(Received for publication, October 27, 1995; and in revised form, November 10, 1995)
From the
The C terminus of the G protein subunit represents an
important site of interaction between heterotrimeric G proteins and
their cognate receptors. We have screened a combinatorial peptide
library based on the C terminus of the
subunit of
G
(340-350) and have identified unique sequences that
bind rhodopsin with high affinity. Six of these sequences, as both
fusion proteins and synthetic peptides, were significantly more potent
than the parent sequence in binding to and stabilization of
metarhodopsin II. These sequences provide information about which
residues are required for appropriate receptor interaction. We observed
that in all the high affinity sequences, a positively charged residue
at position 341 was changed to a neutral one. Thus, it appears that the
receptor-G protein interaction was designed to be low affinity to
ensure efficient catalysis of G protein activation. We also observed
Cys-347 and Gly-348 to be invariant, and hydrophobic residues were
always located at positions 340, 344, 349, and 350, demonstrating the
critical nature of these residues. A composite of the structures of the
high affinity sequences was modeled based upon the structure of
rhodopsin-bound trNOESY NMR of this region of G
(
Dratz, E. D., Fursteneau, J. E., Lambert, C. G., Thireault, D. L.,
Rarick, H., Schepers, T., Pakhlevaniants, S., and Hamm, H. E.(1993) Nature 363, 276-280) and provides insight into the
complementary G protein-binding surface of the receptor.
Understanding the structural basis of receptor-G protein ()interaction is essential to defining a molecular mechanism
of signal transduction. Activated receptors display a high affinity
binding site for heterotrimeric G proteins, which, depending on the
receptor type, appears to consist of one or more of the cytoplasmic
regions of the receptor. Interestingly, however, comparison of the
primary sequences of receptors that are known to interact with the same
G protein has not revealed the underlying consensus sequence. High
affinity interaction of heterotrimeric G proteins with activated
receptors requires the presence of both the G protein
and
/
subunits. Three regions on the
subunit are known to
be important for receptor interaction, the N-terminal 23 residues, an
internal sequence that includes the TCAT region which contacts the
guanine ring, and the C terminus(1) . The extreme C terminus of
G protein
subunits is one of the critical points of contact with
activated
receptors(1, 2, 3, 4, 5, 6, 7) .
This C-terminal region of G protein
subunits is also important in
determining the specificity of receptor-G protein
interaction(9) , although other regions of
subunits are
also involved(10) . Synthetic peptides corresponding to the
last 11 amino acids of
subunits have been shown to mimic the
conformational effect of heterotrimeric G proteins on receptors by
stabilizing the active, agonist-bound form of the receptor, although
with low potency(1, 7, 8) . Additionally,
C-terminal peptides from G
subunits also can
competitively block receptor-G protein
interactions(1, 8) .
The advent of random peptide
libraries displayed on bacteriophage (11, 12, 13) or bacterial plasmids (14) has brought the screening power of very large
combinatorial libraries to the search for potent peptide agonists and
antagonists, as well as to provide detailed structural analysis of the
requirements for peptide-receptor interactions. In this study, we have
used a random ``peptides-on-plasmids'' library approach (14) to identify potent rhodopsin-binding peptide sequences
related to a C-terminal G peptide,
G
-340-350 (1, 7) from a library
of greater than 10
peptides. Using this method, we have
developed several high affinity analogues of
G
-340-350 and have gained insight into the
structural requirements for peptide binding and stabilization of the
activated conformation of rhodopsin, metarhodopsin II (Meta II).
Some
of the MBP fusion proteins were further purified on an amylose column
(New England Biolabs) and eluted with 10 mM maltose as
described by the supplier. The dose response for the ability of each of
these MBP fusion proteins to compete with the
LacI-G-K341R-340-350 fusion protein for binding
to light-activated rhodopsin was measured in an ELISA
format(15) . Microtiter wells were coated with 0.5 µg of
ROS/well and blocked with BSA in the dark. Serial dilutions of purified
MBP fusion proteins were made in HEK/DTT/BSA buffer. After light
activation of the rhodopsin, each dilution was added to duplicate wells
and then incubated for 45 min at 4 °C. The LacI fusion protein
containing G
-K341R-340-350 was then added. The
incubation was continued for 30 min to allow the competition of the two
protein fusions to take place, and the amount of bound LacI was
detected by an anti-LacI antibody(15) . Dose-response curves
were generated, and IC
values were calculated using the
computer programs Ligand and Deltagraph Pro 3.5.
Figure 1:
A, ELISA of
selected MBP fusion proteins binding to activated rhodopsin. In the
dark, 1 µg/well ROS membranes were directly immobilized on Immulon
4 microtiter wells in cold HEK/DTT. The bound ROS was activated and
exposed by light for less than 5 min on ice, and the fusion proteins
were added for 30 min. Subsequent primary and secondary antibody
incubations were performed as described for Table 1. The positive
control for the assay was pELM17, which encodes the MBP fusion protein
of G-340-350 peptide analog (K341R). pELM6,
which expresses a MBP protein fused to a linker only, was used as the
negative control. ``No lysate'' control wells were included
to reflect any intrinsic nonspecific binding within the assay. B, competition of MBP fusion protein binding to activated
rhodopsin by G
. 1 µg/well ROS was directly immobilized
in microtiter wells and 1 µM G
was allowed to
bind to rhodopsin for 30 min at 4 °C before purified MBP fusion
proteins were added in equal volume and incubated for an additional 30
min. The samples were then analyzed by ELISA as described in A.
Figure 2:
A, comparison of the sequences of the
C-terminal 11 amino acids of the known G protein subunits. Darkly shaded amino acids are those conserved throughout G
protein
-subunits, and lightly shaded residues are those
which are similar (homologous). B, comparison of the sequences
of the peptides identified from the fourth and second rounds of panning
of the combinatorial library. Darkly shaded amino acids are
those that remain identical to native G
, and lightly shaded ones are those that have undergone replacement
with similar amino acids. The fourth round was screened using an MBP
ELISA and the second round screened using a LacI ELISA. C,
percent identity and similarity (homology) of the library-derived
sequences from the second and fourth rounds of panning. Based on the
method for generating the oligonucleotides, 50% of the time the
naturally occurring amino acid will be
encoded.
Sequences derived from the fourth round of panning suggest
that 2 of the 11 residues, Cys-347 and Gly-348, are absolutely required
for binding to rhodopsin (Fig. 2). These two amino acids are
conserved among all of the members of the G, G
,
and G
G protein subfamilies (Fig. 2A), and
Cys-347 is the site of pertussis toxin modification in this
subfamily(5) . These 2 residues are also part of a type II`
turn, which is required for Meta II stabilization(7) .
Other residues conserved within the G
/G
subfamily could be replaced only with amino acids having similar
properties. Ile-340 is strictly conserved in this subfamily, and
throughout G proteins it is generally only replaced with Leu (Fig. 2). Even among the most potent of the peptides (see below)
Val, Leu, and Met substitution occurred, suggesting that several
hydrophobic residues are acceptable. The large hydrophobic residues Phe
or Tyr are found at the final position of G
/G
subunits. Phenylalanine occurred almost exclusively in our
study. The one exception, found in two of the sequences, was the much
smaller hydrophobic residue, Leu. This did not appear to be
detrimental, since it appeared in one of the more potent peptides, 9.
Several other amino acids in the C-terminal 11 residues are
generally conserved throughout known G protein subunits, although
G
and G
are more heterogeneous
than the others (Fig. 2A). Two Leu residues, Leu-344
and Leu-349, are absolutely conserved in all G proteins, yet in our
peptides conservative substitutions were possible. Among the high
affinity peptides, Leu-344 was always present, but Leu-349 was switched
to Met in sequences 8 and 24. The conservation of hydrophobic residues
at certain positions (340, 344, 349, and 350) suggests a hydrophobic
component to the conserved binding pocket on the receptor. This, in
fact, has been suggested by mutagenesis studies on muscarinic (18, 19) and
-adrenergic (20) receptors.
C-terminal
peptides of G subunits cannot only bind to their
cognate receptors but can mimic the heterotrimeric G proteins'
ability to affect receptor conformation and stabilize the active
state(1, 7, 8) . The fusion protein,
MBP-G
-K341R-340-350, was able to stabilize Meta
II with an EC
of greater than 100 µM. At
similar concentrations, the MBP protein alone (encoded by plasmid
pELM6) had no effect. The high affinity fusion proteins derived from
the combinatorial library were significantly more potent than
MBP-G
-K341R-340-350 in their ability to
stabilize Meta II (Fig. 3A). Their EC
values ranged from 0.49 to 5.4 µM (Table 1).
Figure 3:
A,
Meta II stabilization by MBP fusion proteins. Dose response of Meta II
stabilization by representative high affinity MBP-peptide fusion
proteins, G and G
-Ac-K341R-340-350.
EDTA-washed rhodopsin (5 µM) was incubated in a 50 mM Hepes buffer, pH 8.2, containing 100 mM NaCl, 1
mM MgCl
, and 1 mM DTT 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 G
(5 µM). The resulting
dose-response curves are presented for G
(
),
G
-K341R-340-350 (
), MBP-8 (
),
MBP-23 (
), and MBP-24 (
). B, Meta II stabilization
by peptides. Dose-response curves for Meta II stabilization by G
(
), G
-K341R-340-350 (
), and
peptides 8S (
), 23S (
), and 24S (
) are
presented.
To examine the sequences directly, in the absence of the 43-kDa MBP
fusion partner, we chemically synthesized the six 11-mer peptides
(denoted ``S'') and compared them with the
G-K341R-340-350 11-mer peptide. Dose-response
studies in the competition ELISA revealed that the 11-mer peptides
displayed higher binding affinity than the parent peptide by 2- (24S)
to 300-fold (23S) (Table 1). The synthetic peptides were then
tested for their ability to stabilize Meta II. The newly identified
peptides were all significantly more potent than
G
-K341R-340-350 in their ability to stabilize
Meta II (Fig. 3B). Their EC
values ranged
from 0.76 (23S) to 152 (24S) µM, which is up to 290 times
higher affinity than the parent sequence (Table 1). Additionally,
these peptide sequences also appear to be potent antagonists of
receptor activation of G proteins, since the peptides competitively
inhibit the ability of light-activated rhodopsin to stimulate GTP
S
binding to G
in a dose-dependent fashion. (
)
It is interesting to note that the relative potency of
the 24S peptide decreased substantially in the absence of the fusion
protein. In general, the MBP fusion proteins all appear to be of higher
affinity than the peptides in both the binding and Meta II assays. This
is probably due to the constraining effect on the N terminus of the
peptide, which would lead to a decrease in conformational freedom. This
difference between the fusion proteins and peptides was not as great in
the Meta II assay, probably due to the higher sensitivity of the ELISA
assay. ``Extra'' Meta II is measured as a tiny absorbance
change (0.02 absorbance unit), and to detect such a small change
the concentration of rhodopsin must be high (>0.05 µM),
thus limiting the dynamic range of the assay.
To examine the effect of the added four random N-terminal amino acids, the full-length 15-mer peptides were also synthesized for clones 18 and 23 (peptides 18L and 23L) (Table 1). Although no obvious consensus sequences were present in the four random residues extended upstream from the core library, this region contributes to the increase in affinity in these two peptides. Peptide 18L is approximately 70- and 16-fold more potent than the corresponding 18S 11-mer peptide in the binding and Meta II assays, respectively, whereas this difference is only 3-fold for peptides 23L and 23S.
By surveying a large number of peptide
sequence possibilities unattainable by standard peptide synthetic
techniques, we found several sequences that bind to rhodopsin with
substantially higher affinity than the native sequence. Presumably,
these analogues bind more tightly and dissociate at a slower rate. It
is interesting that the screening procedure selected against a positive
charge at position 341 but not at position 345 (Fig. 2B). In approximately 70% of the high affinity
peptides from panning round 4 and all of the highest affinity peptides
we fully characterized, the Lys-341/Arg-341 was changed to a
non-charged group. This positive to neutral change correlates well with
higher affinity. Such a change could have implications for the evolved
functional properties of signaling pathways. In order to permit rapid
and amplified information transfer, the receptor-G protein interaction
must allow for rapid dissociation of the activated G protein. We
described an activation-dependent conformational change of
G-Ac-K341R-340-350 in its rhodopsin-bound
conformation by trNOESY and speculated that this conformational change
is important for the highly amplified G
activation
process(7) . Especially marked changes occur in a helix-like
tight turn at the N terminus involving Ile-340, Arg-341, Glu-342, and
Asn-343, with formation of a salt bridge between Arg-341 and the free
carboxyl of the C terminus in the inactive receptor-bound form and
relaxation of this interaction in the activated form(7) . The
hypothesis that the presence of a positive charge at position 341 might
decrease the affinity of binding to receptor and increase the
receptor's catalytic rate is directly testable using
site-directed mutagenesis. Interestingly, a mutation that removes this
positive charge (Arg-385 to His) has been reported to functionally
uncouple G
from its receptor in a patient with Albright
hereditary osteodystrophy(21) . It would be of interest to
examine whether this mutation leads to a higher affinity for receptor.
Other G proteins do not have a positive charge at this position (Fig. 2A), and we predict that these G proteins may
have higher receptor affinity and lower activation rates.
The
crystal structure of G has provided much information
on its overall three-dimensional
conformation(22, 23, 24, 25) .
However, in the crystal structure of G
GDP (which
in combination with the
subunit is the rhodopsin binding
conformation) the C-terminal region was disordered and not visible (23) . Only in the GTP
S-bound form of G
could any structural features of the C terminus be
discerned(22) . Three possibilities are that 1) it is
disordered and extended in the GDP-bound conformation in order to
``sense'' the activated receptor and conform to it by induced
fit, 2) the disorder is an artifact of the crystal packing, or 3) it is
disordered because it is missing an organizing site contributed by the
N terminus, which was deleted from the G
used for
crystal formation.
In the absence of good crystallographic data on
the C terminus, our data can be used to suggest an approximation of the
tolerances of rhodopsin's binding site for the
G-340-350 region by utilizing a combination of
information on the peptide side chains that can be accommodated in each
position and the structural information obtained by
trNOESY(7) . We have constructed surface models of the original
peptide and a composite peptide containing the bulkiest side chain at
each position from the six highest affinity peptides using trNOESY data
from (7) . The resulting models show that both the native and
modified peptides have two distinctly different ``faces,''
one polar and the other hydrophobic. Overall, there are only slight
changes in the shape of the hydrophobic side of the two peptides. The
polar face of the modified peptide does, however, appear to be less
densely packed than the native peptide and more negatively charged
because of the loss of the positive charge at position 341 (Fig. 4). The surfaces of these peptides should be complementary
to their binding site on rhodopsin's cytoplasmic surface. The
actual residues that contact rhodopsin are not known, and their
identification would greatly facilitate our understanding of the
critical point-to-point interactions required for receptor-G protein
interaction.
Figure 4:
Solvent-accessible surfaces of original
and high affinity peptides. The -carbon
/
angles of
the original peptide bound to inactive rhodopsin (determined by trNOESY (7) ) were used in the composite peptide, substituting the
bulkiest side chains at positions that tolerate substitution (I340M,
R341L, D346E, and L349M). The peptides have distinctive polar and
hydrophobic surfaces. A and B show the polar surfaces
of G
-Ac-K341R-340-350 and the substituted
peptide, respectively. The models were constructed using INSIGHT and
GRASP (developed by A. Nicholls and B. Honig, Columbia University). The
amino acids are labeled based on their sequence
position(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11) and the
orientation similar to that previously shown(2) . We have not
accounted for the possibility that the lack of a positively charged
side chain at position 341, which interacts with the free C terminus,
may cause global changes in the backbone
structure.
This paper has demonstrated the power of using a
combinatorial library to develop potent peptide analogues useful in
furthering our understanding of rhodopsin-G interactions,
as well as having broader implications in the study of signal
transduction systems. Additionally, this work will lay the structural
framework by which therapeutic agents can be developed to interfere
with signal transduction mediated via G proteins.