(Received for publication, February 1, 1995; and in revised form, September 28, 1995)
From the
The amino acid sequences of all known cGMP-binding
phosphodiesterases (PDEs) contain internally homologous repeats (a and
b) that are 80-90 residues in length and are arranged in tandem
within the putative cGMP-binding domains. In the bovine lung
cGMP-binding, cGMP-specific PDE (cGB-PDE or PDE5A), these repeats span
residues 228-311 (a) and 410-500 (b). An aspartic acid
(residue 289 or 478) that is invariant in repeats a and b of all known
cGMP-binding PDEs was changed to alanine by site-directed mutagenesis
of cGB-PDE, and wild type (WT) and mutant cGB-PDEs were expressed in
COS-7 cells. Purified bovine lung cGB-PDE (native) and WT cGB-PDE
displayed identical cGMP-binding kinetics, with 1.8 µM cGMP required for half-maximal saturation. The D289A mutant showed
decreased affinity for cGMP (K
> 10
µM) and the D478A mutant showed increased affinity for
cGMP (K
0.5 µM) as
compared to WT and native cGB-PDE. WT and native cGB-PDE displayed an
identical curvilinear profile of cGMP dissociation which was consistent
with the presence of distinct slowly dissociating (k
= 0.26 h
) and rapidly dissociating (k
= 1.00 h
) sites of
cGMP binding. In contrast, the D289A mutant displayed a single k
= 1.24 h
, which was
similar to the calculated k
for the fast site of
WT and native cGB-PDE, and the D478A mutant displayed a single k
= 0.29 h
, which was
similar to that calculated for the slow site of WT and native cGB-PDE.
These results were consistent with the loss of a slow cGMP-binding site
in repeat a of the D289A mutant cGB-PDE, and the loss of a fast site in
repeat b of the D478A mutant, suggesting that cGB-PDE possesses two
distinct cGMP-binding sites located at repeats a and b, with the
invariant aspartic acid being crucial for interaction with cGMP at each
site.
Cyclic nucleotide phosphodiesterases (PDEs) ()constitute a complex family of enzymes which catalyze the
hydrolysis of 3`:5`-cyclic nucleotides to the corresponding nucleoside
5`-monophosphates. The multiple PDEs differ in their substrate
specificities, sensitivities to inhibitors, modes of regulation, and
tissue distributions. Most PDEs are chimeric multidomain proteins,
possessing distinct catalytic and regulatory domains(1) . A
250-amino acid segment of sequence, which is conserved among all
mammalian PDEs and is located in the more carboxyl-terminal portions of
the PDE molecules, contains the catalytic site of these
enzymes(1, 2, 3, 4) . Domains of the
PDEs which interact with allosteric/regulatory factors are thought to
be located within the more amino-terminal
regions(1, 5, 6) .
The cGMP-binding PDEs
comprise a heterogeneous subgroup of PDEs, all of which exhibit
allosteric cGMP-binding sites that are distinct from the sites of
cyclic nucleotide hydrolysis. This group consists of at least three
classes of PDEs: the cGMP-stimulated PDEs (cGS-PDEs, or PDE2s)()(7) , the photoreceptor PDEs (rod
outer segment PDE (ROS-PDE; PDE6A/B) (8) and cone PDE (PDE6C)(9) ), and the cGMP-binding, cGMP-specific PDE
(cGB-PDE; PDE5A)(10) . The stimulatory effect of cGMP
binding to the allosteric sites of cGS-PDE upon cyclic nucleotide
hydrolysis at the catalytic site is well documented(7) , but
the functional role of the allosteric sites in the photoreceptor PDEs
or cGB-PDE is not well understood. However, there is evidence that cGMP
binding at allosteric sites of frog ROS-PDE regulates the interaction
of its catalytic subunits with inhibitory subunits and with the
G-protein, transducin(11) , and that binding of cGMP to the
allosteric sites of bovine lung cGB-PDE allows for the phosphorylation
of the enzyme by cGMP-dependent or cAMP-dependent protein
kinase(12) .
cDNAs encoding each of these cGMP-binding PDEs have now been isolated(13, 14, 15, 16, 17) , and, as predicted(5) , the deduced amino acid sequence of each of these enzymes contains a conserved segment of approximately 340 residues located amino-terminal to the catalytic site. This conserved segment is not present in any PDEs other than the cGMP-binding PDEs, and is proposed to constitute an allosteric cGMP-binding region(5) . Limited proteolysis and photoaffinity-labeling studies of cGB-PDE and cGS-PDE have provided direct evidence to support this proposal(10, 13, 18) . Within their conserved cGMP-binding regions, all known cGMP-binding PDEs contain two internally homologous sequence repeats of 90 amino acids each (a and b), that are arranged in tandem, and share 15-45% sequence identity with the a and b repeats in other cGMP-binding PDEs(13, 14, 15, 16, 17) . cAMP-dependent protein kinase and cGMP-dependent protein kinase, which do not share significant sequence homology with the cGMP-binding PDEs, also contain a tandem repeat pattern of sequence. In these kinases, the repeated sequences, approximately 120 amino acid residues in length, are known to comprise two distinct cyclic nucleotide-binding sites (19) . By analogy, the repeats a and b present in the cGMP-binding region of the cGMP-binding PDEs may also represent two distinct cGMP-binding sites. In support of this proposal, kinetic analyses of cGMP binding to cGS-PDE(18) , ROS-PDE(20, 21) , and cGB-PDE (10) suggest the presence of two classes of allosteric cGMP-binding sites in these enzymes.
If the homologous sequence repeats in the cGMP-binding PDEs represent two distinct cGMP-binding sites, amino acids essential for interaction with cGMP should be conserved among all repeats. Seven amino acid residues are invariant in all repeats in all known cGMP-binding PDEs, and three of these seven residues possess charged side groups (Lys, Asp, Glu) which could be important for interaction with cGMP(13) . In this report, the invariant Asp in repeats a or b of cGB-PDE has been modified by site-directed mutagenesis (see Fig. 1). Analysis of the effect of these mutations on cGMP-binding activity has been utilized to determine if the repeats a and b represent two distinct cGMP-binding sites, and if the conserved Asp is essential for binding at both sites.
Figure 1: A, point mutations within the proposed domain organization of a cGB-PDE monomer. The cGMP-binding region (residues 142-526) is shown as a box with diagonal lines. Internally homologous repeats a(228-311) and b(410-500), within the cGMP-binding region, are shown as black boxes. Single point mutations introduced into repeats a or b are indicated above the diagram. The catalytic region(578-812) is shown as a light gray box, and extends through the putative cyclic nucleotide-binding component of the the catalytic region c(760-812), which is shown as a dark gray box. B, alignment of internally homologous repeats a and b and the putative cyclic nucleotide-binding component of the catalytic region (c) of cGB-PDE. Residues which are identical in each repeat a and b from all known cGMP-binding PDEs are enclosed in boxes. Arrows represent positions in which all residues are chemically conserved among each a and b repeat from all known cGMP-binding PDEs. Residues identical in repeats a and b in cGB-PDE are shown in boldface. The conserved Asp which has been mutated in repeat a of the D289A mutant and in repeat b of the D478A mutant is enclosed in a black box. Residues that are conserved in each region c (putative cyclic nucleotide-binding component of the catalytic region) of all known mammalian PDEs are indicated by stars. Vertical lines illustrate conserved residues among segments a, b, and c. Residues comprising each segment are described in the legend to Panel A.
Oligonucleotide-directed
mutagenesis was performed using standard techniques(22) .
Specifically, a HindIII fragment of cGB-8, spanning bp
712-1546 (encoding the entire cGMP-binding region), was ligated into
the HindIII cloning site of the pBluescript SK(-)
subcloning vector, producing the phagemid construct
cGB-8-H3f-pBSSK(-). Uracil-containing single-stranded
cGB-8-H3f-pBSSK(-) DNA was prepared for use as a template by
transformation of Escherichia coli CJ236 (dut, ung
) with
the phagemid and subsequent infection with M13K07 helper virus. The
following mutagenic oligonucleotides were synthesized on a Cyclone Plus
DNA synthesizer (Millipore): 1) cGBD289A: 5`-GGG ACA TTC ACT
GAA AAA GCC GAA AAG GAC TTT GCT GCT-3` (encoded amino acid sequence:
GTFTEKAEKDFAA), and 2) cGBD478A: 5`-CT TTC AAC CGC AAC GCT GAA
CAG TTT CTG GA-3` (amino acid sequence: FNRNAEQFL). The altered bases
and amino acid residues are underlined. Mutagenic oligonucleotides (100
pmol) were phosphorylated with T4 polynucleotide kinase(22) ,
and 5 pmol of phosphorylated oligonucleotide were annealed to
0.5-1 pmol of uracil-containing single-stranded DNA in 50 mM Tris (pH 7.5), 20 mM MgCl
, and 50 mM NaCl (final volume, 10 µl) by incubating the mixture in a
500-ml beaker filled with 65 °C H
O, and allowing the
temperature to cool to 22 °C over 30-60 min. After cooling, 2
µl of 10
mutagenesis buffer (100 mM Tris (pH 7.5),
50 mM MgCl
, 10 mM ATP, 5 mM dATP, 5 mM dCTP, 5 mM dGTP, 5 mM dTTP,
and 20 mM dithiothreitol), 3 units of T4 DNA polymerase and
200 units of T4 DNA ligase were added to a final volume of 20 µl.
The reactions were incubated for 5 min at 4 °C, followed by
incubations for 5 min at 22 °C and 90 min at 37 °C. One µl
of the resulting mutagenesis reaction products was used to transform E. coli XL1-blue (dut
, ung
). Transformants were screened for the
presence of the appropriate mutation by sequencing miniprepped DNA (23) using Sequenase® Version 2.0 according to the
manufacturer's protocol (U. S. Biochemical Corp.).
In order to create plasmid constructs for expression of mutant cGB-PDEs, the WT HindIII fragment of cGB-8 (bp 712-1546) was removed from the pCDNA-cGB-PDE(WT) plasmid, and replaced with HindIII fragments of cGB-8 containing the desired mutations, creating the mutant expression constructs pCDNA-cGB-PDE(D289A) and pCDNA-cGB-PDE(D478A). E. coli XL1-blue cells were used for all transformations, and DNA was purified from large scale plasmid preparations using QIAGEN Plasmid Mega kits according to the manufacturer's protocol (QIAGEN Inc.). All DNA segments subject to mutagenesis reactions (bp 640-1640 of the cGB-8 sequence in pCDNA- cGB-PDE(D289A) and bp 625-1625 in pCDNA-cGB-PDE(D478A) were sequenced (23) to ensure the presence of the desired mutation, the absence of any spurious mutations, and proper in-frame subcloning at the HindIII sites.
Figure 3:
Effect of [P]cGMP
concentration on binding to native, WT, and mutant cGB-PDEs. Soluble
extracts of COS-7 cells transfected with cDNAs encoding WT or D289A or
D478A mutant cGB-PDE were prepared and cGMP-binding assays were
performed using increasing concentrations of
[
P]cGMP as indicated. Purified bovine lung
(native) cGB-PDE was added to soluble extract from mock transfected
COS-7 cells prior to initiation of the assay. (See ``Experimental
Procedures'' for details.) For Panels A and B,
results are given as percent of maximum ([
P]cGMP
binding/mg of total protein). All results shown are the averages of at
least three separate determinations. Error bars represent
standard deviations. All assays were performed in duplicate. Panel
A, [
P]cGMP binding to extract containing WT
cGB-PDE (
) and native cGB-PDE (
) was compared. Maximum
binding was achieved at 8 µM [
P]cGMP. Panel B,
[
P]cGMP binding to extract containing WT
(
), D289A mutant (
), or D478A mutant (
) cGB-PDE was
compared. Maximum binding was achieved at 8 µM [
P]cGMP for WT cGB-PDE and 4
µM [
P]cGMP for D478A mutant
cGB-PDE. Since the concentration of [
P]cGMP
required for maximum binding to the D289A mutant cGB-PDE could not be
determined (see ``Results''), the
[
P]cGMP binding observed at 24 µM was arbitrarily designated as maximum binding. Panel C,
Scatchard analysis of [
P]cGMP binding to WT
(
) and D478A mutant (
) cGB-PDE. Units for
``bound'' and ``free'' are picomoles of
[
P]cGMP/mg of total protein and µM [
P]cGMP, respectively. Data shown are
representative of at least three separate
experiments.
Rates of cGMP dissociation from
theoretical slow and fast sites on native and WT cGB-PDE were
calculated according to the 2-site model of Doskeland and Corbin, which
was utilized in characterizing the two cyclic nucleotide-binding sites
in cAMP-dependent protein kinase (24) and cGMP-dependent
protein kinase(25) . Linear regression of the data points from
3 to 7 h was used to estimate the slope of the slow component of the
dissociation curves, which represented the rate of cGMP dissociation (k) from the theoretical slow (higher affinity)
cGMP-binding site. The line predicted by this analysis (y = -0.256, x - 0.733) had a y intercept of -0.733, which was consistent with the slow
component representing the dissociation of
48% of the total
[
P]cGMP bound.
The rate of
[P]cGMP dissociation from the theoretical fast
(lower affinity) cGMP-binding site was calculated as follows. First,
the contribution of cGMP binding to the slow site during time points
0-3 h was estimated. The natural log of the fraction of
[
P]cGMP remaining bound to the slow site at each
time point (t),
(ln(B
/B
)
), was calculated by inserting each early time point (x) into the equation of the line representing dissociation
from the slow site (see above). The antilog of
ln(B
/B
)
represented the fraction of
[
P]cGMP remaining bound to the slow site at each
time point. Second, the contribution of cGMP binding to the slow site, (B
/B
)
, was subtracted from total measured (B
/B
) at each time point from
0-3 h in order to calculate the fraction of
[
P]cGMP remaining bound to the fast site:
((B
/B
)
- (B
/B
)
= (B
/B
)
). Third, the natural log of (B
/B
)
for each time point was calculated, and linear
regression of the corrected fast site data was performed in order to
estimate the slope of the fast component of the cGMP-dissociation
curves. This slope represented the rate of cGMP dissociation (k
) from the theoretical fast site. The line
predicted by this analysis (y = -1.00x - 0.665) had a y intercept of -0.665, which
was consistent with the fast component representing the dissociation of
50% of the total [
P]cGMP bound.
[P]cGMP (1500-3000 Ci/mmol) was
purchased from ICN. [
H]cGMP (19.2 Ci/mmol) was
purchased from Amersham Corp. pCDNA1/Amp expression vector, E. coli CJ236, and helper phage M13K07 were purchased from Invitrogen.
Horseradish peroxidase-protein A was purchased from Zymed.
3-Isobutyl-1-methylxanthine, dimethyl sulfoxide, benzamidine, pepstatin
A, leupeptin, Crotalus atrox snake venom, cAMP, cGMP,
8-bromo-cGMP, and protein A-Sepharose CL 4B were purchased from Sigma.
Klenow, T4 DNA polymerase, and T4 DNA ligase were purchased from
Promega. E. coli XL1-blue and pBluescript SK(-) vector
were purchased from Stratagene Cloning Systems. T4 polynucleotide
kinase, 1-kilobase pair DNA ladder, Dulbecco's modified
Eagle's medium, and fetal bovine serum were purchased from Life
Technologies, Inc. Bradford reagent, Coomassie Brilliant Blue stain,
and protein molecular weight markers were purchased from Bio-Rad.
DEAE-dextran was purchased from Pharmacia Biotech Inc.,
Sequenase
Version 2.0 from U. S. Biochemical Corp.,
Nitropure nitrocellulose membrane from Micron Separations Inc., Nu
Serum from Collaborative Biomedical Products, DNA purification columns
from QIAGEN Inc., Ready Safe aqueous scintillation mixture from Beckman
Industries Inc., and Ecoscint-O nonaqueous scintillation mixture from
National Diagnostics. All restriction enzymes were purchased from
Promega and Life Technologies, Inc. COS-7 cells were obtained from the
American Type Culture Collection.
Figure 2:
Expression of WT and mutant cGB-PDEs in
COS-7 cells. COS-7 cells were subjected to mock transfection or were
transfected with cDNAs encoding WT or D289A or D478A mutant cGB-PDEs.
After 48 h, the cells were harvested, and the soluble extracts were
assayed for phosphodiesterase activity using 20 µM [H]cGMP as the substrate. (See
``Experimental Procedures '' for details.) Picomoles of cGMP
hydrolyzed/(min)(mg total protein) was calculated for each extract, and
results are given as the percentage of phosphodiesterase activity
measured in extracts of cells transfected with cDNA encoding WT cGB-PDE
during the same transfection. The results shown are the averages of
five separate transfections. All assays were performed in duplicate. Error bars represent S.D.
D478A mutant cGB-PDE displayed slightly higher affinity for cGMP as
compared to WT and native enzyme (K
0.5
µM) (Fig. 3B). A Scatchard plot of the
data was consistent with the presence of a single class of cGMP-binding
site (Fig. 3C). The D289A mutant cGB-PDE displayed
significantly decreased affinity for cGMP (K
>
10 µM) (Fig. 3B). Binding to the D289A
mutant cGB-PDE was not saturated at up to 24 µM
[
P]cGMP, and measurements of
[
P]cGMP binding at concentrations greater than
24 µM were not reliable due to high nonspecific background
binding and low specific radioactivity of cGMP. Therefore, the range of
[
P]cGMP concentrations at which binding to the
D289A mutant cGB-PDE could be measured was insufficient for K
determinations and/or Scatchard analyses.
Figure 4:
Dissociation of
[P]cGMP from native, WT, and mutant cGB-PDEs.
Soluble extracts of COS-7 cells transfected with cDNAs encoding WT or
D478A mutant cGB-PDEs or extracts of mock-transfected cells containing
native cGB-PDE were incubated under saturating conditions for
[
P]cGMP binding. Since the concentration of
[
P]cGMP required to saturate the cGMP-binding
sites in extracts of cells containing the D289A mutant cGB-PDE could
not be determined, these extracts were incubated with subsaturating
concentrations of [
P]cGMP (8 µM)
(see ``Results'' for the explanation). Binding reactions were
equilibrated to 0 °C and 100-fold excess unlabeled cGMP was added
to initiate exchange. Aliquots were removed at the indicated time
points in order to measure [
P]cGMP binding
(described under ``Experimental Procedures''). Data are
plotted as
ln(B
/B
) versus time where B
/B
represents the ratio of [
P]cGMP
binding at any time t to the
P binding measured
at time = 0 (before addition of unlabeled cGMP). Each data point
represents the natural log of the average of at least three separate
determinations of B
/B
.
Parabolic regression of the data was performed to generate the binding
curves. Panel A, comparison of [
P]cGMP
dissociation from extracts containing WT (
) and native (
)
cGB-PDE. The range of
ln(B
/B
) was
<0.25 for all time points, with the exception of time points 5 h
(native) and 2 and 6 h (WT) for which the range was 0.5. Calculation of
the rates of the slow and fast components of cGMP dissociation is
explained in the text under ``Experimental Procedures.'' In
order to facilitate visual comparison of the rates of cGMP dissociation
from theoretical fast and slow sites of WT/native cGB-PDE and mutant
cGB-PDEs (Panel B), the plots of cGMP dissociation from the
theoretical sites have been corrected such that B
/B
at time
= 0.0 min is equal to 1.0, and
ln(B
/B
)
= 0.0. The true values for
ln(B
/B
) at
time = 0.0 min for the slow and fast component are -0.733
and -0.655, respectively (explained under ``Experimental
Procedures''). Thus, at time = 0.0,
48% of the total
bound [
P]cGMP is bound to the theoretical slow
site, and
50% is bound to the theoretical fast site. Panel
B, comparison of [
P]cGMP dissociation from
extracts containing WT (
), D289A mutant (
), and D478A
(
) mutant cGB-PDEs. For D478A (
), the range of
ln(B
/B
) was
<0.20 for all time points, with the exception of time points 4, 5.5,
and 7 h for which the ranges were 0.6, 0.4, and 0.5, respectively. For
the D289A (
) the range of
ln(B
/B
) was
<0.4 for all time points.
[P]cGMP
dissociated from the D478A mutant cGB-PDE at a single rate of k
= 0.29 h
(Fig. 4B), which was similar to the calculated
rate of dissociation of cGMP from the theoretical slow site of WT and
native cGB-PDEs (Table 1), whereas [
P]cGMP
dissociated from the D289A mutant cGB-PDE at a single rate of k
= 1.24 h
, which was
similar to the calculated rate of cGMP dissociation from the
theoretical fast site of WT and native cGB-PDE (Table 1). Similar
results were obtained when [
P]cGMP dissociation
from WT and mutant cGB-PDEs was measured at 22 °C (not shown); a
curvilinear pattern of cGMP dissociation from WT was observed, whereas
dissociation from the D478A and D289A mutant cGB-PDEs occurred at
single rates which were similar to the calculated rates of cGMP
dissociation from the theoretical slow and fast sites, respectively, of
WT cGB-PDE.
Figure 5: Immunoblot analysis of native, WT, and mutant cGB-PDEs. Samples (625 µg of total protein) of soluble extract from COS-7 cells were analyzed by immunoprecipitation and Western blot detection as described under ``Experimental Procedures.'' COS-7 cells were transfected with cDNA encoding WT cGB-PDE (lane 3), D289A mutant cGB-PDE (lane 4), D478A mutant cGB-PDE (lane 5), or no DNA (mock transfection) (lane 6). Lanes 2 and 7 show approximately 700 ng of native cGB-PDE diluted into soluble extract of mock-transfected COS-7 cells (625 µg of total protein) and 250 µl of PBS buffer, respectively. Lane 1 shows extract from COS-7 cells transfected with WT cGB-PDE which was subjected to immunoprecipitation with preimmune sera in the place of anti-cGB-PDE antisera as a control. All extracts used in this experiment were from a single transfection experiment. This experiment has been repeated with four different sets of extracts (from four separate transfections), and the same results were obtained with each experiment.
The lower molecular mass species of the 99/90-kDa doublet may have been generated by proteolysis of full-length cGB-PDE. However, increasing the number and concentration of protease inhibitors in the buffer in which the COS-7 cells were homogenized did not affect the appearance of the 90-kDa band, nor did inclusion of 0.01% SDS in the homogenization buffer (not shown). It is also possible that the 90-kDa species was produced as a result of initiation of translation of the cGB-PDE mRNA at an alternative AUG start site which is 126 bp downstream from the AUG identified as the start site of translation of the 99-kDa cGB-PDE (nucleotide position 99-101)(13) .
It is unlikely that the presence of the
90-kDa species affected the cGMP-binding properties of WT and mutant
cGB-PDEs. The loss of 10 kDa on either the amino-terminal or
carboxyl-terminal end of cGB-PDE would leave the cGMP-binding region
intact (see Fig. 1). Previous studies showed that the
curvilinear pattern of [H]cGMP dissociation from
a 30-45-kDa fragment of cGB-PDE containing the cGMP-binding
region was identical to the dissociation pattern observed with intact
cGB-PDE(28) , suggesting that the cGMP-dissociation pattern was
an intrinsic property of the cGMP-binding region, and that it was not
altered by the loss of outlying segments of cGB-PDE. The data presented
in this report showed that WT and native cGB-PDEs displayed identical
cGMP-saturation and cGMP-dissociation kinetics ( Fig. 3and 4)
despite the fact that the native cGB-PDE appeared as a single band on
Western blot (Fig. 5). As a control, native cGB-PDE, diluted
into soluble extract of mock-transfected COS-7 cells, was incubated
under the exact conditions used in the cGMP-saturation or
cGMP-dissociation assay, followed by immunoprecipitation and Western
blot detection. The native cGB-PDE still appeared as a single band in
both cases (data not shown), removing the possibility that native
cGB-PDE was degraded into a 99/90-kDa doublet during the course of the
cGMP-binding assay. Therefore, the presence of two species (99/90 kDa)
in the WT cGB-PDE was not responsible for producing cGMP-saturation and
dissociation patterns consistent with the presence of two distinct
cGMP-binding sites, as the identical patterns were obtained when
measuring cGMP binding to native cGB-PDE, which remained as a single
species throughout these assays.
Two internally homologous repeats of sequence (a and b) are conserved within the cGMP-binding region of all cGMP-binding PDEs, and have been proposed to form two distinct cGMP-binding sites in these PDEs(13) . The studies described in this report were designed to test this hypothesis using site-directed mutagenesis of bovine lung cGB-PDE, and the results of the cGMP-saturation and cGMP-dissociation analyses of native, WT, and mutant cGB-PDEs strongly support this hypothesis. WT and native cGB-PDE displayed a higher cGMP-binding affinity than that of the D289A mutant cGB-PDE and a lower cGMP-binding affinity than that of the D478A mutant. Scatchard analysis of cGMP binding to native and WT cGB-PDE produced an upward concave plot which is consistent with the presence of two classes of cGMP-binding sites, whereas Scatchard analysis of the D478A mutant cGB-PDE produced a linear plot, which suggests the presence of a single class of cGMP-binding site. The observed curvilinearity of cGMP dissociation from native and WT cGB-PDE is consistent with the presence of two distinct cGMP-binding sites from which cGMP dissociates at two distinct rates, and the single rates of cGMP dissociation from the D289A and D478A mutant cGB-PDEs were very similar to those calculated for the theoretical fast and slow sites, respectively, of WT and native cGB-PDE (Table 1).
These findings provide compelling evidence for a model of cGB-PDE containing a higher affinity (slow) cGMP-binding site at repeat a and a lower affinity (fast) site at repeat b, with the Asp residue, which is invariant among all repeats a and b of all cGMP-binding PDEs, being crucial to the interaction of cGMP with each site. According to this model, replacement of the Asp residue in site a with Ala (as in the D289A mutant cGB-PDE) diminishes or ablates binding to this site, thereby producing a cGB-PDE which binds to cGMP primarily at lower affinity site b. Therefore, this mutant displays lower cGMP-binding affinity than does WT or native cGB-PDE, and dissociation of cGMP from this mutant occurs at a single rate, which is similar to the predicted rate of dissociation from the fast site of WT and native cGB-PDE. Conversely, the model predicts that the D478A mutant cGB-PDE binds to cGMP primarily at higher affinity site a. Therefore, this mutant displays higher affinity for cGMP binding than does WT or native cGB-PDE, and dissociation of cGMP from this mutant occurs at a single rate which is similar to the predicted rate of dissociation from the slow site of WT and native cGB-PDE. It seems unlikely that the cGMP saturation binding and dissociation kinetics of each of the mutant cGB-PDEs would coincidentally resemble the predicted saturation binding and dissociation kinetics of the respective theoretical sites in WT and native cGB-PDE.
Although the current results provide strong evidence for the existence of two distinct cGMP-binding sites at repeats a and b on cGB-PDE, it is not yet known if these binding sites are formed by the interaction of two identical repeats residing on separate subunits of the homodimer, resulting in a stoichiometry of 2 mol of cGMP per mol of homodimer, or if each repeat on each individual subunit forms a separate binding site, resulting in a stoichiometry of 4 mol of cGMP per mol of homodimer. The stoichiometry of cGMP binding to purified bovine lung cGB-PDE has been reported to be 1.9 mol of cGMP per mol of homodimer(10) , and the reported stoichiometry of cGMP binding to any of the cGMP-binding PDEs has not exceeded 2 mol of cGMP per mol of holoenzyme(9, 18, 21) . Limited proteolysis studies have shown that the dimerization domain of cGB-PDE (10) and cGS-PDE (4) is within or near the cGMP-binding region of the enzyme. Such an arrangement would allow for the formation of cGMP-binding sites through the interaction of two identical repeats residing on separate subunits.
The invariant Asp present in repeats
a and b is the first amino acid residue to be identified as a critical
component of the cGMP-binding sites of the cGMP-binding PDEs. A general
conformational deterioration due to mutation of the Asp seems unlikely,
since cGMP binding to the unmodified site is not affected by mutation
of either Asp. The structural features of the binding sites in two
other families of guanine nucleotide-binding proteins, the E. coli catabolite activator protein (CAP) family (CAP, cAMP-dependent
protein kinase, cGMP-dependent protein kinase, and the cyclic
nucleotide-gated channels), and the GTP-binding protein family, have
now been well characterized, and may serve as useful models in the
analysis of the role of individual amino acid residues of the
cGMP-binding sites of the cGMP-binding PDEs. In the cyclic
nucleotide-binding domains of all of the members of the CAP family, an
invariant Glu forms a hydrogen bond with the 2`OH of the
nucleotide(19) . Although the cGMP-binding region of the
cGMP-binding PDEs shows no sequence homology to the CAP family, it is
possible that this invariant Asp may play a similar role to that known
for the Glu. In support of this hypothesis, cyclic nucleotide analog
studies of cGB-PDE suggest the existence of a hydrogen bond between the
allosteric cGMP-binding sites and the 1-, C-ketone-, and
2`OH-positions of the cGMP molecule(29) . Another possible role
of the Asp could be similar to that played by an invariant Asp in
GTP-binding proteins(30) , which forms hydrogen bonds with both
the 1-NH and the 2-NH
groups of guanosine in GTP. It is of
interest that the conserved sequence of the guanosine-binding component
of G proteins is Asn-Lys-X-Asp, which resembles the
Asn-Lys-(X)
-Asp of the proposed cGMP-binding sites
of cGMP-binding PDEs (Fig. 1), suggesting that the cGMP-binding
domain of the cGMP-binding PDEs and the GTP-binding domain of G
proteins may interact with the guanosine moiety through a common
mechanism. If so, then the conserved Asn of cGB-PDE could form a
hydrogen bond with the 7-N of guanine as is the case for G
proteins(30) .
Since the conserved catalytic domain of PDEs
and the conserved cGMP-binding region of the cGMP-binding PDEs share a
common function, i.e. the ability to bind cyclic nucleotides,
the catalytic and allosteric binding sites may have evolved by
duplication of an ancestral cyclic nucleotide-binding
domain(31) . In support of this proposal, a segment of sequence
located in the carboxyl-terminal portion of the conserved catalytic
domain of PDEs (labeled c in Fig. 1B) shares
limited homology with repeats a and b of all cGMP-binding PDEs. All
published amino acid sequences of mammalian PDEs (as of the listing
compiled in April 1994 for the American Society of Pharmacology and
Experimental Therapeutics, Colloquium and Symposium on Multiple
PDEs(32) ) share the common sequence
K/R(X)F(X)
D(X)E
within segment c. (
)Alignment of region c with repeats a and
b of the cGMP-binding PDEs reveals that the spatial arrangement of
these conserved residues within region c is similar, but not identical,
to that of the corresponding residues in repeats a and b
(K(X)
F(X)
DE) (Fig. 1B). Segment c may represent a cyclic
nucleotide-binding component of the catalytic domain. If so, it could
act in concert with another segment(s), such as the zinc-binding domain (33) , to elicit catalysis.
The functional role of cGMP
binding to sites a and b of cGB-PDE is still unclear. Comparison of the
cGMP-phosphodiesterase activities of the WT and mutant cGB-PDEs
suggests that the loss of cGMP binding at site a or b does not
profoundly affect the K of catalysis. However, the
phosphodiesterase activity measured at 20 µM cGMP was
consistently lower in extracts of cells transfected with the mutant
cGB-PDEs as compared to WT cGB-PDE (Fig. 2). A method for more
precisely quantitating the concentration of cGB-PDE in COS-7 cell
extracts is needed to determine if this small difference in the
cGMP-phosphodiesterase activity is due to a decreased catalytic V
of the mutant cGB-PDEs as compared to WT, or
to a consistently lower level of expression of mutant cGB-PDEs as
compared to WT cGB-PDE.
The studies described in this report provide the first biochemical evidence for the existence of two allosteric cGMP-binding sites located at repeats a and b of a cGMP-binding PDE, and the first evidence for a role of an individual amino acid residue in interacting with the cGMP molecule at the allosteric sites in these enzymes. These findings represent an important first step in characterizing the structural elements that contribute to the function of the allosteric cGMP-binding sites in cGB-PDE and other cGMP-binding PDEs.