From the
The interaction between the GTP-bound form of the transducin
In the phototransduction cascade of enzymatic reactions, the
Despite intensive investigation, the molecular mechanism of
the regulation of PDE activity involving interactions between
G
In this study we have combined synthetic peptide and
mutagenesis approaches to map the functional sites of P
The Carboxyl-terminal PDE Inhibitory Site of P
The
Biochemical, peptide, and mutagenesis
studies have revealed two functional regions of P
Because the interaction of G
To map the
carboxyl-terminal sites of P
The last two isoleucine residues of P
Our data on the
inhibitory binding of P
Studies on
competition between P
In our earlier studies defining
sites on G
Given this information about the molecular details of activation of
cGMP phosphodiesterase, an intriguing question is whether there is a
conserved molecular mechanism of effector activation by G proteins.
Similarities in amino acid sequence make it likely that
The degeneracy of nucleotides at
indicated positions (letters above and below the line) was utilized for
simultaneous statistical introduction of terminator codons
(oligonucleotides I, II, and III) and also for statistical substitution
of Ile
We thank Dr. John Sondek for the gift of P
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-subunit (G
) and the
-subunit (P
) of
cGMP phosphodiesterase (PDE) is a key event in effector activation
during photon signal transduction. The carboxyl-terminal half of P
is involved in interaction with G
as well as in
inhibition of PDE activity. Here we have utilized a combination of
synthetic peptide and mutagenesis approaches to localize specific
regions of the carboxyl-terminal region of P
interacting with
G
and P
and have determined residues
involved in inhibition of PDE activity. We found that synthetic peptide
corresponding to residues 68-87 of P
completely inhibit
trypsin-activated PDE. The peptide P
-63-87 bound to
G
GTP
S with a K
of
2.5 µM, whereas the binding of P
-68-87 to
G
GTP
S was approximately 15-fold less
(K
= 40 µM)
suggesting that carboxyl-terminal P
region 68-87 contains a
site for interaction with P
and also a part of the
binding site. To map G
and
P
sites more precisely within the carboxyl-terminal region, a
set of carboxyl-terminal mutants was generated by site-directed
mutagenesis. Deletion of residues 63-69 and 70-76
diminished the binding of mutants to
while binding to
carboxyl-terminally truncated mutants lacking up to 11 amino acid
residues was unchanged. In contrast, carboxyl-terminal truncations of
P
from
1 to
11 resulted in a gradual decrease of its
inhibitory activity. Thus, the extreme carboxyl-terminal hydrophobic
sequence -Ile
-Ile
together with 9 adjacent
residues provides inhibitory interaction of P
with P
.
The carboxyl-terminal G
GTP
S binding site of
P
is different from but adjacent to its PDE inhibitory site.
During the visual transduction process, G
GTP likely
binds to this region of P
inducing a displacement of the extreme
carboxyl terminus from the inhibitory site on P
, leading to
PDE activation.
-subunit of the rod outer segment cGMP phosphodiesterase (P
)
regulates the catalytic function of the large phosphodiesterase
subunits,
and
(P
) (Hurley and Stryer, 1982). The
retinal rod PDE
(
)
is composed of two catalytic
subunits,
(99.2 kDa) and
(98.3 kDa), and two identical
inhibitory
-subunits (9.7 kDa) (Baehr et al., 1979;
Ovchinnikov et al., 1986, 1987; Lipkin et al., 1990b;
Deterre et al., 1988; Fung et al., 1990). Activation
of the effector enzyme occurs upon binding of G
GTP
(G
*) to P
complexed with P
and
displacement of P
from its inhibitory site on the catalytic
subunits (Wensel and Stryer, 1986, 1990; Deterre et al.,
1986). The activated PDE rapidly hydrolyzes cytoplasmic cGMP resulting
in closure of plasma membrane cationic channels and hyperpolarization
of the rod cell (for review, see Liebman et al.(1987) and
Stryer(1991)). In the absence of photoreceptor membranes or at a low
membrane concentration, the complex of G
* with P
dissociates from P
(Deterre et al., 1986; Yamazaki
et al., 1990; Wensel and Stryer, 1990). In the presence of
high ROS membrane concentrations and under physiological ionic
conditions the G
*
P
P
complex
remains membrane-bound (Clerc and Bennett, 1992; Catty et al.,
1992).
, P
, and P
, is still unclear.
Significant progress has recently been achieved in the understanding of
P
functional structure. Peptide studies have demonstrated that the
central positively charged region of P
located within residues
24-45 (P
-24-45) interacts with both G
(Lipkin et al., 1988; Morrison et al., 1989;
Artemyev et al., 1992, Artemyev et al., 1993a) and
P
(Artemyev and Hamm, 1992). The carboxyl-terminal tryptic
peptide of P
corresponding to residues 46-87
(P
-46-87) also interacts with G
(Artemyev
et al., 1992) and inhibits P
(Artemyev and Hamm,
1992). The last several carboxyl-terminal residues are required for the
inhibitory interaction of P
with P
(Lipkin et
al., 1988; Brown and Stryer, 1989; Brown, 1992). Mutational
analysis of P
has revealed that the positively charged residues
Arg
, Arg
, Lys
, and Lys
are important for binding to P
(Lipkin et al.,
1990a; Brown, 1992; Lipkin et al., 1993). Despite the number
of P
mutants with impaired PDE inhibition activity, only two
mutants so far have been reported to have diminished G
binding properties: the triple mutant K41Q,K44Q,K45Q (Brown,
1992) and P
W70F (Otto-Bruc et al., 1993). It is likely
that the length of the spacer sequence linking the central and
carboxyl-terminal sites for P
interaction as well as
Pro
are important for their optimal orientation (Brown,
1992; Lipkin et al., 1993). Much less data have been
accumulated concerning functional regions of G
and
P
that are involved in interaction with P
. We have
recently reported that the synthetic G
peptide
corresponding to residues 293-314 activates PDE (Rarick et
al., 1992) through interaction with a carboxyl-terminal fragment
of P
, residues 46-87 (Artemyev et al., 1992). This
region of G
GTP
S is a part of a surface that,
according to its recently solved crystal structure (Noel et
al., 1993), forms a contiguous patch of exposed residues. Another
three adjacent regions that are localized on one face of G
and change conformation upon GDP-GTP exchange, switch I
(Ser
-Thr
), switch II
(Phe
-Thr
), and switch III
(Asp
-Arg
) (Lambright et al., 1994)
may also be potential PDE binding sites of G
.
Mutational analysis of recombinant G
produced in
Sf9/baculovirus expression system have shown that tryptophan 207 is
involved in the activation-dependent binding to P
(Faurobert
et al., 1993). The information regarding regions of PDE
catalytic subunits interacting with P
is limited to the finding
that synthetic peptides, representing distinct amino-terminal sites of
P
and P
, exhibited P
binding in solid-phase
radioimmunoassay (Oppert et al., 1991; Oppert and Takemoto,
1991).
located
within its carboxyl-terminal region. We have determined the ability of
the peptides P
-63-87 and P
-68-87 as well as
several carboxyl-terminal P
mutants to inhibit PDE activity and
bind to G
. We have assigned the carboxyl-terminal
hydrophobic sequence -Ile
-Ile
plus nine
adjacent residues as a P
inhibitory site and a region
internal to this, residues 63-76, as a carboxyl-terminal
G
binding site. Binding of G
* to this
site relieves the inhibition normally imposed on PDE by its
-subunit and leads to PDE activation.
Materials
GTP, GTPS, cGMP, calf intestinal
alkaline phosphatase, and all restriction and DNA modification enzymes
were obtained from Boehringer Mannheim. Blue-Sepharose CL-6B and Fast
Flow SP-Sepharose were products of Pharmacia Biotech Inc. Trypsin and
soybean trypsin inhibitor were from Worthington.
[
-
P]ATP (600 Ci/mmol) was from ICN Inc. All
other analytical grade reagents were from Sigma or Fisher.
Preparation of G
Bovine ROS membranes were prepared by the method of
Papermaster and Dreyer(1974) with some modifications (Mazzoni et
al., 1991). The GGTP
S, PDE, and
tPDE
GTP
S was extracted from ROS
membranes with GTP
S and purified using chromatography on a
Blue-Sepharose CL-6B column as described by Kleuss et al. (1987). PDE was extracted from ROS membranes according to the
procedure described by Baehr et al.(1979). PDE was purified
and tPDE was prepared using the protocol of Artemyev and Hamm(1992).
The purified proteins were kept in 40% glycerol at -20 °C for
several months with no loss of functional activity.
Expression of P
Recombinant P in E. coli
was expressed under the control of the T7 promoter in E. coli BL21/DE3. The P
expression vector was constructed and
generously provided by J. Sondek (Yale University, New Haven, CT). This
vector is a derivative of pET11a, where polymerase chain
reaction-amplified P
gene flanked with NdeI and
BamHI sites was inserted between corresponding sites of the
parent plasmid. The pLcIIFXSG plasmid DNA containing the synthetic
P
gene (Brown and Stryer, 1989) was the PCR template. The
expression product was authentic full length P
at 87 amino acids.
Construction of P
The genes encoding
P Mutants
3, -
4, -
5, -
8 and P
1, -
2,
-
1I86F,
1I86N were constructed by inserting degenerate
phosphorylated oligonucleotides I or III, correspondingly, into the
expression vector cut with SacI and BamHI.
P
11 and -
17 were constructed by replacement of the
NcoI-SacI fragment of the P
gene with degenerate
phosphorylated oligonucleotide II. P
70-76 and
P
63-69 were made by insertion of the synthetic duplex I
or II, respectively, into the expression vector cut with KpnI
and SacI. P
G85A was prepared by replacing the P
gene
SacI-BamHI fragment with phosphorylated
oligonucleotide IV (see for a list of the
oligonucleotides). All DNA manipulations were performed using standard
techniques (Maniatis et al., 1989). The sequences of all
mutant P
genes were confirmed by dideoxy DNA sequencing (Sanger
et al., 1977).
Purification of Recombinant P
E. coli cells, after a 4-h induction with 0.5
mM isopropyl-1-thio- and
Mutants
-D-galactopyranoside, were
spun down, resuspended in 1:20 of a cell culture volume of buffer
containing 50 mM Tris-HCl, pH 7.5, 2 mM EDTA, 50
mM NaCl, 1 mM dithiothreitol, 10 µg/ml pepstatin
A, 5 µg/ml aprotinin, and 100 µM phenylmethylsulfonyl
fluoride (buffer A). The cell suspension was sonicated, and insoluble
material was spun out at 100,000
g for 60 min. The
supernatant was loaded onto a SP-Sepharose fast flow cation exchange
column equilibrated with buffer A and eluted off the column with buffer
A containing 0.5 M NaCl. Recombinant proteins were
additionally purified by reversed-phase chromatography on HPLC Vydac
C-4 column using a gradient concentration of acetonitrile in 0.1%
trifluoroacetic acid/H
O. The purified proteins were
lyophilized, dissolved in 20 mM Tris-HCl, pH 8.0, 100
mM NaCl, 2 mM MgCl
, 1 mM
dithiothreitol, aliquoted and stored at -80 °C. The final
yield of the different P
s ranged from 15 to 30 mg of more than 95%
pure protein/liter of culture.
Peptide Synthesis
Peptides corresponding to
residues 63-87 and 68-87 of P were synthesized by the
solid-phase Merrifield method on an Applied Biosystems automated
peptide synthesizer. The amino and carboxyl termini of the peptides
were not modified. Peptides were purified by reversed-phase
chromatography on a preparative Aquapore octyl column (25
1 cm)
(Applied Biosystems). The purity and chemical formula of peptides were
confirmed by fast atom bombardment mass spectrometry, analytical
reversed-phase HPLC, and amino acid analysis.
PDE Activity Assay
Different amounts of P
peptides or mutants were added to 50 pM tPDE in 200 µl of
20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 2 mM
MgCl
, and 1 mM dithiothreitol. The reaction was
initiated by the addition of 0.5 mM cGMP. Bacterial alkaline
phosphatase (1 unit) was added to the reaction mixture immediately
after substrate addition, and incubation was continued at 37 °C for
10 min. The reaction was stopped by adding 800 µl of 0.012% Triton
X-100. Inorganic phosphate release was determined using ammonium
molybdate as described by Eibl and Lands (1969). To prevent adsorption
of proteins to the walls during the enzymatic reaction, siliconized
glass test tubes were used.
Fluorescent Assay of Competition between P
For fluorescent
measurements, PLY and
P
Mutants or Peptides for Binding to
G
GTP
S
was specifically labeled at its single Cys
using the sulfhydryl specific reagent lucifer yellow vinylsulfone
as described by Artemyev et al.(1992). Fluorescent assays were
performed on a Perkin Elmer LS5B spectrofluorimeter in 10 mM
HEPES pH 8.0, 100 mM NaCl, and 1 mM MgCl
(buffer B) with excitation at 430 nm and emission at 520 nm.
Fluorescence of P
LY (50 nM) in the presence of
G
GTP
S (100 nM) was measured before and
after additions of increasing concentrations of P
mutants or
synthetic peptides. K
values for all
P
mutants and peptides were determined from their competition
curves based on calculated EC
values and
K
= 36 nM for the
P
LY
G
GTP
S complex (Artemyev et
al., 1992). The affinity of the unlabeled recombinant P
to
G
GTP
S was slightly higher then that for P
LY
(K
= 14 nM, see
``Results'' for details).
Miscellaneous Methods
Protein concentration was
determined by the method of Bradford(1976) using bovine serum albumin
as a standard. The concentrations of P and its mutants containing
both Trp
and Tyr
were determined
spectrophotometrically using
= 6,700. The
extinction coefficients for Tyr
lacking mutants
(P
4, -
5, -
8, -
11, -
17) and Trp
lacking mutant P
70-76 were 5,300 and 1,400,
respectively. Curve-fitting of the experimental data was performed with
nonlinear least squares criteria using GraphPad InPlot 4.0 software.
Is Located
within Region 68-87-Previous experiments have shown that
the carboxyl-terminal half of P
corresponding to residues
46-87 possesses PDE inhibitory activity
(K
= 0.8 µM)
(Artemyev and Hamm, 1992; Artemyev et al., 1992), and the last
5 carboxyl-terminal amino acid residues are necessary for inhibitory
binding to P
(Brown, 1992). To understand how PDE inhibitory
function is distributed within this region of P
, we studied the
functional properties of the synthetic peptides P
-63-87 and
P
-68-87. Peptide P
-68-87 was capable of
completely inhibiting tPDE with K
0.8 µM (Fig. 1A). The
K
values obtained for both
P
-63-87 and P
-46-87 were similar to that of
peptide P
-68-87 (data not shown), indicating that the
carboxyl-terminal PDE inhibitory region lies mainly within amino acid
residues 68 and 87. The Region 63-87 of P
Contains the Carboxyl-terminal
G
Binding Site-The binding of
P
-63-87 and P
-68-87 to
G
GTP
S was estimated by competition with P
LY
for binding to G
GTP
S. The synthetic peptides had
no effect on the fluorescence of P
LY (data not shown). However,
addition of either peptide reversed the fluorescent increase of
P
LY upon their binding to G
GTP
S due to their
ability to compete with P
LY for binding to
G
GTP
S (Fig. 1B). A
K
of 2.5 µM for the
P
-63-87
G
GTP
S complex was
calculated based on the K
for the
P
LY
G
GTP
S complex (36 nM)
(Artemyev et al., 1992) and the concentration of
G
GTP
S in the assay (50 nM). This
K
value was similar to that of
P
-46-87 (K
= 1.5
µM) and a shorter carboxyl-terminal peptide
P
-58-87 (K
= 2
µM) (Artemyev et al., 1993b) indicating that the
P
region 46-62 does not contribute significantly to binding
to G
GTP
S. The peptide P
-68-87 showed
15-fold weaker binding to G
GTP
S
(K
= 40 µM) compared
to P
-63-87. Mutational Analysis of P
Functional Regions within Residues
63-87-To determine carboxyl-terminal sites of P
interaction with P
and G
more precisely, we
generated a set of mutants containing deletions and substitutions of
amino acid residues at the carboxyl terminus of P
(see
for details). The P
mutants were analyzed for their
ability to inhibit tPDE. The calculated K
value for recombinant P
from fitting of the inhibition curve
(Fig. 2A) was approximately 10 pM, similar to
the K
reported earlier (Wensel and
Stryer, 1986, 1990). Removing even one carboxyl-terminal amino acid
residue (Ile
) resulted in a 7-fold decrease of P
inhibition activity (K
= 75
pM); however, this mutant was still able to inhibit tPDE
completely. Subsequent truncations of 2 to 5 amino acids not only
further reduced the ability of P
mutants to inhibit tPDE
(K
values ranged from 220 to 250
pM), but also gradually decreased their maximal inhibitory
activity from 80% (P
2) to 50% (P
5)
(Fig. 2B). The mutants with more profound truncations,
P
8 and P
11, showed an additional decrease in PDE
inhibition activity (K
of 330 pM
and 650 pM and a maximal effect of 50 and 40%, respectively).
The P
17 had the weakest inhibitory activity, and its maximal
inhibitory effect was only 20% (Fig. 2, A and
B). The mutants P
70-76 and
P
63-69, containing internal deletions of amino acid
residues but an intact extreme carboxyl-terminal sequence, were more
than 20-fold weaker PDE inhibitors compared to P
(K
values of 230 and 270 pM
correspondingly), but were able to inhibit tPDE completely
(I).
Figure 1:
A, effect of synthetic peptide
P-68-87 on tPDE activity. The activity of tPDE (50
pM) was determined in the presence of increasing
concentrations of synthetic peptide. Percent of maximal tPDE activity
is plotted as a function of peptide concentration. B,
competition between P
LY and synthetic peptides P
-63-87
or P
-68-87 for binding to G
GTP
S. The
fluorescence of P
LY (50 nM) in the presence of
G
GTP
S (100 nM) was measured before and
after addition of increasing concentrations of P
-63-87
(triangles) or P
-68-87 (circles), and the
relative fluorescent change (F/F
) is
plotted as a function of the peptide
concentration.
Figure 2:
Inhibition of PDE activity by P
mutants. The activity of tPDE (50 pM) in the presence of
increasing concentrations of P
(filled circles),
P
1 (filled diamonds) (A), and also
P
2 (open circles), P
4 (filled
triangles), P
8 (open diamonds), and
P
11 (open triangles) (B) was determined.
Percent of maximal tPDE activity is plotted as a function of P
or
P
mutant concentration.
The hydrophobicity of the carboxyl-terminal
Ile-Ile cluster implies a hydrophobic interaction of this region with
corresponding site(s) on the catalytic subunits that may form a
complementary pocket. To test this idea, we constructed a set of
carboxyl-terminal mutants to vary hydrophobicity as well as the steric
pattern at this inhibitory site. Replacing -Ile-Ile
with Phe partially restored the inhibitory activity of
P
2 and made this mutant very similar to P
1
(I). Substitution of the Ile-Ile with hydrophilic Asn
revealed similar inhibitory properties of this mutant as P
2
(I). The role of the small flexible glycine at the third
position from the carboxyl terminus was examined by substitution with
the less flexible alanine. This mutant showed a 7-fold decrease in the
potency of PDE inhibition (I) suggesting that for maximal
inhibition of PDE the Ile-Ile hydrophobic tail of P
must be
flexible. The Carboxyl-terminal Site of Interaction of P
with G
Is Located within Amino Acid Residues 63-76-To
determine the ability of P
mutants to bind to G
,
we studied their ability to compete with P
LY for binding to
G
GTP
S in a fluorescent assay. In control
experiments, the calculated K
for P
binding to G
GTP
S in competition with P
LY was
14 nM. The binding of carboxyl-terminally truncated P
mutants
1,
2,
3,
4,
5, and
8 to
G
GTP
S was not changed (I),
indicating that the carboxyl-terminal eight amino acid residues of
P
are not involved in binding to G
GTP
S. The
binding of P
11 to G
GTP
S was only
slightly decreased (K
= 30
nM, Fig. 3), suggesting that residues 77-79 are
also not very significant for G
binding. A more
significant change in affinity to G
GTP
S occurred
when 17 carboxyl-terminal residues were deleted (P
17),
resulting in a 6-fold decrease in affinity to
G
GTP
S (K
=
90 nM, Fig. 3). Peptide binding studies had implicated
residues 63-67 as well as adjacent residues 68-87 in
binding to G
(Fig. 1B). Data on binding
of carboxyl-terminally truncated P
mutants to
G
GTP
S allow the G
binding site
within region 68-87 to be restricted to residues 68-76. To
test the role of amino acids in the 63-76 region, a series of
internal deletion mutants were constructed. Deletion of amino acids
63-69 caused a drastic decrease in affinity for G
(K
= 230 nM,
Fig. 3
), while deletion of residues 70-76 resulted in a
further drop in affinity (K
= 460
nM, Fig. 3). Comparison of the primary structure of
P
70-76 and P
17 () would suggest
that the only meaningful difference in their sequence, assuming that
region 77-87 is not critical for transducin binding, is
Trp
which is the last amino acid residue in P
17
and is deleted in P
70-76. The 5-fold difference in the
affinity for G
GTP
S between P
70-76
and P
17 would implicate Trp
in binding of P
to G
GTP
S. The decreased affinity of P
17
and P
63-69 to G
GTP
S indicates
that amino acids surrounding Trp
within region 63-76
compose the carboxyl-terminal G
binding site of
P
.
Figure 3:
Competition between PLY and P
mutants for binding to G
GTP
S. The fluorescence of
P
LY (50 nM) in the presence of
G
GTP
S (100 nM) was measured before and
after addition of increasing concentrations of P
(open
squares), P
11 (filled squares), P
17
(open triangles), P
63-69 (filled
triangles), and P
70-76 (diamonds). The
fluorescent change (F/F
) is plotted as a
function of P
mutant concentration. The K values for all
mutants were calculated from the competition curves as described under
``Experimental Procedures.''
-subunit of PDE is a small molecule consisting of 87
amino acid residues. Despite its small size, P
possesses three
essential functions that play critical roles in visual signal
transduction: interaction with P
and PDE inhibition;
interaction with transducin and PDE activation; and, finally, in a
membrane complex G
*
P
P
,
stimulation of transducin GTPase activity (Arshavsky et al.,
1994; Angleson and Wensel, 1994). The important functional roles of
P
in the regulation of the light-induced enzymatic reactions in
ROS combined with the simplicity of its structure make P
a very
attractive object for the study of molecular mechanisms of
protein-protein interaction and provides insight into the detailed
mechanism of PDE activation.
(central,
residues 24-45, and carboxyl-terminal, residues 46-87)
involved in interaction with both P
and G
.
In solution, P
by itself displays both sites for interaction with
G
. From the peptide studies it appears that the
binding energy of P
G
interaction in
solution (K
= 14 nM) is
distributed approximately equally between these two sites based on
K
values for the central and
carboxyl-terminal peptides (0.7 µM and 1.5 µM
respectively, Artemyev et al. (1992)). However, in the complex
with P
, the affinity of P
to activated
G
, based on its K
for
PDE activation by G
GTP
S (1 µM), is
significantly lower. This can be explained by an interaction of one or
both of these sites with P
in the P
complex, leading to inaccessibility for
G
GTP
S interaction. Our earlier data suggest that
binding of G
-293-314 to the carboxyl-terminal
half of P
causes PDE activation (Rarick et al., 1992;
Artemyev et al., 1992) while the central region binds both PDE
catalytic subunits (Artemyev and Hamm, 1992) and G
.
This suggests that the central region may not be available for
G
binding in the PDE holoenzyme, contributing to the
low K
of PDE activation in solution.
with the
carboxyl-terminal region leads to PDE activation, in this study we
investigate in more detail the binding sites within this region with
P
and G
. We utilized synthetic peptides
corresponding to residues 63-87 and 68-87 of P
to
gradually narrow down P
and G
binding sites
within the carboxyl-terminal region 46-87. Both peptides
completely inhibited tPDE with a K
similar to that of the tryptic peptide P
-46-87. This
observation defines the carboxyl-terminal inhibitory domain within
residues 68-87 and suggests that residues 46-67 are not
critical in this function. The fluorescent studies on competition
between P
LY and synthetic peptides for binding to
G
GTP
S have revealed a significant difference in
their affinity to G
GTP
S. The binding of
P
-63-87 was similar to that of the previously reported
longer carboxyl-terminal peptides P
-58-87 (Artemyev et
al., 1993b) and P
-46-87 (Artemyev et al.,
1992) suggesting that the P
region between residues 46-62 is
not critical for binding to G
GTP
S. The peptide
P
-68-87 bound to G
GTP
S approximately
15-fold weaker in comparison with the P
-63-87. Thus, we
conclude that P
region 63-67 participates in the interaction
with G
GTP
S; however, part of the G
binding site is located within region 68-87.
interaction with P
and
G
more precisely, we constructed 13 mutants containing
systematic deletions and substitutions of carboxyl-terminal amino acid
residues. We determined their ability to inhibit tPDE and bind to
G
GTP
S in a competition fluorescent assay. All
P
mutants had impaired inhibitory function. A deletion of even one
amino acid residue (P
1) caused a 7-fold decrease in
inhibitory activity. Subsequent deeper truncations of carboxyl-terminal
residues resulted in a gradual decrease in the inhibitory activity of
mutants. P
11 and P
17 were the weakest inhibitors of
tPDE. Earlier data have shown that correct spacing and orientation of
the central region (residues 24-45) and carboxyl-terminal
inhibitory site are required for full inhibitory activity of P
.
The P
mutant P69L had impaired inhibitory activity, presumably
because of a change in the orientation of two sites of binding to
P
(Lipkin et al., 1993). Pro
is a part
of the deleted region in mutant P
63-69 and may be
responsible for the decrease in its inhibitory activity. The decrease
in the inhibitory activities of P
63-69 and
P
70-76 may also be caused by shortening of the
necessary spacer length similar to the very weak inhibitor
P
54-61 (Brown, 1992). These observations indicate that
the P
carboxyl-terminal site interacting with P
is
composed of the last 11 residues and support data obtained with
synthetic peptides.
are
important for its inhibitory binding to P
. Their removal
causes more than a 20-fold decrease in inhibitory activity. Replacing
the double isoleucine for phenylalanine did not completely restore the
authentic inhibition of P
, indicating that there is a tight
hydrophobic fit of the -Ile
-Ile
cluster,
presumably into a hydrophobic pocket on P
. Previous data on
the decreased affinity of a P
variant, containing six additional
hydrophobic residues on the carboxyl terminus, to P
(Brown,
1992) supports this idea. Decreased inhibitory activity of the G85A
mutant to P
may mean that Gly
provides
flexibility of the carboxyl-terminal hydrophobic region necessary to
match its partner site. Another explanation of this effect might be
that Gly
is involved in
-turn formation. Glycine
residues are frequently found in the second and third position of
-turns which are often present in regions of molecular recognition
(Wilmot and Thorton, 1988, 1990). Protein termini are also often sites
of recognition. For example, the carboxyl termini of G
-subunits
are involved in recognition of receptors (Conklin et al.,
1993). A recent NMR study on the structure of the carboxyl-terminal
peptide 340-350 has identified that a similar
structural motif -G-L-F-COOH has a
-turn critical for its binding
to rhodopsin (Dratz et al., 1993).
mutants make it attractive to predict some
of the molecular events that take place upon PDE inhibition. The
carboxyl-terminal 11 amino acid residues of P
function as an
inhibitory domain. The region 77-87 interacts with P
likely in close proximity to its catalytic site(s) and induces a
conformational change that imposes an inhibitory constraint. The
Ile
-Ile
domain anchors in a complementary
hydrophobic pocket on P
and locks the inhibitory conformation
of the P
complex. Such a model satisfactorily explains
the functional properties of the truncated mutants.
LY and P
mutants for binding to
G
GTP
S revealed that the carboxyl-terminal site
interacting with
is located in close proximity to the
P
binding site, but is not identical or overlapping. While
G
GTP
S binding of mutants with truncations up to
11 residues was practically unchanged, deeper truncation
(P
17) and deletion of regions 70-76 and 63-69
significantly diminished their affinity to G
GTP
S.
The truncation of amino acid residues 70-76 can be viewed as a
deletion of a carboxyl-terminal part of the G
binding
core, and, thus, decreased affinity of P
70-76 to
G
reflects the removal of a part of the
carboxyl-terminal G
binding site. The decreased
affinity of P
63-69 may be partially caused by a
shortening of the distance between the 70-76 and the central
regions of P
. We cannot distinguish whether diminished affinity of
P
63-69 is simply a result of the partial removal of one
G
binding site or a composite effect of a change in
the geometry of two binding sites combined with a deletion of a part of
the G
binding site. These data, together with data on
synthetic peptide functions, also indicate that the residues within
region 63-76 form the carboxyl-terminal site of P
important
for interaction with G
. This observation explains why
peptide P
-68-87 has more than a 15-fold lower affinity to
G
GTP
S compared to peptides P
-46-87 and
P
-63-87, because it lacks several amino acid residues from
the G
binding site. A diminished affinity of a
P
W70F mutant to G
GTP
S (Otto-Bruc, 1993) is
in good agreement with our data.
of interaction with PDE we determined that
a synthetic peptide encompassing residues 293-314 of G
can activate PDE (Rarick et al., 1992) while directly
binding to the carboxyl-terminal half of P
(Artemyev et
al., 1992). Our current data allow us to narrow the interface of
such an activating interaction between G
and PDE to
residues 63-76 of P
. This region of P
is located in
close proximity to its inhibitory site but does not overlap it and thus
is available for coupling with G
GTP
S. The primary
event of PDE activation appears to include interactions between the
G
region 293-314 and P
region 63-76.
We suggest that this interaction impairs the inhibitory binding of
P
region 77-87 to P
, resulting in the physical
removal of the carboxyl terminus of P
from its inhibitory site on
PDE. Thus, of the two G
binding sites on P
, our
current and previous data indicate that the carboxyl-terminal site is
sufficient for activating interaction with G
.
Alternatively G
GTP
S may bind to a composite site
which includes also lysine residues 41, 44, and 45 (Brown, 1992) of the
central region. The close agreement between the peptide binding and
activation studies and the mutant studies make it unlikely that the
carboxyl-terminal region is only secondarily involved in the PDE
activation process. A conformational change at carboxyl terminus may
cause a more profound perturbation in P
structure leading to
exposure of its central region for binding to
G
GTP
S or reorientation of the central and
carboxyl-terminal sites in an optimal way for G
binding. Under some conditions, G
GTP
S can
completely displace P
from its complex with P
in the
absence of ROS membranes (Wensel and Stryer, 1990). In the presence of
ROS membranes, the residual interaction of P
with P
via
the central region as well as the anchoring of
G
GTP
S and P
on the membrane can hold
the G
GTP
S
P
P
complex
together (Clerc and Bennet, 1992; Catty et al., 1992).
-subunits
of all heterotrimeric G proteins share the same basic three-dimensional
structure. The recent finding of overall structural similarity of
G
and G
(Noel et al., 1993;
Lambright et al., 1994; Coleman et al., 1994) support
this idea. The fact that one of the G
regions involved
in activation of adenylyl cyclase (residues 349-356) (Berlot and
Bourne, 1992) corresponds to residues 307-314 of G
important in PDE activation (Rarick et al., 1992) may
indicate that the cognate regions of all G
-subunits form effector
interacting surfaces. Available information does not indicate any
primary structure similarity among known effectors; however,
conservative mechanisms of G protein activation/inhibition of effector
via structurally similar recognition surfaces may be considered as a
working hypothesis. Thus, the molecular details of G protein-effector
interaction that are best understood for the visual signal transduction
system may be applied to study relevant partners in other signaling
systems.
Table:
Oligonucleotides used for construction of
carboxyl-terminal P mutants
for phenylalanine or asparagine (oligonucleotide
III).
Table:
Carboxyl-terminal amino acid sequence of P
mutants
Table:
Functional properties of P mutants
S, guanosine 5`-3-O-(thio)triphosphate; HPLC,
high performance liquid chromatography.
expression vector, Dr. John Mills for valuable discussions, and
Ferdinand Belga for technical assistance.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.