has been previously modified to asparagine (
PP(H76))
(25). The H76N mutation resulted in a 105-fold reduction in
kcat toward pNPP, a 40-fold increase in the Km for Mn2+, the divalent metal ion
activator used in assay buffers, yet little change in
Km for substrate. In a study by Lee and colleagues
(32), the comparable residue in PP1 (His-125) was mutated to a number
of residues. In that study it was found that most of the substitutions
resulted in the production of insoluble protein except for two
mutations, H125A and H125S, where a fraction of the protein was soluble
and could be purified by affinity chromatography. Although neither PP1
mutant exhibited any detectable phosphatase activity, the upper limit
for activity and/or fold reduction relative to wild type PP1 was not
reported.
Similar to the results noted in the PP1 study, the level of expression
of H151Q soluble protein was also lower than that found for wild type
calcineurin A. Hence, the yield of the CN(H151Q) heterodimer was lower
(4-fold) than wild type reconstituted enzyme prepared in an
identical fashion.3 As with
wild type calcineurin (21), growth of E. coli expressing the
calcineurin A subunit at lower temperatures (23 °C) improved the
yield of soluble protein in crude extract. Reconstitution of the mutant
calcineurin A subunit with calcineurin B allowed purification of
sufficient protein for biochemical studies and spectroscopic analysis
using EPR.
To assess the affect of mutagenesis on catalytic activity, two
different substrates were used for calcineurin, pNPP and
[P]-RII peptide.4 Furthermore,
MnCl2 was included in buffers to obtain the maximum activity for both enzymes (5, 33, 34). Manganese is known to
incorporate into one or both metal sites in PP1 (9, 10). Preliminary
data indicate that a spin-coupled dinuclear Mn2+ cluster is
also assembled in PP.5
In terms of catalytic activity, mutagenesis of His-151 to glutamine resulted in a 460-fold decrease in kcat using pNPP as substrate. In comparison, the kcat for CN(H151Q) decreased 1,300-fold compared with wild type calcineurin using [P]-RII peptide as substrate. For either substrate, Km was either not affected or slightly improved by mutagenesis. The kcat values for wild type calcineurin measured in this study are comparable or slightly higher than previously reported (24, 34, 35); these differences may reflect slight variability between rat versus bovine isoforms.
Using pNPP as substrate, PP(H76N) also exhibited little difference
in substrate Km and a decrease in
kcat of 590-fold relative to wild type
PP.
This difference is more than 100-fold lower than the 105
noted previously (25) primarily because of a 30-fold higher activity
measured for the
PP(H76N) mutant in this study but also because of a
slightly lower activity of the wild type enzyme used in this study (
5-fold; see Table II). In fact, variability in kcat has been documented for wild type
PP (5,
25, 36); we have also observed about a 2-3-fold difference in activity for several different preparations of wild type
PP prepared by similar procedures. Kinetic parameters of wild type
PP and
PP(H76N) for a second substrate, phenyl phosphate, paralleled the
results using pNPP. Thus
PP(H76N) exhibited a decrease in
kcat of 530-fold and a Km
similar to wild type enzyme.
Based on primary sequence homologies, PP is thought to be a member
of the family of metallophosphatases including calcineurin, PP1, and
PP2A (6). We now show that
PP can accommodate a binuclear metal
center as predicted for enzymes containing the phosphoesterase motif.
Therefore, it is likely that His-76 in
PP and His-151 in calcineurin
have similar functions in phosphate ester hydrolysis. Proposed roles
for this conserved histidine include an active site nucleophile, a role
in orienting substrate, a role in general acid catalysis involving
protonation of the leaving group, or a role in general base catalysis
by deprotonation of an iron-coordinated solvent molecule. All of these
would be consistent with the 102-103-fold
decrease in activity observed for wild type versus mutant enzymes.
The fold decreases for His-151/His-76 are comparable to the decrease of
103 found for serine-to-alanine and serine-to-leucine
mutations for the nucleophilic serine residue of alkaline phosphatase
(37). However, experiments with purple acid phosphatase, a member of the metallophosphatase superfamily with a strikingly similar active site as calcineurin (8) demonstrated that hydrolysis proceeded by
direct transfer of the substrate phosphoryl group to solvent (38). By
analogy, therefore, it seems unlikely that this histidine participates
in nucleophilic catalysis. Similar substrate Km values for mutant and wild type enzymes also argue against a necessary role in substrate binding.
In the crystal structure of calcineurin with phosphate bound at the
active site, His-151 is within H-bonding distance of the most
solvent-exposed oxygen atom of phosphate, close enough to assist in
leaving group protonation (11). Similar orientations are observed in
the crystal structure of human PP1 with bound tungstate ion (10),
rabbit PP1
with phosphate modeled in the active site
(9), and in the purple acid phosphatase structures with phosphate and
tungstate bound at the active site (16). If His-151/His-76 were
involved in proton donation to the leaving group, the
kcat for each mutant enzyme relative to wild
type should show a marked dependence using substrates that have leaving
groups of disparate pKa values. In fact, only a
~3-fold difference in relative kcat (wild type
versus CN(H151Q)) for pNPP versus [P]-RII peptide is observed, even though the products of
the reaction have acidities that differ by >106
(p-nitrophenol has a pKa of 7.2 compared
with
14 for serine). With
PP, there is no difference between
relative kcat values of wild type
versus H76N using pNPP and phenyl phosphate (pKa = 9.95). It seems likely therefore that this
histidine is not required for protonation of the leaving group.
Although the kcat values for CN(H151Q) and
PP(H76N) represent significant decreases compared with wild type
enzyme, they are 105-107-fold greater than the
noncatalyzed rate of
hydrolysis.6 A significant
amount of this remaining catalytic activity is most likely derived from
the presence of the active site dinuclear metal center, which has been
proposed to lower the pKa of a coordinated water
molecule, the putative nucleophile in the reaction. His-151/His-76
could be functioning in concert with this solvent molecule to either
position a lone pair on the oxygen atom for optimum in-line attack on
the phosphorus atom of the substrate or to serve as a general base to
take up a proton concomitant with solvent nucleophilic attack. At least
in one crystal structure model of calcineurin, the N
atom of His-151
was H-bonded to one of two solvent molecules coordinated to the iron
atom (13). In the crystal structure of PP1 with microcystin bound, the
N
atom of the analogous histidine, His-125, was also within
H-bonding distance of a water molecule, but that water was modeled
3.2 Å away from the metal ions (9). Further evidence for this model is provided by mutagenesis studies of PP1 examining the influence of a
conserved aspartic acid residue, Asp-95, on catalytic efficiency. This
conserved aspartate residue is part of the phosphoesterase consensus
motif (6, 7). PP1 residue Asp-95 is within H-bonding distance of the
conserved histidine, and mutagenesis to asparagine resulted in a
71-fold decrease in activity compared with wild type using
phosphorylase a as substrate (32). The analogous mutant in
PP, D52N, resulted in a 36-fold reduction in activity using pNPP as
substrate (25). The corresponding residue in calcineurin, Asp-121, is
also within H-bonding distance of His-151 (Fig. 1). Thus, the
interaction of this conserved histidine/aspartate pair with a solvent
molecule is analogous to the catalytic aspartate/histidine/serine motif
of serine proteases and could be thought of as a "catalytic tetrad"
with the metal ion serving as a Lewis acid to lower the pKa of the nucleophile.
If His-151 participates in a hydrogen bond with a metal-coordinated
solvent, mutagenesis will disrupt this interaction and is likely to
affect the spectroscopic properties of the dinuclear metal center. We
have reconstituted calcineurin and PP with iron to generate an
active site dinuclear iron center for use as a spectroscopic probe of
the active site. The Fe3+-Fe2+ oxidation state
of this cluster gives rise to a signature EPR resonance with
gav < 2.0 (14, 15) which is sensitive to
changes in the metal environment via perturbation of zero-field
splitting (Di, Eii) and spin
coupling (J) constants in Equation 1 (39). The EPR spectrum
of iron-reconstituted CN(H151Q) exhibited g values
consistent with the formation of a Fe3+-Fe2+
center, indicating that the H151Q mutant enzyme is still able to
support a dinuclear metal center. However, the overall shape of this
spectrum was quite different from that of wild type calcineurin. In
comparison, the EPR spectrum of the iron-reconstituted
PP(H76N) also
exhibited the characteristic gav < 2.0 signal
with a shape distinct from the corresponding spectrum of wild type
PP.
The fact that phosphate addition to iron-reconstituted CN(H151Q) caused a change in the shape of the EPR resonance indicates that it arises from an active site metal center. Interestingly, in both wild type calcineurin and purple acid phosphatase, phosphate binding to the mixed valence cluster led to a broadening of the corresponding EPR signal, a result of a decrease in the spin coupling constant, J, caused by the phosphate ion bridging the two metal ions of the cluster (39).2 With CN(H151Q), on the other hand, phosphate caused a sharpening of the EPR resonance. Further spectroscopic analysis is required to understand the structural basis for these differences.
These results demonstrate that the conserved histidine in the
metallophosphatases calcineurin and PP is an essential component of
the active site since disruption led to significant decreases in
activity. Loss of activity may have resulted from removal of an active
site base and the disruption of an essential H bond to a
metal-coordinated solvent molecule. Future experiments to confirm this
are in progress.
We acknowledge gratefully Jack Dixon's
laboratory for providing the plasmids containing the clones for PP
wild type and
PP(H76N). We also thank Jim Griffith and colleagues at
Vertex for providing the coordinates of calcineurin, John Kuriyan and
co-workers for PP1 coordinates, and Melissa Snyder and Sergei
Venyaminov for help with circular dichroism experiments.