(Received for publication, September 18, 1995; and in revised form, October 23, 1995)
From the
The G protein transducin has been an often-used model for
biochemical, structural, and mechanistic studies of G protein function.
Experimental studies have been limited, however, by the inability to
express quantities of mutants in heterologous systems with ease. In
this study we have made a series of G/G
chimeras differing at as few as 11 positions from native
G
. Ten chimeras are properly folded, contain GDP, can
assume an AlF
-induced activated
conformation, and interact with
and
light-activated rhodopsin. They differ dramatically in their affinity
for GDP, from G
-like (initial rates 225 µmol/mol s) to
G
-like (initial rates 4.9 µmol/mol s).
We have used
these chimeras to define contact sites on G with the
effector enzyme cGMP phosphodiesterase. G
GTP but not
G
GDP activates it by removing the phosphodiesterase
(PDE)
inhibitory subunit. In solution, G
GTP
interacts with PDE
(K
12
nM), while G
GDP binds PDE
more weakly (K
0.88 µM). The interaction
of G
GDP with PDE
is undetectable, but
G
GDP-AlF
interacts
weakly with PDE
(K
2.4
µM). Using defined G
/G
chimeras, we have individuated the regions on G
most important for interaction with PDE
in the basal and
activated states. The G
sequence encompassing
helix 3 and the
3/
5 loop contributes most binding energy to
interaction with PDE
. Another composite P
interaction site is
the conserved switch, through which the GTP-bound G
as
well as G
interact with P
. Competition studies
between PDE
and truncated regions of PDE
provide evidence for
the point-to-point interactions between the two proteins. The
amino-terminal 1-45 segment containing the central polycationic
region binds to G
's
3 helix and
3/
5 loop, while the COOH-terminal region of P
,
63-87, binds in concert to the conserved switch regions. The
first interaction provides specific interaction with both the GDP- and
GTP-liganded G
, while the second one is conserved
between G
and G
and dependent on the
activated conformation.
G proteins are key intermediates in signal transduction by a
large class of G protein-coupled receptors that respond to sensory,
hormonal, and neurotransmitter signals in the environment. The
important roles of G proteins in shaping the specificity and temporal
features of cellular responses to a variety of signals are under
intense investigation (1, 2, 3, 4) .
Receptor-mediated GTP/GDP exchange on G protein subunits
regulates the production of both activated G
GTP and free
G
subunits, which in turn regulate the activity of a variety
of effector enzymes and channels; GTP hydrolysis by G
then
determines the turn-off of the response. Four classes of mammalian
G
subunits with sequence homology from 45 to 80% along with an
unknown number of
s (five
and at least eight
genes) (5, 6) make up heterotrimeric G proteins that
interact with a large number of different receptors and a smaller
number of effectors(7, 8, 9) .
One of the best studied G protein-mediated transmembrane signaling cascades from the physiological, biochemical, and structural points of view is the visual transduction system of rod outer segments. The abundance of the signaling proteins including the receptor, rhodopsin, the G protein, transducin, and the effector, cGMP phosphodiesterase, has led to a range of studies that makes them prototypical members of these classes of signaling proteins(10, 11, 12, 13) . Another reason for the intense scrutiny of this signaling system is that a single species of receptor, G protein, and effector is present, so the confounding influence of closely related isoforms in other cell types is eliminated. A third reason for the fascination with the rod visual signal transduction system is its highly specialized function as a single photon detector and consequent high sensitivity(14) .
To understand the molecular details of interaction between
G and its partners G
, rhodopsin,
and cGMP phosphodiesterase, relatively large amounts of recombinant
functionally active G
are required for mutational
analysis of the molecule and functional testing in biochemical assays.
Numerous unsuccessful attempts to express functional G
in various expression systems have been made. The only expression of
functional G
has been reported by Faurobert et al.(15) in the Sf9/baculovirus system, but protein yields
were relatively low (50 µg/liter). The Escherichia coli expression system to express functional G
was
tried extensively in several labs without success. A high yield of
insoluble protein was obtained, mainly in inclusion bodies. Refolding
of recombinant G
from urea-solubilized material did
not result in proper protein folding.
In this work we have obtained
functionally expressed G variants using a chimera
approach. G
, with 68% homology of amino acid
sequence, has a very similar overall three-dimensional structure (19) to
G
(16, 17, 18) , although
functionally, the two proteins couple different receptors to different
effectors. The fact that recombinant G
is soluble and
functional (20) , but recombinant G
is not,
provides a strategy to ``rescue'' recombinant G
by systematic replacement of G
regions, which
inhibit proper folding, with G
regions, which are
permissive for proper folding. We have constructed a set of
G
/G
chimeras, which are soluble and
functional and express to high levels in E. coli. The most
G
-like chimera contains only 11 amino acids different
from native G
and is functionally almost
indistinguishable from G
.
We have used these
chimeric G/G
proteins to investigate
the structural basis of high affinity interaction of G
with its effector, cGMP PDE. (
)The inactive retinal
rod cGMP PDE is composed of two membrane-anchored catalytic subunits,
(99.2 kDa) and
(98.3 kDa), and two identical inhibitory
subunits (9.7
kDa)(21, 22, 23, 24, 25) .
Normally, PDE becomes activated when light-activated rhodopsin
activates GTP/GDP exchange on G
.
G
GTP interacts with phosphodiesterase and
activates it by displacing P
from inhibitory site on catalytic
subunits P
(26, 27) . The activated PDE
rapidly hydrolyzes cytoplasmic cGMP, resulting in closure of plasma
membrane cationic channels and hyperpolarization of the rod cell (10) .
Sites on G subunits of effector interaction have
been investigated for both G
and G
.
We have demonstrated that the synthetic G
peptide
corresponding to residues 293-314 activates PDE (28) through interaction with a COOH-terminal region of P
:
residues 46-87(29) . Another important site for P
interaction is Trp-207 of G
(15) . Mutational
analysis of G
has revealed regions involved in
interaction with adenylyl cyclase(30, 31) .
The
chimeric G/G
molecules described in
this study defined three regions of G
involved in
interaction with the PDE
effector molecule. The presence of single
or combinations of these regions in a systematic series of chimeras led
to increasing affinity for PDE
. Competition studies with fragments
of PDE
have allowed us to define the point-to-point interactions
that are important for productive binding of activated G
to PDE
leading to enzyme activation.
Chimeric genes were
constructed by introduction of unique restriction enzyme sites,
flanking target fragments, into G and G
cDNAs using PCR amplification with corresponding oligonucleotide
primers-mutagenes and then replacement of G
cDNA
fragments with corresponding G
cDNA fragments and vice versa. In no case did insertion of novel restriction
sites change protein sequences. Table 2shows the location of
restriction enzyme sites along the G
sequence which
are the joining points for a combinatorial arrangement of the
G
and G
sequences in
G
/G
chimeras. The quadruple
G
mutant, L232M/A235V/E238D/M240V (Chi1), changed
G
's switch III region to that of
G
. Two regions of G
cDNA encoding
residues 1-240 and 241-354 were PCR-amplified in separate
reactions. The downstream primer for synthesis of 5`-terminal fragment
was an oligonucleotide mutagene directing not only substitutions of
indicated 4 amino acids but also introduction of SphI site.
The upstream oligonucleotide primer for PCR amplification of the
3`-terminal fragment directed introduction of the SphI site as
well. The resulting fragments were cut with EcoRI and SphI (5`-terminal fragment) and SphI and HindIII (3`-terminal fragment), and simultaneously ligated
with the large fragment of pHis
G
digested
with EcoRI and HindIII. Chi1 plasmid DNA was used as
an intermediate construct for making Chi8, Chi5, Chi4, Chi7, and Chi10,
where G
switch III region was replaced with the
corresponding G
region. The DNA sequence of all
chimeric genes around joining points (usually 150-200 base pairs)
was confirmed by DNA sequencing. Chi6 and Chi9 genes were sequenced
nearly completely. No misincorporations of deoxynucleotides in
PCR-amplified fragments were detected.
P-1-45 expression
vector was constructed by PCR amplification of the corresponding P
gene fragment using the P
expression vector DNA (32) as a
template. The downstream oligonucleotide primer directed an insertion
of a TAG terminator codon after Lys-45 codon of P
gene, following
by BamHI site. The resulting PCR product, cut with NdeI and BamHI, was ligated with the large fragment
of pET11
cut with the same restrictases. The DNA sequence of this
construct was confirmed by DNA sequencing over the PCR-amplified
region.
G, G
, G
GDP,
G
GTP
S, G
, and rhodopsin
containing ROS membranes treated with urea were prepared as described
in(33) .
SDS-polyacrylamide gel electrophoresis of proteins was performed according to the method of Laemmli(34) . For immunoblotting analysis, proteins were transferred from SDS-polyacrylamide gel to nitrocellulose using standard semi-dry transfer method. Peroxidase-labeled secondary antibodies immobilized on membranes were detected using a luminol-based chemiluminescent detection system (LumiGLO substrate kit, Kirkegaard & Perry Laboratories, Gaithersburg, MD). Curve-fitting of the experimental data was performed with nonlinear least squares criteria using GraphPad Prism software.
Our experimental strategy took advantage of the significant
structural similarity of G and G
(see Fig. 1A for structure-based alignment) and
functional divergence to use them as parent proteins for construction
of chimeric G
proteins that fold properly. We systematically
replaced regions of G
that inhibit proper folding with
corresponding G
regions that are permissive for
proper folding.
Figure 1:
A, alignment of amino acid sequences of
G and G
. Sequences of G
and G
were taken from Yatsunami and Khorana (64) and Nukada et al.(65) , respectively. Yellow shows
helixes, blue shows
sheets,
and boxes indicate locations of the conformational switch
regions. B, G
G
chimera. Panel is a ribbon drawing highlighting regions that inhibit folding in
G
(red). C, G
(red) and G
(blue). Panel shows superposition of G
GTP
S (Noel et
al., 1993) and G
GTP
S (Coleman et
al., 1994).
Chimeras 9 and 10, which contained
region 237-270 of G, had significantly reduced
solubility. Only approximately 5-10% of the expressed polypeptide
was located in the soluble fraction when cells were induced at room
temperature with lowered concentration of IPTG (30 µM).
The final yield of purified chimeras from this group was 0.1-0.25
mg/liter of cell culture. The region 237-270 is composed of the
3 helix, the
3/
5 loop, and the
5 sheet; the
3
helix and the
3/
5 loop contain the bulk of differences in
amino acid sequence (Fig. 1A). Thus, the decreased
solubility of chimeras containing this G
region may be
explained by substantial inhibition of their assembly caused by either
misfolding of the
3 helix or improper interaction of the
3-
3/
5 motif with other parts of polypeptide upon
synthesis in bacterial cells.
The rate-limiting step of G protein activation
is the release of GDP. The intrinsic GDP release rate of the chimeras
was measured by determining the rate of
[S]GTP
S binding and compared to literature
values(38, 39) . Fig. 2A shows that at
25 °C native G
does not appreciably bind GTP
S
and thus the dissociation of GDP that precedes GTP
S binding is
very slow (initial rate of 4.9 µmol/mol s, Table 3).
Recombinant His
-G
showed significantly
faster nucleotide exchange rate (initial rate of 225 µmol/mol s, Table 3; K
0.05 min
, Fig. 2A), in good agreement with data for authentic
NH
-terminally acylated G
and recombinant
non-myristoylated protein (39) . Thus either lack of
NH
-terminal acylation or presence of an unrelated extra
sequence at its NH
terminus (His
tag) does not
significantly alter guanine nucleotide exchange properties of the
protein. The rate of intrinsic guanine nucleotide exchange for chimeras
ranged from very low, similar to G
, to high, similar
to G
. Chimera 2, containing the 56 COOH-terminal
amino acids of G
in a G
context had
a high GTP
S binding rate, similar to G
(Fig. 2A, Table 3, K
of 0.056 and initial rate of 227 µmol/mol s). The replacement
of the NH
-terminal G
fragment 1-215
with that of G
(Chi6) resulted in a significant
decrease in the rate of GTP
S binding (Fig. 2A, Table 3, initial rate of 15.9 µmol/mol s). Chimera Chi9
containing additional residues 237-270 of G
had
a lower GTP
S binding rate approaching G
(Fig. 2A, Table 3, initial rate of 7.9
µmol/mol s).
Figure 2:
Time course of GTPS binding to
G
(squares), G
(circles), chi2 (diamonds), chi6 (triangles), and chi9 (reversed triangles). Proteins
(1 µM) alone (A), in presence of 100 nM rhodopsin and 1 µM G
(B), or in presence of 100 nM rhodopsin alone (C) were incubated at room temperature in buffer G containing
5 µM [
S]GTP
S. Duplicate
aliquots were withdrawn at the indicated times, filtered, and counted.
Binding of GTP
S to proteins is expressed as percent of maximal,
calculated based on protein concentration and fit using nonlinear least
squares criteria to the equation B = B
(1 - e
).
Data shown are the mean ± S.E. of four
experiments.
The rates of rhodopsin-catalyzed exchange of GDP
for [S]GTP
S were measured in the chimeras
to determine their ability to interact with G
and
activated rhodopsin. The addition of equimolar concentrations of
G
(1 µM) and 100 nM light-activated rhodopsin to G
,
G
, and chimeras caused a stimulation of GTP
S
binding to all proteins (Fig. 2B). For G
and Chi2, the rhodopsin-catalyzed increase in GTP
S binding
was just 2-5-fold (initial rates: 1304 and 1352 µmol/mol s; K
of 0.095 and 0.097 min
,
respectively) and less than for G
(780-fold) (initial
rate of 3798 µmol/mol s, Table 3; K
= 0.3/min, Fig. 2B). Chi6 and Chi9 had
significantly increased but similar rates of nucleotide exchange in the
presence of Rho and G
(K
0.047 and 0.032 min
; initial rates 738 and 554
µmol/mol s, respectively; Fig. 2B and Table 3) but 6-8-fold lower than for ROS
G
. Rhodopsin alone in the absence of G
stimulated the nucleotide exchange of only G
,
due to low levels on contamination with G
.
Figure 3:
Binding of GGDP,
G
, and chimeras to P
LY in the presence of
AlF
. The relative increase in
fluorescence (F/F
) of P
LY
(50 nM) was measured after addition of increasing
concentrations of G
(filled circles),
G
(open triangles), Chi6 (diamonds), Chi9 (squares), Chi10 (filled
triangles), and Chi8 (open circles). The solid lines represent the best fit to a four parameter logistic equation
(sigmoidal curve) where the fluorescence at each point has been
corrected for dilution. K
values for the
binding of indicated proteins to P
LY were determined based on
calculated EC
parameters.
Chimeras from the second group (Chi4, Chi5,
Chi6, Chi7, and Chi8) contain various combinations of two effector
interaction regions, and have a 2-4-fold increased affinity to
PLY compared to G
(Fig. 3, Table 2). The presence of any two of the regions (295-314,
switch III, and the amino-terminal 1-215 region) thus appear to
collaborate to form a composite P
binding site. Chi8 contains all
three of these G
regions, and had a
4-fold
increase in affinity to P
LY (K
550
nM, Fig. 3, Table 2).
Chimeras from the third
group are represented by Chi9 and Chi10. Their sequence contains only
15 and 11 amino acid residues derived from G,
respectively (Table 2, Fig. 1A), and the rest of
the sequence originates from G
. These two chimeras
both formed a high affinity complex with P
LY in the presence of
AlF
(K
70 and 38
nM, respectively; Fig. 3, Table 2), just
3-6-fold lower than the affinity of wild type
G
GDP-AlF
for P
.
Comparison of the primary structures of these two chimeras to the
structure of Chi6 indicates that the additional G
region 237-270 increases the affinity of the chimera for
P
by 13-fold. This region consists of the
3 helix and
3/
5 loop as well as the well conserved
5 sheet and the
NKXD guanine ring binding element. Comparison of the sequences
in this region shows that the main differences between the two proteins
occur in the
3 helix and the
3/
5 loop (Fig. 1A). By ruling out the conserved regions, we can
narrow down the most important determinant for high affinity P
binding to residues 237-257 (blue residues in Fig. 6).
Figure 6:
High affinity effector binding surface of
G. Highlighted residues indicate: pink,
COOH-terminal P
site interface (
4-
4/
6 and switch
III); blue, site of interaction with central region of P
(
3-
3/
5); tan, switch II; maroon,
switch I.
Chi9 and Chi10, similar to GGDP,
were able to form a low affinity complex with P
LY in the absence
of AlF
(K
3.7 and
3.6 µM, respectively; Table 2). Neither
G
or any of the other chimeras had detectable
affinity for P
in their GDP-bound inactive state (Table 2).
This indicates that the main determinant for binding of
G
GDP to P
is contained in this area. To test this
idea, we synthesized a peptide encompassing this region (residues
232-259) and tested its binding to P
. The peptide increased
the fluorescence of P
LY in a dose-dependent manner (Fig. 4). The binding was completely reversible by adding an
excess of unlabeled P
to the
G
(232-259)-P
LY complex. The K
for this complex calculated from the binding
curve was 4.3 µM.
Figure 4:
Binding of the synthetic peptide
G(232-259) to P
LY. Peptide binding to
P
LY (100 nM) was estimated by the relative increase in
fluorescence (F/F
) after
recording the fluorescence of P
LY plotted as a function of peptide
concentration. The kinetic parameters calculated from sigmoidal binding
curve are EC
= 4.35 mM, F/F
max = 1.
94.
The chimeras Chi9 and Chi10 that
contain this sequence both had 4-fold lower affinity than
GGDP for P
LY, suggesting that another P
interaction site is found within one of the problem regions.
Figure 5:
Binding
of P fragments to parent proteins and chimeras. A,
competition between P
LY and P
-1-45 for binding with
G
and Chi9. The fluorescence of the complexes of
P
LY (50 nM) with G
GDP (800 nM) (triangles),
G
GDP-AlF
(50
nM) (circles), and Chi9-AlF
(75 nM) (reversed triangles) was measured
before and after addition of increased concentrations of
P
-1-45. B, competition between P
LY and
P
-63-87 for binding to chimeras in the presence of
AlF
. The fluorescence of P
LY (50
nM) in the presence of Chi6 (1 µM) (diamonds), Chi7 (750 nM) (squares), and
G
(2.5 µM) (triangles) as well
as all other chimeras and G
(see Table 2for
details) was measured before and after addition of increased
concentrations of P
-63-87. The fluorescent change is
expressed as a percent of maximal change (100% is fluorescence of
P
LY-G
complex before adding P
fragment; 0% is
fluorescence P
LY alone) and plotted as a function of P
fragment concentration. The K
values were
calculated from the competition curves as described under
``Experimental Procedures.''
On the other hand, G and all
the chimeras interacted, to different degrees, with the COOH-terminal
region of P
, but only in their activated state (Fig. 5B, Table 2). The relative affinities of
interaction shown in Table 2match quite closely with affinities
for full length P
, except for chimeras 9 and 10. This suggests
that the several effector regions cooperate for binding to
P
's carboxyl-terminal region. The presence of region
295-314 adds about 3-fold strength of interaction (compare
chimeras 7 and 1); likewise, residues within the first half of the
molecule add about 3-fold (compare chimeras 1 and 5). Interestingly,
the switch III region of G
plays an important role in
binding the COOH terminus; those chimeras lacking the transducin
sequence in switch III (chimeras 2, 3, 6) have lower affinity for this
region of P
. Two of the three regions are sufficient for binding
the carboxyl terminus, and adding the third region does not help (for
example, compare chimeras 8 and 5 or chimeras 5 and 4). The most
surprising result is that the affinity of chimera 7 for the
COOH-terminal region of P
is indistinguishable from
G
GDP-AlF
(0.7
µM). This chimera differs from G
at only
about 10 residues within the G
Switch III and
4-helix
4/
6 loop.
The soluble chimeric G
subunits are functional by several criteria. They are stably folded and
soluble, they contain GDP and are able to undergo
AlF
binding and assumption of an
activated conformation measured by an increase in intrinsic
fluorescence of Trp-207. The fact that the maximal intrinsic
fluorescence change is similar to native
subunits suggests that
both the inactive and active conformations are similar. With addition
of increasing numbers of G
residues, chimeric proteins
become progressively more transducin-like in biochemical properties
such as interaction with P
and intrinsic rate of GDP release. In
the presence of G
, rhodopsin stimulated GDP/GTP
exchange for G
, G
, and all chimeras
as expected from reconstitution experiments(44, 45) .
The stimulation was much more significant for G
, Chi6,
and Chi9 than for G
and Chi2, which have significant
spontaneous exchange. The fact that G
was
strictly necessary to promote GDP/GTP exchange above spontaneous GDP
release suggests that free
subunits have very little ability to
interact on their own with activated receptors under conditions in
which no
contamination can occur.
The structural basis for the
different GDP off-rates is of great interest, since this is the
rate-limiting step in G protein activation (47) . G and G
have quite similar overall folds in their
activated, GTP
S-bound forms (16, 19) ; the
GDP-bound, inactive form of G
has a very similar
global fold, with differences in conformation concentrated around the
and
phosphates(17) . One rather striking difference
between G
and G
is the intimacy of
packing of the two domains and, consequently, the buried nature of the
guanine nucleotide, with G
having a slightly more
open structure. In fact, the two domains of the structures are not
superimposable unless one of the domains is shifted by a 5° angle ((19) ; Fig. 1C). Of course the binding of the
guanine nucleotide in solution is a dynamic process, and the crystal
structures, while providing a detailed framework for understanding the
stereochemistry of guanine nucleotide binding, do not give complete
insight into regions of protein flexibility that may contribute to GDP
release. Second, there are probably many ways to affect the GDP release
rate, as mutagenesis studies have
shown(48, 49, 50) . The functional studies
show a 15-fold decreased initial rate of spontaneous GDP release caused
by replacing the G
helical domain, along with
helices 1 and 2 and
sheets 1-3 (residues 1-215), by
that of G
(Chi6). This suggests that the main
determinants for GDP affinity are within this portion of the molecule
and could be further delineated by future study.
The functional
implications for the differences in spontaneous GDP release between
different G proteins are quite dramatic. Since GDP release is the
rate-limiting step for G protein activation, the spontaneous release
rate should correspond to a tonic activation rate in the absence of
activated receptors. The signaling cascades of each physiological
system must adapt to or control the spontaneous activation properties
of the particular G protein, which will effect receptor independent
activation of channels or inhibition of adenylyl cyclase. The
subunits can modulate this rate. In highly sensitive
detection systems like the visual and olfactory systems, on the other
hand, any spontaneous activation of any step in the cascade would
increase the background noise and decrease sensitivity. This leads to
the prediction that G
, which mediates olfactory
activation of adenylyl cyclase, should have strikingly lower
spontaneous GDP release than G
, which is >95%
identical.
The chimeric approach allows expression and mutagenesis
studies of G, but cannot differentiate functions in
regions that are highly conserved. Overall, the two proteins are quite
homologous, and in regions of nucleotide binding or conformational
switches this homology increases (Fig. 1A). The
``problem'' regions of G
that do not support
folding cannot be examined directly because they lead to insoluble
protein. Site-directed mutagenesis of the 11
G
-specific residues in these regions should help to
define roles of these residues in folding or other functions.
PDE is a potent
inhibitor of PDE activity. The structural basis for this inhibition is
a two-site interaction including a COOH-terminal inhibitory region and
a central site for tight binding affinity; G
perturbs
both of these sites to cause PDE activation(29, 32) .
Mutational analysis of P
has revealed that positively charged
residues located in the central region (Arg-24, Arg-25, Lys-29, and
Lys-31) are involved in interaction with P
(42, 56, 57) , while 3 lysines at positions
41, 44, and 45 contact G
GTP(42) . The
interaction between the P
-24-45 region and G
has been studied using a photocross-linking
approach(58) . The site of cross-linking of P
fragment was
localized to G
region 306-310. It was suggested
that the central P
region may rather contact the
3-
3/
5 region of G
but reaches the
covalent attachment site via the 12-Å cross-linker. The second,
COOH-terminal G
binding site of P
is located
within residues 63-76 and is adjacent to, but not overlapping
with, the PDE inhibitory site encompassing residues 77-87 (32, 59) . Removal of 1, 2, or 3 residues from
P
's COOH terminus dramatically decreases the inhibitory
potency of
, leading to the idea that G
GTP may
cause PDE activation by displacing these residues(32) .
GGDP can interact with low affinity with PDE
;
however, its ability to activate effector has not been detected (40) . The recent report by Kutuzov and Pfister (60) that at high concentration G
GDP may
activate PDE (K
50 µM) implies that
at very high concentration an activating region of
G
GDP can activate PDE. GTP binding to G
causes a conformational switch, leading to a 70-fold increased
affinity for PDE
. At least two putative regions of effector
interaction on G
have been identified. The synthetic
peptide corresponding to residues 293-314 of G
activates PDE (28) by coupling to a carboxyl-terminal
fragment of P
located within residues 46-87(29) .
This region, corresponding to the surface exposed
4 helix and the
4/
6 loop, does not change its conformation upon
activation(16, 17) . The increased affinity of
G
GTP for PDE
may serve to increase the local
concentration of this activating region. The Switch II (15) and
III regions (this work) interact with PDE
only in their activated
conformations. Coordinated interaction between these
G
GTP regions and PDE
may be critical for effector
activation.
Our studies define at least
two effector binding surfaces, each of which is composed of several
linear sites of G. G
GDP forms a low
affinity complex with P
through a major contact with the central
region of PDE
(K
5.3 µM) and a
weaker one with its COOH-terminal region (K
25
µM). G
activation increases its affinity
to P
approximately 70-fold, as a result of interaction with the
conserved conformational switch regions. More than 10- and 30-fold
increased affinity of the central and COOH-terminal P
regions to
activated G
suggests that both P
sites acquired
new contacts with G
switch regions.
A complementary study on binding
of the synthetic peptide encompassing residues 232-259 of
G to P
LY (K
4.3
µM) additionally proves the involvement of this region in
effector interaction. Cunnick et al.(61) have found
that synthetic peptide G
-250-275, which contains
a sequence corresponding to the
3/
5 loop, was able to bind to
P
. The presence of this region establishes a basal low affinity
binding for inactive Chi9 and Chi10, which is, however,
5-fold
lower than for G
GDP. Activation of G
leads to a 10-fold higher affinity for the P
central region,
presumably involving switch regions.
It was shown earlier that synthetic peptide
G-293-314 activates PDE (28) as well as
binds to a COOH-terminal fragment of P
located within residues
46-87(29) . Eight-fold weaker potency of PDE activation
for this peptide (K
8 µM) compared to
that of G
* (1 µM) in the absence of
membranes implies that G
region 293-314 is at
least a part of an effector activating surface of G
.
Our current data allow us to examine this surface in detail. The
293-314 region is exposed in G
GDP and able to
bind to the COOH terminus of P
, however, very weakly. The
approximately 10-fold lower affinity of this region as a part of the
whole molecule (25 µM) to the COOH-terminal fragment of
P
compared to the affinity of G
-293-314
alone (K
2 µM, (29) ) may be
explained by the greater accessibility of the flexible peptide for
contact with P
while separated from overall structural context of
G
. Analysis of substituted peptides derived from
G
-293-314 for their ability to activate PDE has
revealed that Asn-297, Val-301, Glu-305, Met-308, and Arg-310, located
mostly on one exposed face of the
4 helix and the immediately
adjacent portion of the
4/
6 loop, are directly involved in
G
-P
interaction(62) . We propose, based
upon results obtained with the chimeras, that this activating region
may form with Switch III an effective alignment for binding the P
COOH-terminal region, resulting in 30-fold tighter contact and PDE
activation. These two regions are colored pink in Fig. 6.
Chimera 5, which does not contain the 295-314
region but contains the amino-terminal half of G corresponding to the extra domain, the phosphate binding regions,
and switches I and II, also binds to the COOH-terminal P
region
with similar affinity as chimeras 6 and 8. This suggests a composite
binding site, which must contain at least two of three regions, since
Chi8 with all three regions does not bind with higher affinity.
In
the high affinity G-P
complex, Erickson et
al.(63) have recently estimated the distance between
Cys-68 of P
and Lys-267 of G
GTP
S to be 45
Å, based on the efficiency of energy transfer between these
fluorescently labeled residues. In their proposed model of the
G
-P
complex, the contacts of the COOH-terminal
and central sites of P
were assigned to G
regions
106-116 and 300-310 (
4-
4/
6), respectively.
The involvement of the 106-116 region in interaction with P
does not contradict our data that a P
binding determinant is found
within residues 1-215. However, our results prove the
participation of the conformational switch regions of G
in binding with the COOH-terminal and central sites of P
as
well as the existence of contact sites between the
3-
3/
5
region of G
and the central region of P
. This
contradicts the data of Erickson et al. The basis for
differences in the two studies is not known. Hopefully, in the future,
the crystal structure of the G
-P
complex should
resolve this difference.
Fig. 6summarizes the
effector-interacting surfaces we have described. The picture that
emerges from this study and previous data is of a concerted set of
mechanisms underlying: 1) selectivity of G protein effector interaction
imparted by the 3 and
3-
5 loop (blue region);
2) the formation by the GTP-dependent conformational switch of a
composite effector surface, which increases affinity for the effector,
without imparting high selectivity, because the switch must be
extremely conserved among G proteins (Switch III, left pink
region; Switches I and II, tan and purple
regions); and 3) an ``activating region'' (right
pink region; (28) ), which is highly specific for cognate
effector(62) , but does not significantly contribute to the
affinity of the interaction. We suggest that this activating region is
essentially ``presented'' to the appropriate site on the
effector by the switch-dependent and independent mechanisms (mechanisms
1 and 2).