From the Institute for Neuroscience, Northwestern
University, Chicago, Illinois 60611, the § Affymax Research Institute,
Palo Alto, California 94304, and the ¶ Department of Chemistry and
Biochemistry, Montana State University,
Bozeman, Montana 59717
Received for publication, March 24, 2000, and in revised form, September 29, 2000
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ABSTRACT |
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The retinal receptor rhodopsin undergoes a
conformational change upon light excitation to form metarhodopsin II
(Meta II), which allows interaction and activation of its cognate G
protein, transducin (Gt). A C-terminal 11-amino acid
peptide from transducin, Gt Heterotrimeric guanine nucleotide-binding proteins (G
proteins)1 are critical
regulatory proteins in a variety of cell signaling pathways.
Stimulation of a G protein-coupled receptor by an appropriate agonist results in conformational changes leading to its interaction with a heterotrimeric G protein, catalysis of GDP release, and subsequent G protein activation (for reviews see Refs. 1-3). In
the visual system, the retinal light receptor rhodopsin is activated by
absorption of a single photon of light followed by interaction with and
activation of the G protein transducin (Gt), leading to
stimulation of 3',5' cyclic GMP phosphodiesterase (for review see Ref.
4). In the absence of additional guanine nucleotides, the G
protein enhances agonist binding to a G protein-coupled receptor.
Binding of either GDP or GTP to the G protein disrupts the high
affinity complex or active state of the G protein-coupled receptor (5,
6).
The receptor-G protein interface has been defined in some detail and
involves portions of the intracellular loops and juxtamembrane regions
of the G protein-coupled receptor with several regions on
heterotrimeric G proteins (for review see Ref. 7). Distinct regions on G For rhodopsin-transducin interactions, all three intracellular loops of
rhodopsin have been implicated (22-28) as well as the C-terminal tail
(29). The receptor appears to interact with both the Important insights into the mechanism of G protein-mediated signal
transduction have been provided by the crystallization and high
resolution structure determination of G protein In an alternative approach to defining important determinants in the C
terminus, Martin et al. (54) used a combinatorial peptide
library to identify high affinity analogs of
Gt In this study we have examined the structural basis for the invariance
of Gly and Cys at the Peptide Synthesis--
Gt Protein Preparation--
Washed bovine rod outer
segments-containing rhodopsin were prepared from fresh bovine retinas
using sucrose gradient centrifugation and washing of the membranes with
EDTA to remove peripherally bound proteins (55). Heterotrimeric
Gt was prepared as described previously by Stryer et
al. (56).
"Extra" Meta II Assay--
The absorbance spectra of washed
rod outer segments (5 µM) were measured in the
presence of varying concentrations of Gt Decay Assay--
The absorbance spectra of washed rod
outer segments (10 µM) were measured in an SLM Aminco
DW2000 spectrophotometer in decay buffer (10 mM
K2PO4, pH 6.5, 0.1 M KCl, 0.1 mM EDTA, 1 mM dithiothreitol) in the presence
of either 1 or 2 mM Gt Molecular Dynamics--
Calculations were carried out on Silicon
Graphics workstations using the program DISCOVER (MSI, Inc.) and the
CVFF forcefield. After amino acid substitutions the energy was
minimized using steepest descents, and then 250 ps of molecular
dynamics were run at 300 K, pH 7 with a
distance-dependent dielectric constant.
The three-dimensional structures of heterotrimeric G proteins have
provided detailed information about their subunit and receptor interactions and have suggested mechanisms for GTP hydrolysis. In most
of the crystal structures the final residues of the G NMR studies indicate that the C-terminal 11-amino acid peptide of
Gt For an alternative approach, we identified potent high affinity
sequences related to the C terminus of Gt Rhodopsin can be measured spectrophotometrically in many of its
light-induced conformational states (for review see Ref. 4). The
binding of Gt to light-activated rhodopsin stabilizes an
active signaling state of the receptor (R*) that can be measured
spectrophotometrically (60, 61). The active state can be stabilized by
the R*-catalyzed loss of GDP from Gt, leading to an empty
guanine nucleotide binding pocket (62). Addition of either GTP or GDP
promotes the loss of the active R* state, as measured by the loss of
Meta II stabilization (62-64). The biologically active Meta II state
can be differentiated from its precursor, Meta I, by the extra Meta II
assay. This assay makes use of the observation that under conditions of
slightly alkaline pH and low temperatures Meta I is strongly favored in the absence of Gt. Meta II is stabilized in the presence of
Gt and can be measured spectrophotometrically. We have
exploited the ability of Gt The Role of Free Amino Groups in the Peptide Stabilization of Meta
II--
The native Gt
We have shown previously that the increase in potency of
Gt
We have also investigated the role of the free N terminus on
activity using two of the high affinity analogs, peptide 23 (VLEDLKSCGLF) and peptide 24 (MLKNLKDCGMF), identified by combinatorial
screening (54). Peptides 23 and 24 contain free N-terminal amino
groups. Acylation of the N terminus of peptide 23 to neutralize the
free N-terminal amino group decreases the EC50 only
slightly, whereas acylation of peptide 24 decreases its
EC50 16-fold (Fig. 2, Table I). Substitution of the Lys at position 345 with Arg in the acetylated peptide 23 or substitutions at positions 342 and 345 in acetylated peptide 24 do not affect the EC50 values significantly
(Fig. 2, Table I). Taken together, the data in Table I suggest that
removal of a localized positive charge by acylation of the N terminus or delocalization of the positive charge that occurs when Lys is
replaced with Arg can enhance the affinity of certain
Gt The Role of the Negatively Charged C Terminus in the Peptide
Stabilization of Meta II--
NMR studies of the Gt Roles of Cys-347 and Gly-348 in the Peptide Stabilization of Meta
II--
The Cys residue at position 347 of Gt
To identify the role of the Cys side chain in its interaction with Meta
II, we initially synthesized a Cys to Ala analog, Ac-Gt
To further test the role of the side chain of Cys-347, we synthesized a
Cys to Ser analog,
Ac-Gt
To test the requirement for a Gly residue at position 348, we
substituted this position with an Ala. A structural feature of the
NMR-based model of the rhodopsin-bound peptide is the presence of a
The Role of Lys-341 in the Peptide Stabilization of Meta
II--
The six highest affinity peptide analogs from the
combinatorial library had all replaced the positive charge at position
341 with a hydrophobic residue (54). To further test the idea that a
hydrophobic residue at position 341 increases the affinity for Meta II,
we substituted a Leu for Lys at position 341 in the
Ac-Gt
Similarly, to characterize the hydrophobic site at Cys-347 in the
context of the high affinity Ac-peptide 23 peptide sequence, which also has a Leu at position 341, we introduced substitutions (Ac-peptide 23-C347Abu, Ac-peptide 23-C347M,
Ac-peptide 23-C347S). Table III indicates that the Cys-347
side chain can be substituted with any of those residues, confirming
that it interacts in a hydrophobic manner with the receptor. However,
the changes did cause a decrease in the potency (11.0 µM
EC50 for Ac-peptide 23-C347M, 2.0 µM EC50 for Ac-peptide 23-C347V,
and 10.6 µM EC50 for Ac-peptide 23-C347Abu compared with 0.5 µM EC50 for
Ac-peptide 23). Therefore, even though these peptides were
all more potent than the native peptide at binding Meta II, changes at
the critical 347 position diminished the affinity of the
Ac-peptide 23 analogs for the receptor. Thus, the detailed
fit of many residues within the peptide binding site on rhodopsin
appear to be critical for stabilization of Meta II.
Our results corroborate that the Lys at position 341 decreases the
affinity of native Gt The Role of Random Linker in the Peptide Stabilization of Meta
II--
To examine the effect of the random 4-amino acid linker
present on the high affinity peptide analogs identified in the
combinatorial screening, we synthesized and tested the full-length
15-mer peptides for clones 23 and 24 (peptides 23 and 24). The 15-mer
of peptide 24 long was found to be 15-fold more potent than the
corresponding 11-mer peptide (Fig. 2, Table
IV), whereas the difference is only 1.5-fold for the 15-mer and 11-mer of peptide 23 (Table IV). Thus, although no obvious consensus sequences were present in the 4-amino acid linker region of the sequenced clones (54), the region may
contribute to the affinity for light-activated rhodopsin in some
cases.
The results presented here confirm that the C terminus of
G-(340-350), has been
shown to both bind and stabilize the Meta II conformation, mimicking
heterotrimeric Gt. Using a combinatorial library we
identified analogs of Gt
-(340-350) that bound light-activated rhodopsin with high affinity (Martin, E. L.,
Rens-Domiano, S., Schatz, P. J., and Hamm, H. E. (1996)
J. Biol. Chem. 271, 361-366). We have made peptides
with key substitutions either on the background of the native
Gt
-(340-350) sequence or on the high affinity sequences
and used the stabilization of Meta II as a tool to determine which
amino acids are critical in G protein-rhodopsin interaction. Removal of
the positive charge at the N termini by acylation or delocalization of
the charge by K to R substitution enhances the affinity of the
Gt
-(340-350) peptides for Meta II, whereas a decrease
was observed following C-terminal amidation. Cys-347, a residue
conserved in pertussis toxin-sensitive G proteins, was shown to
interact with a hydrophobic site in Meta II. These studies provide
further insight into the mechanism of interaction between the
Gt
C terminus and light-activated rhodopsin.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
that are involved in receptor recognition,
GTP binding and hydrolysis, guanine nucleotide-induced conformational changes, and effector interaction have been elucidated using diverse studies including disruption by ADP ribosylation (8), binding of
antibodies (9-11), proteolytic mapping (12), alanine scanning (13,
14), peptide or minigene studies (15-17), and studies of
chimeric G
proteins (18-21). Researchers have
determined that the N terminus, C terminus, and parts of the
5 helix
of G
are important sites for receptor recognition.
(30, 31) and
subunit (32-34) of heterotrimeric Gt. On the
subunit of Gt, multiple sites of contact have been identified (reviewed in Ref. 35). Three regions, the N-terminal 23 residues, an internal sequence (Gt-(311-323)), and the
C-terminal 11 amino acids, were identified by peptide competition (31). Using chimeric proteins, the C terminus and residues 299-314 of Gt were shown to contribute to rhodopsin binding (36-38).
Of these sites of interaction, the C terminus of G protein
subunits
has been the most extensively investigated. Rhodopsin-Gt
interaction can be disrupted by a number of treatments that block the C
terminus of Gt
, including pertussis toxin-catalyzed ADP
ribosylation (8) and binding by an antibody (39). The C-terminal
peptide Gt
-(340-350) has been shown to directly bind to
and stabilize Meta II (31), mimicking the entire holo-G protein.
Furthermore, selective mutagenesis of this C-terminal Gt
region leads to alterations in G protein function (14, 40, 41).
(42-47) and
(48) subunits as well as heterotrimeric
G
complexes (49, 50).
However, in many of the crystal structures the final residues of the
G
C terminus are disordered and not visible. NMR studies
indicate that the C-terminal 11-amino acid peptide of Gt
has no structure in solution, but it takes on significant structure
when it is bound to either excited (light activated) or unexcited
rhodopsin (51-53), suggesting a direct physical interaction between
the C-terminal residues of Gt
and rhodopsin. However,
detailed structures of the Gt
peptide-receptor or
Gt
peptide-R* complexes are still uncertain because of methodological
limitations.2
-(340-350) that bound to light-activated rhodopsin.
Sequences derived from panning the biased library demonstrated the
presence of certain positions in which amino acids were absolutely
conserved (Cys-347 and Gly-348). Based on work by Dratz et
al. (51), as well as peptide substitutions, these residues are
predicted to be part of a type II'
turn, which is thought to be
required for establishing Meta II stabilization. Also highlighted by
the combinatorial library screening procedure was that the Lys-341 in
the native sequence was selected against. In nearly 70% of the
sequences obtained from the panning, and in all of the highest affinity
sequences, Lys-341 was changed to a noncharged group (54). It was
hypothesized that a positive charge at position 341 in Gt
might decrease its affinity for rhodopsin, leading to a faster
"off" rate and a higher rate of G protein activation. Given that
other G proteins do not have a positive charge at this position, the
presence of a charged residue in Gt has implications for
the evolved properties of the signaling mechanisms.
3 and
4 positions, respectively, and the loss
of a positively charged residue at the
10 position in the high
affinity analogs from the combinatorial library. The structural
features predicted by NMR were also further explored with analog
peptide studies of the functional interaction of these peptides with
activated rhodopsin leading to Meta II stabilization.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
-(340-350) analogs were
synthesized by the solid-phase Merrifield method using Fmoc chemistry
on Milligen 9050 or Applied Biosystems peptide synthesizers. Peptides
were purified by reverse-phase HPLC using C4 or C18 preparative columns
and an acetonitrile, 6 mM HCl, water gradient. The
purified peptides were subjected to fast-atom bombardment or
electrospray mass spectrometry to determine authenticity and analytical
reverse-phase HPLC to determine purity. Fmoc-2-amino butyric
acid was purchased from Advanced ChemTech (Louisville, KY).
-(340-350) analogs or heterotrimeric Gt using an SLM Aminco DW2000
spectrophotometer as described previously (51). Samples were mixed in
Meta II buffer (50 mM HEPES, pH 8.2, 100 mM
NaC1, 1 mM dithiothreitol, 1 mM
MgC12). The samples were maintained at 5.4 °C using a
water-jacketed cuvette holder and refrigerated circulator. After
a dark-adapted spectrum was measured, a flash of light bleaching 10%
of the rhodopsin was presented, and after a 1-min incubation a second
spectrum was measured. The difference between the two spectra was then calculated. Extra Meta II was calculated as the difference
between the absorbance at 390 nm and that at 440 nm. Measurements were done in duplicate for each individual experiment and calculated as a
percentage of the extra Meta II produced by 2 µM
heterotrimeric G protein measured on the same day. The average ± the standard error of the mean were calculated using GraphPad PRISM
(Version 3.0). Dose-response curves for the
Gt
-(340-350) peptide analogs or heterotrimeric
Gt were generated by nonlinear regression using the
following sigmoidal dose-response (variable slope) equation, also known
as a four-parameter logistic equation, to obtain the EC50
values and slope (Hillslope).
Bottom is the Y value at the bottom
plateau, which was set to 0, and Top is the Y
value at the top plateau. LogEC50 is the logarithm of the
EC50, the concentration that gives a response halfway
between Bottom and Top. The variable
Hillslope controls the slope of the curve such that (i) when
Hillslope equals 1, the equation generates a standard
dose-response curve, (ii) when Hillslope is less than 1.0, the curve is more shallow, and (iii) when Hillslope is more
than 1.0, the curve is steeper.
(Eq. 1)
-(340-350) analog
peptides. The spectra were measured in the dark, then completely
bleached in room light. Spectra for the bleached samples were measured at 30-min intervals over a 6-h time period. Finally, 260 mM
HCl was added to protonate the retinal Schiff base in rhodopsin and leave free retinal unprotonated.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
C
terminus are disordered and not visible. However, in one of the crystal
structures of Gt
·GTP
S (45) the ordered C-terminal residues 343-349 form an
-helix and make van der Waals contacts with residues of the
2/
4 loop. It is not clear whether this is a
crystal-packing artifact or an indication of a preferred conformation
of the C terminus. Additionally, for both Gs
(47) and
the Gi
·RGS4 complex (57), the C terminus of G
is an extension of the
5 helix. In the
Gi
·RGS4 complex (57) the extended
5 helix was
stabilized by the N-terminal helix as well as by crystal contacts,
whereas for Gs
(47) the extended
5 helix is in close
proximity with the
4-
6 loop. Sunahara et al.
(47) suggest that the divergence of the G
C-terminal
structures may contribute to receptor selectivity.
has little or no structure in solution, but it takes on significant structure when it is bound to rhodopsin (51-53), indicating a direct physical interaction between the C terminus of
Gt
and its receptor. However, detailed structures of the Gt
peptide-receptor or Gt
peptide-R*
complexes are still uncertain because of methodological
limitations.2 It has not been possible to refine the
published bound peptide structures (51, 52) by comparing experimental
NMR data to that calculated from the bound structures, indicating that
there are significant errors in the NMR distance constraints. In
addition, many of the predictions for amino acid substitutions that
would be tolerated or favorable for the Kisselev et al. (53)
proposed R*-bound structure were not born out by experiment (58). When more accurate NMR data are obtained, it may be possible to refine the
protein-bound peptide structures to within the uncertainty in 1.6-Å
resolution x-ray structures.2
using a
"peptides-on-plasmids" combinatorial technique (59) in which a
library with greater than 109 different peptide sequences
was tested for binding to light-activated rhodopsin (54). We have now
expanded on observations from the combinatorial screening by making
specific point mutations within the C-terminal peptide sequence to
clarify how the C terminus of Gt
interacts with and
stabilizes the activated rhodopsin species Meta II.
-(340-350) C-terminal
peptide analogs to stabilize Meta II in the same manner as
Gt to investigate the interface between G proteins and
their agonist-activated receptors.
-(340-350) peptide is of
relatively low potency in its ability to
interact with and stabilize Meta II, with an EC50 of 1209 µM (Fig. 1, Table I). Two
similar peptide analogs were found to have increased potencies at
stabilizing Meta II; Gt
-(340-350)-K341R, with one fewer
amino group, displayed an EC50 of 180 µM, and
Ac-Gt
-(340-350)-K341R, with two fewer amino
groups, had an EC50 of 163 µM. The potencies
of Gt
-(340-350)-K341R and
Ac-Gt
-(340-350)-K341R are 6.7- and 7.4-fold
higher, respectively, than that of native Gt
-340-350
peptide (Table I). Substitution of the second Lys at position 345 with
Arg (Ac-Gt
-(340-350)-K341R-K345R) decreased
the EC50 even further to 35 µM, resulting in
a 35-fold increase in potency as compared with the native
Gt
-(340-350) peptide (Fig. 1, Table I).
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Fig. 1.
The role of the N and C termini in
Meta II stabilization by
Ac-Gt -(340-350)
analogs. Dose-response curves of Meta II stabilization by analogs
of Ac-Gt
-(340-350). Washed rod outer
segments membranes (5 µM rhodopsin) were incubated in
Meta II buffer (50 mM HEPES, pH 8.2, 100 mM
NaCl, 1 mM MgCl2, 1 mM
dithiothreitol) at 5.3 °C, and extra Meta II was measured as
described under "Materials and Methods." Maximal Meta II
stabilization (100%) was considered to be the amount of Meta II
stabilized by a saturating amount of heterotrimeric Gt (2 µM). The dose-response curves are presented for
heterotrimeric Gt (
),
Ac-Gt
-(340-350)-K341R-K345R (
),
Ac-Gt
-(340-350) (
),
Gt
-(340-350) (
), and
Ac-Gt
-(340-350)-K341R-amide (
).
Data presented are the average of at least three independent
experiments ± the standard error of the mean.
Role of the charge at the N and C termini in Meta II stabilization by
Gt-(340-350) analogs
analogs to stabilize Meta II was measured as described under
"Materials and Methods." Bold letters indicate amino acid residues
that differ relative to the native Gt
-(340-350) sequence.
Dose-response curves were analyzed by non-linear regression using a
sigmoidal dose-response variable slope equation (GraphPad Prism) to
obtain the EC50 values, S.E., and slope. The number of
independent experiments, done in duplicate, is listed as n.
The EC50 obtained for heterotrimeric G protein was 0.28 ± 1.12 µM, with a slope of 1.19 (n = 8).
-(340-350)-K341R as compared with the native
Gt
-(340-350) peptide is associated with a slower rate
of Meta II decay in the presence of a Lys to Arg change at position 341 (51). It was hypothesized that the N terminus of the native
peptide can attack the retinal Schiff's base linkage of Meta II
nonspecifically (51). Thus, we compared the rate of Meta II
decay in the presence of various peptide analogs to ascertain whether
the increase in potency observed might be associated with changes in
the rate of Meta II decay. The half-life for Meta II in the presence of
2 mM Ac-Gt
-(340-350)-K341R was
654 min versus a half-life of 702 min in the presence of 2 mM Ac-Gt
-(340-350)-K341R-K345R,
indicating that there was little difference in their rate of Meta II
decay over a 6-h period (data not shown). This is different
from the results obtained for unacetylated
Gt
-(340-350)-K341R and Gt
-(340-350) peptides, which attack the retinal Schiff's base and result in a much
shorter half-life for Meta II when compared with
Ac-Gt
-(340-350)-K341R (51). Thus, the
increased potency for the peptide in which both Lys residues have been
changed to Arg (Ac-Gt
-(340-350)-K341R-K345R) appears to be due to an increase in affinity for Meta II rather than an
increased stability of Meta II.
-(340-350) peptide analogs for their interaction with
Meta II.
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Fig. 2.
The role of N termini and random
linker sequence in peptide 24-mediated Meta II stabilization.
Dose-response curves of Meta II stabilization by the 11-mer and 15-mer
high affinity analogs of peptide 24. The resulting
dose-response curves are presented for heterotrimeric Gt
( ), peptide 24 (
), Ac-peptide 24 (
),
Ac-peptide 24 long (
), and Ac-peptide
24-K342R-K345R (
). Data presented are the average of at least three
independent experiments ± the standard error of the mean.
peptide analogs bound to rhodopsin indicate substantial structural
changes upon light excitation, suggesting that the C-terminal carboxyl
group shifts its orientation upon interaction with the activated
receptor (51-53). We tested the importance of the free carboxyl group
of Gt
in maintaining Meta II stabilization by amidating
the Ac-Gt
-(340-350)-K341R peptide to
neutralize the C-terminal negative charge. This resulted in a peptide
(Ac-Gt
-(340-350)-K341R-Amide) that
stabilized Meta II but was 2-fold less potent than the same peptide
with a free C terminus (Fig. 1, Table I). Screening of the peptide library showed that the C-terminal final seven amino acids were the
most invariant, with the fourth round clones having identities at these
positions ranging from 72 to 100% (54). These are the same residues
that are disordered in most of the crystal structures. It is reasonable
to speculate that the C terminus of Gt
directly contacts
the activated receptor, and our data supports the idea that the
negative charge at the C terminus may participate in this interaction.
has long
been known to play a critical role in the interaction between
Gt
and rhodopsin. It is the site of pertussis
toxin-catalyzed ADP ribosylation, which leads to an uncoupling of
G
i family G proteins from their cognate
receptors (8). That Cys-347 and Gly-348 are critical residues in
rhodopsin binding was apparent from the combinatorial library study
(54), in which all of the Meta II-binding peptide analogs sequenced
from the fourth round were conserved at these positions. This
phenomenon suggests that other amino acids could not substitute
effectively for the Cys-347 and Gly-348 residues when the peptides were
being selected for binding to activated rhodopsin. To test this idea,
substitutions were made at positions 347 and 348, and the peptides were
then tested for their ability to stabilize Meta II.
-(340-350)-K341R-C347A. This analog
showed a substantial decrease in its ability to stabilize Meta II. Meta
II could be stabilized maximally to 77% of control by 1.8 mM
Ac-Gt
-(340-350)-K341R-C347A peptide with an EC50 of 579 µM (Fig. 3, Table
II). This loss of Meta II stabilization
is similar to results obtained by Osawa and Weiss (41), who showed that
the same mutation in the whole G
resulted in a
loss of rhodopsin binding to recombinant mutant Gt.
However, Garcia et al. (40) have reported that in their
recombinant assay system, this same mutation had no deleterious effect
on rhodopsin binding and rhodopsin-stimulated G protein activation.
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Fig. 3.
The role of Cys-347 in Meta II
stabilization by Gt -(340-350)
analogs. Dose-response curves of Meta II stabilization by
Gt (
), Ac-Gt
-(340-350)-K341R
(
), Ac-Gt
-(340-350)-C347Abu (
),
Ac-Gt
-(340-350)-K341R-C347A (
),
Ac-Gt
-(340-350)-K341R-C347D (
), and
Ac-Gt
-(340-350)-K341R-C347S (
) are
presented. Data presented are the average of at least four independent
experiments ± the standard error of the mean.
Role of Cys-347 and Gly-348 in Meta II stabilization by
Gt-(340-350) analogs
analogs to stabilize Meta II was measured as described under
"Materials and Methods." Bold letters indicate amino acid residues
that differ relative to the native Gt
-(340-350) sequence.
The
symbol represents 2-aminobutyric acid (Abu). Dose-response
curves were analyzed by non-linear regression using a sigmoidal
dose-response variable slope equation (GraphPad Prism) to obtain the
EC50 values, S.E., and slope. The number of independent
experiments, done in duplicate, is listed as n.
-(340-350)-K341R-C347S, because Ser has
a size similar to the sulfhydryl moiety. To ascertain whether the
anionic form of the sulfhydryl moiety is important in the
binding pocket environment, a Cys to Asp analog,
Ac-Gt
-(340-350)-K341R-C347D, was synthesized. At 1.8 mM the
Ac-Gt
-(340-350)-K341R-C347S peptide
stabilized only 40% of Meta II, and the
Ac-Gt
-(340-350)-K341R-C347D peptide
stabilized only 25% of Meta II, as compared with the control Ac-Gt
-(340-350)-K341R peptide (Fig. 3). The
EC50 values obtained for both peptides
(Ac-Gt
-(340-350)-K341R-C347S and
Ac-Gt
-(340-350)-K341R-C347D) were greater
than 1 mM (Table II). To determine whether it is the
hydrophobicity of Cys that is important, we substituted Cys-347 with
2-aminobutyric acid (Abu), a compound that replaces the sulfhydryl group of the cysteine with a methyl group and approximately mimics cysteine in both size and hydrophobicity. This -SH to -CH3
peptide (Ac-Gt
-(340-350)-K341R-C347Abu) was able to
stabilize Meta II with essentially the same potency as the
Ac-Gt
-(340-350)-K341R parent peptide, with
an EC50 of 127 µM versus 163 µM (Fig. 3, Table II). Therefore, the data suggest that
the Cys-347 side chain of Gt
interacts in a hydrophobic
manner with Meta II.
-turn between Cys-347 and Phe-350 with Gly-348 in the n + 1 position
in the dark-bound conformation (51) and the light-bound conformation
(52, 53). Consistent with this observation, peptides in which Gly was
substituted with L-Leu, which would be predicted to break
the
-turn, lost functional activity (51). Meanwhile, D-Ala, predicted to maintain the peptide structure, was
almost as potent as the parent peptide (51). Using molecular dynamics calculations to estimate the relative energy to form the
turn, with Gly being taken as 0 kcal mol
1, we found
that the energy required would be 0 kcal mol
1
for a Gly at position 348, 3.2 kcal mol
1 for
D-Ala, 12 kcal mol
1 for
L-Ala, and 88.2 kcal mol
1 for
L-Leu. We therefore made a synthetic peptide with a Gly to Ala substitution
(Ac-Gt
-(340-350)-K341R-G348A). The
L-Ala substitution at this position is much milder than
that of L-Leu, and it was of interest to see whether this
analog could stabilize Meta II. As predicted by the energy calculation,
the peptide with the Gly to Ala substitution at position 348 was only
minimally capable of stabilizing Meta II, with an EC50
value of nearly 1 mM and maximal stabilization of 15% at
1.8 mM (Fig. 4, Table II). Thus, the Gly at position 348 appears to be essential for the ability
of the C-terminal peptide to effectively stabilize Meta II. This result
is consistent with the presence of a type II'
-turn at the C
terminus of the peptide. Alternatively, the binding pocket on rhodopsin
may not be able to tolerate bulky side chains at position 348. This
alternative interpretation is not as likely, because substitution of
the Gly at this position with D-Ala is equipotent at
stabilizing Meta II.
View larger version (13K):
[in a new window]
Fig. 4.
The role of Gly-348 in Meta II
stabilization by Gt -(340-350)
analogs. Dose-response curves of Meta II stabilization by
Gt (
), Ac-Gt
-(340-350)-K341R
(
), and Ac-Gt
-(340-350)-K341R-G348A (
)
are presented. Data presented are the average of at least three
independent experiments ± the standard error of the mean.
-(340-350)-K341R-C347Abu peptide and
tested its ability to stabilize Meta II (Fig.
5). We found that these changes
substantially enhanced the EC50 such that the
EC50 of
Ac-Gt
-(340-350)-K341R-C347Abu is 127 µM, whereas that of
Ac-Gt
-(340-350)-K341L-C347Abu is 44 µM. When peptides were tested with the K341L substitution
that had also been substituted with a hydrophic residue at position 347 (Met or Val), we found EC50 values of 19.6 µM
for Ac-Gt
-(340-350)-K341L-C347M and 43.6 µM for
Ac-Gt
-(340-350)-K341L-C347V (Table
III). These values were 4-8-fold more
potent than the native acetylated peptide, indicating the importance of
a hydrophobic residue at position 341.
View larger version (18K):
[in a new window]
Fig. 5.
The role of Lys-341 in Meta II
stabilization by Gt -(340-350)
analogs. Dose response of Meta II stabilization by Gt
(
), Ac-Gt
-(340-350)-K341R (
),
Ac-Gt
-(340-350)-K341L-C347M (
),
Ac-Gt
-(340-350)-K341L-C347V (
), and
Ac-Gt
-(340-350)-K341L-C347Abu (
). Data
presented are the average of at least three independent
experiments ± the standard error of the mean, except for
Ac-Gt
-(340-350)-K341L-C347V, which was only
tested twice.
Role of Lys-341 in Meta II stabilization by Gt-(340-350)
analogs
-(340-350) sequence. The
symbol represents 2-aminobutyric acid (Abu). Dose-response curves were
analyzed by non-linear regression using a sigmoidal dose-response
variable slope equation (GraphPad Prism) to obtain the EC50
values, S.E., and slope. The number of independent experiments, done in
duplicate, is listed as n.
for rhodopsin. One might wonder why such a disruptive residue at the site of receptor interaction would
be present. Perhaps it is important for the G protein to have a lower
affinity for the receptor so that the receptor can be a more efficient
catalyst and thus more rapidly catalyze activation of more G proteins
following ligand activation. We are currently testing this idea in
full-length G
by making a K341L point mutation.
We predict that a G protein with this single mutation will have a
higher affinity for rhodopsin.
Role of the random linker sequences in Meta II stabilization by
Gt-(340-350) analogs
-(340-350) sequence. Dose-response
curves were analyzed by non-linear regression using a sigmoidal
dose-response variable slope equation (GraphPad Prism) to obtain the
EC50 values, S.E., and slope. The number of independent
experiments, done in duplicate, is listed as n.
is critical in stabilizing the active conformation of
the receptor. Understanding the changes that can be tolerated in this region is essential to furthering our knowledge of how G
protein-coupled receptors interact with their cognate G proteins.
Previous work from our laboratory indicates that C-terminal
G
peptides can compete with G protein for binding
receptor and therefore potently block signal transduction (16). By
screening a receptor of interest, it should be possible to select for
peptide sequences with high affinity for the receptor that will
competitively inhibit receptor-G protein interaction and potently block
signal transduction through that receptor. Peptide sequences so
obtained can then be expressed in cells as minigenes (17, 65),
providing a facile approach for studying G protein signaling.
![]() |
FOOTNOTES |
---|
* This work was supported by Grants EY06062 and EY10291 (to H. E. H.) and EY06913 (to E. A. D.) from the National Institutes of Health, a Distinguished Investigator Award from the National Alliance for Research on Schizophrenia and Depression (to H. E. H.), and Postdoctoral Training Grant HL07829 (to A. G.) from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Vanderbilt University School of Medicine, Dept. of
Pharmacology, 942 Robinson Research Bldg., Nashville, TN 37232-6600. Tel.: 615-343-3533; Fax: 615-343-1084; E-mail:
heidi.hamm@mcmail.vanderbilt.edu.
Published, JBC Papers in Press, October 3, 2000, DOI 10.1074/jbc.M002533200
2 E. A. Dratz and D. Gizachew, submitted for publication.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
G protein, guanine nucleotide-binding protein;
Gt, transducin;
Meta
II, metarhodopsin II;
R*, activated receptor;
Fmoc, N-(9-fluorenyl)methoxycarbonyl;
HPLC, high pressure liquid
chromatography;
Ac, acetylated;
GTPS, guanosine
5'-O-(thiotriphosphate).
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