From the
Polyclonal anti-idiotypic antibody raised to a synthetic
discontinuous peptide derived from the human
Human
Two small
regions within the cytoplasmic domain have been identified as important
to the function of this receptor (Farrar et al., 1991, 1992;
Cook et al., 1992). The extracellular domain is believed to
interact directly both with ``accessory factors'' required to
generate the full complement of transduction signals (Hibino et
al., 1992; Gibbs et al., 1992) and with ligand (Garotta
et al., 1990). Studies on the extracellular domain by Stuber
et al. (1993) suggest that all of the disulfide bonds may be
required for full ligand binding capacity. Using proteolytic digestion,
Fountoulakis et al. (1991) found that the shortest fragment
capable of retaining full
A neutralizing anti-idiotypic antibody which binds
to the extracellular domain of huIFN-
To further probe the receptor peptide-ligand interactions, we have
examined the rhuIFN-
Overlapping octamer peptides were synthesized on
polyethylene pins in a 96-pin format using the method of Geysen et
al. (1987). The peptides were synthesized using
Fmoc/ t-butyl protecting groups; the amino acids coupled were
highly activated pentafluorophenyl and oxo-benzotriazine esters.
Approximately 20-50 pmol of peptide were synthesized on each pin.
NOESY contour expansions for the free peptide depicting
the amide to aliphatic and amide to amide regions are shown in Figs.
6 A and Fig. 7 A for a mixing time of 100 ms at 15
°C, pH 7.0. Fig. 6 A shows intense sequential
d(
NOESY contour expansions for the
peptide in the presence of rhuIFN-
The TRNOE
data suggest that there is an interaction between the peptide and the
rhuIFN-
Previous studies have suggested that two regions on
huIFN-
The anti-idiotypic antibody has identified a
portion of the receptor (120-141) which resides at the
ligand-receptor-binding site. Most interestingly, peptide
120-141(Tyr)acm is an antagonist of biological activity, having
an IC
The human growth hormone
receptor intermolecular contact regions fall in proximal regions on
either side of a Pro-Pro region described as a hinge between the two
domains and which appears to be common to the hematopoietic growth
factor family (deVos, 1992; Sprang and Bazan, 1993). Interestingly, the
receptor peptide 120-141 consists of residues in the immediate
regions on either side and including this cytokine-conserved sequence
suggested by Bazan (1990) to be the segment connecting the two
extracellular domains. Most of the TRNOEs described here arise from the
IG, DIRK, and EK portions of the molecule. This suggests that these
regions bind most strongly, and that in comparison with the rest of the
molecule, they are the most immobile segments of the peptide when bound
to rhuIFN-
Chemical shifts were referenced internally to
deuterated TSP. Solution conditions were 4.3 m
M peptide in 20
m
M phosphate, pH 7.0, at 15 °C.
We thank Tami Maiore for excellent technical
assistance, Dr. S. Baldwin and S. Paliwal for sequencing and amino
acids analysis, J. Durkin and Drs. R. Zhang and W. Windsor for
synthesis of the 123-138 peptide, R. Syto for the purification of
the rhuIFN-
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-interferon
(huIFN-
) sequence recognizes soluble human
-interferon
receptor (Seelig, G. F., Prosise, W. W., and Taremi, S. S. (1994)
J. Biol. Chem. 269, 358-363). We sought to use this
reagent to identify a ligand-binding domain within IFN-
-receptor.
To do this, the neutralizing anti-idiotypic antibody was used to probe
overlapping linear peptide octamers of the extracellular domain of the
huIFN-
receptor. A 22-amino-acid residue receptor segment
120-141 identified by the antibody was synthesized. CD and NMR
analysis indicates that peptide 120-141 has no apparent secondary
structure in water or in water containing 50% trifluoroethanol. The
synthetic receptor peptide inhibited huIFN-
induced expression of
HLA/DR antigen on Colo 205 cells with an approximate IC
of
35 µ
M. Immobilized peptide specifically bound recombinant
huIFN-
but did not bind human granulocyte-macrophage
colony-stimulating factor on a microtiter plate in a direct binding
enzyme-linked immunosorbent assay. The binding results are supported by
two-dimensional transferred nuclear Over-hauser effect (TRNOE) NMR data
obtained on the peptide in the presence of recombinant huIFN-
.
Characterization of the conformation of the bound peptide by TRNOE
suggests that this peptide assumes a distinct conformation.
Intramolecular interactions within the bound peptide were detected at
two non-contiguous regions and at a third region comprising a
-turn formed by the sequence DIRK. We believe that this represents
the structure of the receptor within the ligand-binding domain.
-interferon huIFN-
(
)
is a
protein which expresses antiviral, antiproliferative, and
immunomodulatory activities (for review see Georgiades et al.,
1984 and Trichieri and Perussia, 1985). The actions of this cytokine
are believed to be mediated through interaction with specific receptors
expressed on the surface of responsive cells (Langer and Pestka, 1988;
Farrar and Schreiber, 1993). The cDNA of the huIFN-
receptor has
been cloned and expressed (Aguet et al., 1988; Gray et
al., 1989). The receptor consists of an extracellular domain (228
amino acids), a 23-amino-acid transmembrane region, and a cytoplasmic
domain (221 amino acids). A report by Fountoulakis et al. (1991) indicates that one huIFN-
receptor binds to one
huIFN-
dimer; however, more recent reports (Greenlund et
al., 1993; Fountoulakis et al., 1992; Dighe et
al., 1993) provide significant data suggesting that two
huIFN-
receptors bind a single dimer of huIFN-
.
-interferon binding was a 25-kDa
fragment including residues 6-227. They suggest that a smaller
15-kDa fragment (residues 94-227) which bound to immobilized
huIFN-
with low affinity carries part of the receptor-binding
domain. Monoclonal antibodies raised against huIFN-
receptor
possessing inhibitory activity have been broadly mapped to amino acid
residues between 26-133 and 70-210 (Garotta et
al., 1990).
receptor has previously been
described (Seelig and Prosise, 1992). This anti-idiotypic antibody was
derived from a neutralizing anti-huIFN-
antibody which had been
raised against a synthetic peptide containing tethered segments from
both the amino terminus and the carboxyl terminus (Seelig and Prosise,
1992; Seelig et al., 1994). In this paper, we use this unique
antibody tool to probe specific regions of the receptor which are
responsible for the ligand-receptor interactions. Using the method
described by Geysen et al. (1987), immobilized overlapping
octamers of the rhuIFN-
receptor were synthesized, peptide regions
were identified, free peptides were synthesized, and analyzed both for
their ability to bind rhuIFN-
and for their antagonist activity.
-bound structure of the peptide using
two-dimensional transferred nuclear Overhauser enhancement spectroscopy
(TRNOE). The TRNOE experiment has been applied to a wide range of small
ligands complexed with macromolecules to gain information about the
structure of the bound ligand (Otting, 1993). This technique is an
extension of the nuclear Overhauser enhancement (NOE) experiment to
systems involved in chemical exchange. It has been described for the
one-dimensional case (Clore and Gronenborn, 1982) and more recently for
the two-dimensional case (Sykes and Campbell, 1991). In the free
ligand, negative NOEs are normally weak or absent, reflective of both
the ligand's small size and short correlation time (Noggle and
Schirmer, 1971). When bound to a large molecule, however, the fast
exchange situation permits transfer of magnetization from the bound
state to the free ligand population. Large negative NOEs will be
observed and are reflective of the ligand's conformation in its
bound state. Significant information can be obtained about the bound
conformation of the ligand and compared with the solution conformation
of the ligand (Neuhaus and Williamson, 1989). The binding of the
120-141 peptide to rhuIFN-
seemed well suited for this type
of study. We discuss the peptides, their respective antibodies, and the
implications from the NMR data in relation to the binding of
huIFN-
to its cellular receptor.
Materials
Kirkegaard and Perry Laboratories,
Inc. (Gaithersburg, MD) and Jackson Immunoresearch Laboratories
(Avondale, PA) supplied horseradish peroxidase-conjugated goat
anti-mouse IgG and goat anti-rabbit IgG, respectively. Cambridge
Research Biochemical (Valley Stream, NY) was the source for the
derivatized pins, control pins, antibodies, and Fmoc-
L-amino
acids used in the Geysen immobilized peptide synthesis. Mouse
monoclonal anti-HLA/DR antibody was from Becton-Dickinson.
Anti-idiotypic antibody was raised in BALB/c mice followed by
intraperitoneal injections with rabbit polyclonal antibody P616 (raised
against peptide 15-21-GGG-132-138) as described before
(Seelig and Prosise, 1992).
Cytokines and Cytokine Receptors
Rh-GM-CSF
( Escherichia coli; nonglycosylated; Schering-Plough/Sandoz)
was purified to a constant maximal specific activity by a methodology
similar to that described for recombinant murine GM-CSF (Trotta et
al., 1987). Expression of rhuIFN- was as described previously
(Lunn et al., 1992) utilizing standard techniques of
recombinant DNA methodology described by Nagabhushan and Leibowitz
(1985). In each case, cloned variants were purified to constant
specific activity by a methodology similar to that detailed by
Nagabhushan et al. (1988). Recombinant murine
-interferon
was purified as described by Trotta et al. (1986).
Peptide Synthesis
Peptides 120-141acm and
120-141(Tyr)acm were synthesized by Multiple Peptide Synthesis
(San Diego, CA) as the HCl salt with the acetamidomethyl modification
of the cysteine side chain. In the case of 120-141(Tyr)acm, a
conservative replacement of the Val-121 in the native sequence for a
Tyr-121 was also made. The presence of the Tyr-121 provides a facile
method both for quantitation and for conjugation as desired. All other
peptides were synthesized using the solid-phase method described by
Merrifield (1963). The t-butyloxy carbonyl amino protecting
group, symmetrical anhydrides, and the solid-phase peptide synthesizer
(model 430A) were from Applied Biosystems (Foster City, CA). Following
the loading of the phenylacetamido (polystyrene) starting resin as well
as the complete assembly, the NH-terminal t-Boc
group was removed with 65% trifluoroacetic acid. The resin was then
neutralized, washed with ethanol, and dried; the peptide was cleaved
from the resin with a 10:1.5 ratio of liquid hydrogen fluoride-anisole
at 0 °C for 60 min. The cleaved, deprotected peptides were
solubilized with aqueous acetic acid, washed with t-butyl
methyl ether, and then lyophilized. All peptides were purified by
reversed-phase high performance liquid chromatography on a C-4 Dynamax
400 Å widebore column (Rainin Associates, Woburn, MA). Amino acid
sequencing by automated Edman degradation was employed to confirm the
peptide sequences. Fast atom bombardment mass spectral analysis was
carried out on a VG ZAB-SE double focusing mass spectrometer operating
at an accelerating voltage of 8 kV. Circular dichroism measurements
were made on an IBM-interfaced Jasco 500C spectropolarimeter at room
temperature using 1.0-cm path length cells with a protein concentration
of 1.0 mg/ml.
Antibody Production
Rabbits were injected
intradermally with 0.1 ml of sample/site of injection. Each sample
consisted of 0.5-1.0 mg of peptide in 500 µl of sterile PBS
emulsified with 500 µl of Freund's complete adjuvant. Boosts
were performed with Freund's incomplete adjuvant at 4-week
intervals or as needed judged by ELISA response to peptide and
huIFN- receptor.
ELISA
Rabbit antibodies were screened for specific
binding of antigens by employing a direct solid-phase ELISA at room
temperature. A 96-well microtiter plate (NUNC; Intermed; Roskilde,
Denmark) was coated with 100 µl of antigen/well for 1 h at room
temperature. The plate was washed five times with Tris-buffered saline
containing 0.05% Tween 20 (TBS). The plate was subsequently blocked
with 1% bovine serum albumin for 1 h and again washed five times with
TBS. The wells were then coated with the antibody of interest for 1 h,
washed five times with TBS, and coated with 2.5 ng of horseradish
peroxidase-conjugated goat anti-rabbit IgG. Following incubation for 1
h, the plate was washed five times with TBS. The plate was then
developed by adding either
2-2`-azino-bis[3-ethyl-benzthiazoline sulfonate] (ABTS)
or 3,3`,5,5`-tetramethyl benzidine (TMB) and hydrogen peroxide to each
well. The horseradish peroxidase reaction product was detected
colorimetrically at 414 nm (ABTS) or at 450 nm (TMB) 20 min after the
addition of the enzyme substrates. Control wells were also developed in
which one of the three assay components (antigen, antibody, or
peroxidase-labeled antibody) was deleted.
Immobilized Peptide Immunosorbent
Assay
Immobilized pins were blocked for 1 h by inverting the
pins onto a standard 96-well microtiter plate and incubating in PBS
containing 1% bovine serum albumin and 1% ovalbumin. The pins were
incubated overnight at 4 °C in the appropriate mouse or rabbit
antisera diluted 1:500 in the above PBS solution, followed by a wash
with PBS containing 0.05% Tween 20. The pins were incubated with the
appropriate horseradish peroxidase-labeled conjugate, washed, and then
developed with the colorimetric detection methods described in the
previous section.
Protein Determination
The method of Lowry (1951)
or Bradford (1976) was employed with bovine serum albumin as a standard
for the proteins and the species appropriate IgG as a standard for the
antibodies.
Assay for HuIFN-
The bio-ELISA assay for HLA/DR induction by huIFN--induced Expression of HLA/DR
Antigen
was carried out according to the method of Gibson et al. (1989). The Colo 205 cell line was obtained from the American Type
Culture Collection (CLL 222). Stock cultures were grown to confluence
in RPMI 1640 containing 10% fetal calf serum. The cells were
trypsinized and seeded in 96-well tissue culture plates at a density of
at least 10
cells/well in 0.1 ml of RPMI 1640 containing
10% fetal calf serum. The cells were incubated overnight in the wells
at 37 °C in a 5% CO
incubator. Culture media in the
presence of polypeptides and a fixed amount of interferon (150
p
M) were added in a 0.1 ml volume to the wells containing Colo
205 cells, and then incubated for 1 h at 37 °C. Following
incubation, the media was removed from each well and the wells were
washed three times with culture media. Aliquots (0.2 ml) of culture
media were added to the wells and the plates incubated for 48 h at 37
°C to allow for induction of HLA/DR antigen. The wells were next
washed with PBS and then fixed for 2 min with ice-cold anhydrous ethyl
alcohol. After the alcohol was removed, the wells were washed with PBS
and then incubated for 1 h at room temperature with mouse monoclonal
anti-HLA/DR antibody diluted in PBS containing 0.5% bovine serum
albumin. PBS was used to wash the wells, and peroxidase-labeled goat
anti-mouse IgG was added to each well for 1 h at room temperature. The
wells were washed three times with PBS and then developed as described
for the ELISA plates.
Nuclear Magnetic Resonance Sample
Preparation
RhuIFN- NMR samples were prepared by dialyzing
0.5 m
M protein against 20 m
M phosphate buffer, pH
7.0, containing 0.015% sodium azide. Rh-GM-CSF NMR samples were
prepared in the same buffer at a concentration of 0.45 m
M protein. Peptide and peptide-protein solutions were prepared by
dissolving 5.0 mg of the solid peptide into either the 20 m
M phosphate buffer or into the dialyzed protein solution to yield a
final peptide concentration of 4.3 m
M. The pH of all NMR
samples was adjusted by adding trace amounts of either dilute sodium
hydroxide or phosphoric acid. The final volumes in the NMR tubes were
brought to 0.55 ml by diluting with approximately 60 µl of
deuterium oxide (Cambridge Isotopes, Andover, MA). The final pH of the
NMR samples used for the transferred NOE studies was 7.0.
Nuclear Magnetic Resonance Measurements
All NMR
experiments were carried out at 500 MHz on a GN-500 Omega NMR
spectrometer with the temperature of the probe maintained to within
±0.2 °C. Phase-sensitive double-quantum filtered correlation
spectroscopy (DQF-COSY) (Rance et al., 1983) and total
correlation spectroscopy (TOCSY) (Bax and Davis, 1985) data sets as
well as phase-sensitive NOESY data sets in the hypercomplex mode
(States et al., 1982) were acquired at 15 °C, pH 6.0, for
the assignment of the proton peptide resonances. The mixing times used
in the TOCSY and NOESY experiments were 55 and 150 ms, respectively.
For the transfer NOE experiments, phase-sensitive NOESY data sets in
the hypercomplex mode were collected at 15 °C, pH 7.0, for the free
peptide and for the peptide plus rhuIFN-, at mixing times of 80,
100, and 225 ms. The two-dimensional data sets were collected with
either 256 or 512 free induction decays (FIDs) using a spectral width
of 7000 Hz, 2048 data points, and 64 scans/FID with the carrier
frequency set on the residual water line. Water suppression was
accomplished by presaturation with minimal power for 1.5 s prior to the
first 90
pulse. Digitized data were exported to a remote
SparcII workstation (Sun Microsystems) and analyzed using the Felix
2.10 NMR processing program (Biosym Technologies, Inc., San Diego, CA).
All data sets were processed with squared sine-bell window functions
followed by Fourier transformation. Data sets were zero filled to yield
final two-dimensional matrices with sizes of either 1K
1K or 2K
2K real points. NOESY data sets were subject to first-order
polynomial base line correction in both dimensions after Fourier
transformation.
Epitope Mapping of Anti-idiotypic Antibody Recognizing
HuIFN-
Murine polyclonal anti-idiotypic antibody was
previously raised against rabbit polyclonal antibody to discontinuous
peptide 1, an inhibitory peptide comprised of regions from huIFN- Receptor by the Method of Overlapping Synthetic
Peptides
corresponding to amino acid residues 15-21 and
132-138
(
)
and tethered by a Gly-Gly-Gly
linkage (Seelig et al., 1994). This anti-idiotypic antibody
specifically recognizes rhuIFN-
R and prevents binding of the
receptor to rhuIFN-
(Seelig et al., 1994). The epitope of
the anti-idiotypic antibody was examined using the method described by
Geysen et al. (1987). Neutralizing anti-idiotypic antibody
recognized octapeptides in the region 120-141
(
)
of rhuIFN-
R (Fig. 1). Neither murine preimmune serum
or rabbit polyclonal antibody to discontinuous peptide 1 showed any
recognition of these peptide regions (data not shown). To confirm this
observation, and to explore the three regions which were weakly
recognized by the anti-idiotypic antibody, peptides corresponding to
regions 34-49, 51-62, 103-120, 120-141(Tyr)acm,
and 126-139 were synthesized. ELISA analysis of the ability of
anti-idiotypic antibody to bind each of these peptides resulted in the
recognition of only two peptides, 120-141(Tyr)acm and
126-139. Recognition above background was not observed for any of
the other peptides. In addition, recognition of huIFN-
by the
anti-idiotypic antibody can be completely prevented by addition of 300
n
M peptide 120-141(Tyr)acm (data not shown). This
further supports the thesis that the site recognized on the intact
receptor, like that on the overlapping octapeptides, is the receptor
region 120-141.
Inhibition of Biological Activities by Rabbit Polyclonal
Antibodies Raised against Synthetic Peptides
Four synthetic
peptides corresponding to regions within the extracellular portion of
the huIFN- receptor, one corresponding to an intracellular region,
as well as the soluble rhuIFN-
protein, were used to elicit rabbit
polyclonal antibody response. Each antibody was tested for its ability
to prevent huIFN-
-induced expression of HLA/DR antigen on the
surface of Colo 205 cells (Table I). Of the six peptides examined, only
two peptide-derived polyclonal antibodies (Ab 493-89 and Ab
495-89 raised against peptides 120-141(Tyr)acm and
123-138, respectively), and the protein-derived antibody SP68
inhibited expression of the HLA/DR antigen on the surface of Colo 205
cells. The fact that the peptide recognized by the anti-idiotypic
antibody was able to generate antibody with an inhibitory response is
consistent with the observed antagonist activity of the anti-idiotypic
antibody. This further supports the observation that the region
120-141 of the receptor may represent a portion of the epitope of
the anti-idiotypic antibody.
Inhibition of HuIFN-
The inhibitory activity and
ability of the anti-idiotypic antibody to recognize huIFN--induced Expression of HLA/DR
Antigen by Synthetic Peptides
receptor suggests that this antibody possesses the ``internal
image'' of huIFN-
. Thus, the epitope for the anti-idiotypic
antibody on huIFN-
receptor is likely to exist on or near the
binding site for huIFN-
. It was hypothesized that a synthetic
peptide corresponding to the epitope might interfere with the ability
of the huIFN-
to induce biological activity. In order to further
explore this possibility, the antagonistic property of the peptide
120-141(Tyr)acm was examined (Fig. 2). A concentration of 35
µ
M reduced the huIFN-
induction of HLA/DR antigen on
Colo 205 cells by 50%. In contrast, concentrations of greater than one
m
M were required for inhibition by peptides corresponding to
regions 51-62, 197-213, 214-223, and 224-240 on
huIFN-
receptor (data not shown). The observation that the peptide
identified by the anti-idiotypic antibody inhibits biological activity
indicates that the peptide may directly bind to huIFN-
. Direct
ELISA binding studies were performed to study the interaction between
the peptide and huIFN-
.
ELISA Binding of rHuIFN-
Receptor peptide 120-141(Tyr)acm, an irrelevant
peptide corresponding to a region of huIFN- to Synthetic
Peptides
, a
scrambled
(
)
version of peptide
120-141(Tyr)acm, or no peptide at all was coated on the ELISA
plate. After blocking, rhuIFN-
or rh-GM-CSF was then incubated on
the plate. Polyclonal anti-huIFN-
or anti-GM-CSF antibodies and
the appropriate secondary antibody were then incubated to detect
huIFN-
and GM-CSF binding, respectively (Fig. 3). Only peptide
120-141(Tyr)acm was recognized in this format and it was only
recognized by rhuIFN-
. Van Volkenburg et al. (1993) noted
a related murine IFN-
receptor peptide binds to murine IFN-
.
Due to the sequence similarity of the murine and human receptors,
recombinant murine
-interferon was also examined in the system.
Under the conditions examined, murine
-interferon recognized the
peptide slightly (approximately 10% of the absorbance by ELISA that was
observed for an equivalent amount of rhuIFN-
). Thus, there may be
some cross-recognition by murine and human
-interferon for binding
sites on the receptor. This has been suggested in a very recent paper
by Szente et al. (1994). The peptide 120-141(Tyr)acm was
not recognized by rh-GM-CSF, and rhuIFN-
did not bind to an
irrelevant peptide. These data further support the premise that peptide
120-141(Tyr)acm may bind directly to rhuIFN-
. However,
another method was sought to probe the binding of the peptide to
huIFN-
.
NMR Studies: NMR of the Peptide Plus
RHuIFN-
One-dimensional proton spectra for the peptide plus
rhuIFN- and for the free peptide
(
)
are shown
in Fig. 4, A and B, respectively. At 15 °C,
addition of rhuIFN-
to the peptide caused line broadening of some
of the peptide resonances, but single species multiplets were still
observed. In general, the resonances showing the largest degree of line
broadening were in the amide,
, and
proton regions. Proton
spectra were acquired for the peptide plus rhuIFN-
at temperatures
ranging from 5 to 40 °C. The resonances become sharper and narrower
with increasing temperature. These data as well as the observation of
the TRNOE peaks in Figs. 6 B and 7 B support the
assumption that the peptide is in fast exchange between its free and
its bound state. Rapid exchange between the free and bound states
causes one set of peptide chemical shifts to be observed, which are
virtually identical to those observed for the peptide in the absence of
rhuIFN. The similarity to the free peptide shifts is due to the large
molar excess of peptide used in the TRNOE experiment relative to the
rhuIFN concentration and to the low concentration of peptide bound.
Thus, the resonance assignments made for the free peptide will be
applicable to the TRNOE experiment.
Assignment of Peptide Resonances
In the discussion
of the two-dimensional NMR data and results for the peptide, the
sequence and numbering of the peptide is as follows:
AY
C
R
D
G
K
I
G
P
P
K
L
D
-I
R
K
E
E
K
Q
I
.
Proton sequence-specific assignments of the peptide backbone and the
majority of the side chain resonances at pH 6.0 and 15 °C in the
absence of protein were made using a combination of DQF-COSY, NOESY,
and TOCSY data sets according to established methods (Wuthrich, 1986).
At pH 7.0, exchange broadening of the amide resonances in the peptide
precluded observation of some of the
to amide cross-peaks.
Lowering the temperature to 15 °C and the pH to 6.0 slowed the
amide exchange so that 19 of the expected 20 backbone cross-peaks were
observed in the free peptide. The NH-terminal alanine amide resonance
was not observed in the DQF-COSY spectrum but was identified in the
NOESY spectrum of the peptide plus rhuIFN-
. At both pH 6.0 and
7.0, the intraresidue d(
N)
(
)
cross-peak of
Lys-131 and Lys-136 are overlapped. An expansion of the
to amide
proton region from the DQF-COSY spectrum under these conditions is
shown in Fig. 5. Assigning the resonances at pH 6.0 served as a basis
for making nearly complete assignments at pH 7.0. At the higher pH,
some of the amide resonances shift relative to their value at pH 6.0,
but the changes are small enough so that they are still identifiable at
pH 7.0. It was easy to distinguish the side chain resonances of Arg-123
and Arg-135, but impossible to separate those of Lys-131 and Lys-136 at
pH 7.0. There was also extensive overlap of the Tyr-121, Asp-133,
Arg-135, and Glu-138 amide resonances. Chemical shifts at pH 7.0, 15
°C are referenced to the internal standard TSP and are listed in
Table II.
N) NOEs, and medium to weak intraresidue d(
N) and d(
N)
NOEs. This NOE pattern is typical of a peptide which adopts an extended
structure in solution. Fig. 7 A shows a very weak d(NN)
cross-peak between residues Ile-134 and Arg-135. The presence of this
NOE cross-peak, although very weak, may indicate a nascent turn
structure inherent in this particular peptide (Dyson et al.,
1988). However, attempts to induce secondary structure by examining the
peptide at 5 °C or by dissolving the peptide in a mixture of
TFE/buffer (50:50, v/v) failed to induce a folded conformation as
determined by both NMR and CD spectroscopies (data not shown).
Figure 6:
NOESY
contour plot expansions of the peptide in the absence and presence of
rhuIFN-. Contour plot expansions from the NOESY data sets
corresponding to the amide/aliphatic regions of peptide
120-141(Tyr)acm, 4.3 m
M, 20 m
M phosphate
buffer, pH 7.0, at 15 °C ( A) and for the peptide in the
presence of rhuIFN-
( B). The NMR experimental details are
described in the text. The numbering system corresponds to the
following peptide proton assignments for both A and
B: 1, I134 H
-R135 NH; 2, L132
H
-D133NH; 3, P130 H
-K131 NH; 4, R123
H
-NH; 5, R123 H
-D124 NH; 6, G125 H
-NH;
7, K126 H
-NH; 8, I127 H
-NH; 9,
G128 H
-NH; 10, G125 H
-K126 NH; 11, D133
H
-I134 NH; 12, Q140 H
-I141 NH; 13, L132
H
-NH; 14, D133 H
-NH; 15, I134 H
-NH;
16, R135 H
-NH; 17, K136 H
-NH; 18,
E137 H
-NH; 19, E138 H
-NH; 20, K139
H
-NH; 21, Q140 H
-NH; 22, I141 H
-NH;
23, I134
H-NH; 24, I127
H-NH; 25,
K126
H-NH 26, L132, K136
H-NH; 27, K136
H-NH; 28, R135
H-NH; 29, R135
H-NH;
30, K131
H-NH; 31, E138
H-NH; 32 and 33, D133
H-NH; 34, D124
H-NH;
35, A120 H
-NH; 36, A120 CH3-NH; 37,
I134 CH3-NH; 38, I134
H-NH; 39, I134
H-NH;
40, I127
H-NH; 41, I127
H-NH; 42,
K126
H-NH; 43, L132 CH3-NH; 44, L132
H-NH;
45 and 46, R135
H-K136 NH; 47, R123
H-D124 NH; 48, E138
H-K139 NH; 49, K131,
K139
(
)H-NH; 50, K131, K139
H-NH; 51,
E138
H-K139 NH; 52, R135
H-NH; 53, Y121
H-NH; 54, R135
H-NH; 55 and 56,
R135
H-NH; 57 and 58, E137
H-NH;
59, E138
H-NH; 60, K139
H-Q140 NH;
61, R123
H-PG; 62, I134 CH3-R135 NH;
63, R123
H-NH; 64, R123
H-NH. PG is the acetamidomethyl protecting group
Figure 7:
NOESY contour plot expansions of the
peptide and peptide-peptiderhuIFN-
complex. Contour plot
expansions of the NOESY data sets corresponding to the amide to amide
regions of peptide 120-141(Tyr)acm (4.3 m
M in peptide
concentration in 20 m
M phosphate buffer, pH 7.0 at 15 °C)
( A) and for the peptide in the presence of rhuIFN-
( B) are shown at a molar ratio of 9:1 peptide to rhuIFN-
.
The NMR experimental details are described in the text. The labels
refer to the following amide to amide NOEs and TRNOEs: I/R in
both A and B refer to the Ile-134/Arg-135; in
B, I/G refers to Ile-127/Gly-128, L/D to
Leu-132/Asp-133, R/K to Arg-135/Lys-136, and E/K to
Glu-138/Lys-139.
Transferred NOE NMR of the Peptide Plus
RhuIFN-
In the 225 ms NOESY data set (pH 7.0, 15 °C)
collected on the peptide in the presence of rhuIFN-, there is a
significant reduction in some of the signal intensity as well as the
appearance of new cross-peaks that are not present in the data sets
with mixing times of 80 and 100 ms. Due to these observations, the
225-ms data set was assumed to be reflecting spin diffusion effects. A
high ratio of peptide to rhuIFN was selected to minimize spin diffusion
effects. The 100-ms data set was selected for the analysis of the TRNOE
effects because the signal intensities were more intense than those
observed in the data set with the 80-ms mixing time. Thus, it was
assumed that the 100-ms data set displays maximum signal to noise with
minimal spin diffusion effects.
are shown in Figs. 6 B and 7 B. Selected interresidue side chain to amide TRNOES
for the peptide in the presence of rhuIFN-
are shown in Fig. 8.
The appearance of numerous cross-peaks which are not present in the
NOESY contour plots of the free peptide in Figs. 6 A or 7 A are evidence that there is a large transferred NOE effect. The
observed interresidue NOEs in the free peptide and the TRNOEs observed
for the peptide in the presence of rhuIFN are summarized in Fig. 9. The
numerous intraresidue and interresidue side chain TRNOEs are indicative
of a molecular interaction between the peptide and the rhuIFN-
.
Further support of this statement comes from the presence of three
sequential d(NN) TRNOEs in Fig. 7 B between residues
Leu-132/Asp-133, between Ile-134/Arg-135, and Ile-127/Gly-128. Two
somewhat weaker d(NN) TRNOEs were also observed between Arg-135/Lys-136
and also near the COOH terminus of the peptide between Glu-138/Lys-139.
As a control for specificity, GM-CSF was also examined. No TRNOEs were
observed with the 120-141(Tyr)acm peptide-rh-GM-CSF, further
indication that the interaction between the peptide and rhuIFN-
is
specific and that the interaction sites are likely to represent
specific contact points between the peptide and protein.
in three distinct regions of the peptide. Since TRNOE
cross-peaks typical of helices were not observed (Wuthrich, 1986), it
is likely that the peptide is folding into
turn or bulge-like
structures. Inducement of these types of structures into peptide
ligands upon binding to large molecules has been observed in both x-ray
crystallographic (Stanfield et al., 1990) and NMR studies
(Bushweller and Bartlett, 1991; Landry et al., 1992; Scherf
et al., 1992; Dratz et al.; Stradley et al.,
1993). In the experiment described above, there is some evidence for
the presence of a
turn structure in the DIRK region of the
peptide. Examination of Fig. 9shows that sequential d(NN) TRNOEs
were observed between both Ile-134 and Arg-135 and between Arg-135 and
Lys-136 as well as a sequential d(
Ni,i+2) TRNOE between
Ile-134 and Lys-136. This NOE patterns suggests the presence of a type
I
turn. A turn with Ile and Arg at the corners could accommodate
the observed Ile-134 (
,
, CH
) side chain
interactions with the amide proton of Arg-135. In addition, a long
range TRNOE between the methyl group of Ile-134 and the amide proton of
either Lys-131 or Lys-139 (resonances are overlapped at pH 7.0) is
further evidence of a bend in the structure. These side chain to amide
TRNOEs are shown in Fig. 8. The other observed dNN, d
N, and
d
N interactions observed for residue pairs Ile-127/Gly-128 and
Glu-138/Lys-139 raise the possibility that type II
turns may be
present in these regions. These TRNOEs are indicative of the presence
of dynamic turn-like conformations which lack long range order (Dyson
et al., 1988). More stable turns would be accompanied by
additional d(
Ni,i+2) and dNN(i,i+2) TRNOEs
(Wüthrich, 1986).
Figure 9:
NOE summary diagram of the interresidue
NOEs observed for the peptide and the peptide in the presence of
RhuIFN-. The NOEs observed for the 120-141(Tyr)acm peptide
( A) and TRNOE connectivities ( B) observed for the
peptide in the presence of rhuIFN-
at a molar ratio of 9:1 peptide
to protein are detailed. A solid bar between 2 residues
represents an NOE between them. The height of the bar is reflective of
the NOE intensity. In B, the label d(SN) refers to
observed side chain to amide TRNOEs, which are depicted in Fig.
8.
Figure 8:
NOESY contour plot expansion of the
peptiderhuIFN-
complex. Contour plot expansion of the NOESY
data set corresponding to the peptide 120-141(Tyr)acm in the
presence of rhuIFN-
at a molar ratio of 9:1 peptide to protein.
The figure depicts selected interresidue TRNOEs observed between side
chain and amide resonances. The superscripts refer to the following
cross-peaks: 1 and 2, Arg-123
H to Asp-124 NH;
3, Arg-135
H to Lys-136 NH; 4 and 5,
Glu-138
H to Lys-139 NH; 6, Ile-134 methyl to Arg-135 NH;
7, Ile-134
H to Arg-135 NH; 8, Lys-139
H to
Gln-140 NH; and 9, Lys-139
H to Gln-140 NH; 10,
Ile-134 CH
to Lys-131(139) NH.
The lack of significant TRNOE contribution by
Tyr-121 suggests that it is unlikely this conservative replacement for
Val-121 was responsible for the binding contacts observed. This was
confirmed by subsequent studies in which native peptide was examined.
Identical TRNOEs are observed when this native peptide is used. Thus,
this peptide and modifications of the peptide remain fertile areas for
further study. To summarize qualitatively, the results observed in the
TRNOE experiment are consistent with the surface type of interaction
expected for an epitope peptide binding to a large protein. The
appearance of intra- and interresidue side chain TRNOEs along the
Ile-128/G-129 and Glu-138/Lys-139 regions of the peptide in the
presence of rhuIFN- as well as the observation of a turn-like
structure between residues Asp-133 and Lys-136 are indicative of close
molecular contact with the protein.
may contribute to the binding of this ligand to its
receptor (Seelig et al., 1989, 1994; Seelig and Prosise,
1992). Anti-idiotypic antibody generated from an antibody raised
against a peptide mimicking juxtaposed regions of the amino and
carboxyl regions of huIFN-
has been shown to specifically
recognize rhuIFN-
receptor, and to inhibit the binding of
huIFN-
to its receptor (Seelig et al., 1994). We now
present evidence which identifies a region on the rhuIFN-
receptor
which is likely to be a portion of the epitope of the anti-idiotypic
antibody. The linear synthetic peptide corresponding to the region
identified by the anti-idiotypic antibody is itself capable of
generating an antibody having neutralizing activity. Since this would
be expected if the peptide represented an epitope of the anti-idiotypic
antibody, the data are consistent with the selection of the peptide by
the overlapping peptide epitope mapping technique. This anti-idiotypic
antibody appears to be another example in which anti-idiotypic
antibodies can function as a molecular mimic of the ligand, and in this
case appears to be a true mimic of the ligand huIFN-
(Garcia
et al., 1992).
value of approximately 35 µ
M. This
peptide is also shown to bind rhuIFN-
both in ELISA studies and in
TRNOE NMR studies. Significantly, while the free peptide has no
apparent secondary structure, it is shown by TRNOE studies to adopt
considerable structure when it binds to the ligand. It is possible that
the observed structure is that which this region has in the unbound
full-length receptor. Alternatively, the peptide may adopt this
structure as part of the mechanism by which its acts to bind
huIFN-
and inhibit its activity. Indeed, huIFN-
receptor is
believed to be a member of the hematopoietic superfamily of receptor
structures (Bazan, 1990). The crystal structure of another member of
this family, growth hormone, in complex with its receptor is known
(deVos et al., 1992) and may serve as a useful model of
cytokine-receptor interactions. Interactions of human growth hormone
with its homodimer receptor occurs at two distinct regions of the
ligand. In this case, binding of the ligand to the receptor is believed
to be accompanied by some conformational changes in the receptor which
occur upon contact (deVos, 1992). Analogous to the growth hormone
model, huIFN-
is believed to bind as a homodimer to two
huIFN-
receptors. The receptor-binding peptide described here was
identified through an anti-idiotypic antibody response which was
derived from a peptide sequence (Seelig et al., 1993)
corresponding to the AB loop of huIFN-
and the carboxyl segment
beyond the last helix which is not resolved by the huIFN-
crystal
structure (Ealick et al., 1991). Both of these regions are
believed to be themselves very flexible regions of the molecule and,
thus, capable of structural movement. The possibility that the receptor
peptide 120-141 may adopt its secondary structure upon binding to
the ligand and that flexible portions of the ligand may move to bind
the receptor is consistent with conformational changes which have been
seen in the human growth hormone and other systems upon complexation
(de Vos 1992; Rini et al., 1992). It is interesting that
additional players such as the cofactor molecules identified for the
murine and human receptors may also contribute to the conformations
adopted by both the receptor and ligand (Soh et al., 1994;
Hemmi et al., 1994). The identification of such accessory or
cofactor molecules is strong indication that the overall picture of
this cytokine system is quite complex which may be best revealed in
segments which ultimately fit together. In any event, the data obtained
by TRNOE studies provide a powerful picture of one segment: that is,
the structure of a portion of the ligand-receptor binding region of the
receptor as it is bound to huIFN-
.
. Evidence from the TRNOE data indicates that the DIRK
segment of the peptide forms a
-turn in the bound conformation.
Given the level of understanding of
-turn mimetics (Rizo and
Gierasch, 1992), this region could be a reasonable target for
peptidomimetic design. The TRNOE information about this region presents
itself as an excellent starting point for structural modeling and
peptide design in order to gain further insight into the binding of
rhuIFN-
to its receptor.
Table: Response of rabbit polyclonal antibodies
raised to -interferon receptor and its peptides
Table: Proton NMR
chemical shifts for the rhuIFN- receptor peptide
antagonist
, human
-interferon; rhuIFN-
, recombinant human
-interferon;
rhuIFN-
R, recombinant human
-interferon receptor;
120-141acm, receptor peptide in 120-141 in which the
cysteine side chain is modified with an acetamidomethyl group;
120-141(tyr)acm, receptor peptide 120-141 in which Val-121
is replaced by Tyr and the cysteine side chain is modified with an
acetamidomethyl group; DIRK, receptor peptide 133-136
(Asp-Ile-Arg-Lys); IG, receptor peptide 127-128 (Ile-Gly); EK,
receptor peptide 138-139 (Glu-Lys); ELISA, enzyme-linked
immunosorbent assay; IgG,
-immunoglobulin; Fmoc,
9-fluorenylmethoxycarbonyl; TBS, Tris-buffered saline; PBS,
phosphate-buffered saline; ABTS,
2-2`-azino-bis[3-ethyl-benzthiazoline sulfonate]; TMB,
3,3`,5,5`-tetramethylbenzidine; NOE, nuclear Overhauser effect;
DQF-COSY, double quantum filtered correlation spectroscopy; TOCSY,
total correlation spectroscopy; NOESY, nuclear Overhauser enhancement
spectroscopy; TRNOE, transferred nuclear Overhauser enhancement; TSP,
sodium salt of 3-trimethylsilylpropionate; FID, free induction decay;
1K, 1024; 2K, 2048; GM-CSF, human granulocyte-macrophage
colony-stimulating factor; t-Boc,
t-butoxycarbonyl.
peptides and protein was derived from the full-length
146 amino acid species of huIFN-
as predicted from the cDNA
sequence (Gray and Goeddel, 1983).
R peptides and protein was derived from the
cDNA sequence described by Aguet et al. (1988) including the
14-amino-acid signal sequence.
H-
H distances in the peptide
follow the notation outlined in Wuthrich (1986).
receptor and murine
-interferon, Dr. C. Lunn for
the purified
-interferon, and S. Mittelman for the CD analysis of
the peptides. We also thank Prof. J. Prestegard for his continued
support of this project. In addition we express thanks to Drs. C. A.
Evans, H. Eaton, H. Le, Nick Murgolo, Chuck Lunn, and A. Frederick for
critical review of the manuscript and helpful advice.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.