©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Development of a Receptor Peptide Antagonist to Human -Interferon and Characterization of Its Ligand-bound Conformation Using Transferred Nuclear Overhauser Effect Spectroscopy (*)

Gail F. Seelig (1)(§), Winifred W. Prosise (1), Julio C. Hawkins (1), Mary M. Senior(§) (2)

From the (1) Department of Structural Chemistry and the (2) Department of Physical Analytical Chemical Research and Development, Schering-Plough Research Institute, Kenilworth, New Jersey 07033-0539

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Polyclonal anti-idiotypic antibody raised to a synthetic discontinuous peptide derived from the human -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 ICof 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.


INTRODUCTION

Human -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-.

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 -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).

A neutralizing anti-idiotypic antibody which binds to the extracellular domain of huIFN- 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.

To further probe the receptor peptide-ligand interactions, we have examined the rhuIFN--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.


EXPERIMENTAL PROCEDURES

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.

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.

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--induced Expression of HLA/DR Antigen

The bio-ELISA assay for HLA/DR induction by huIFN- 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 10cells/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% COincubator. 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 90pulse. 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.


RESULTS

Epitope Mapping of Anti-idiotypic Antibody Recognizing HuIFN- Receptor by the Method of Overlapping Synthetic Peptides

Murine polyclonal anti-idiotypic antibody was previously raised against rabbit polyclonal antibody to discontinuous peptide 1, an inhibitory peptide comprised of regions from huIFN- 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--induced Expression of HLA/DR Antigen by Synthetic Peptides

The inhibitory activity and ability of the anti-idiotypic antibody to recognize huIFN- 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- to Synthetic Peptides

Receptor peptide 120-141(Tyr)acm, an irrelevant peptide corresponding to a region of huIFN-, 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: AYCRDGKIGPPKLD-IRKEEKQI. 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.

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(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.

NOESY contour expansions for the peptide in the presence of rhuIFN- 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.

The TRNOE data suggest that there is an interaction between the peptide and the rhuIFN- 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, dN, and dN 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 CHto 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.


DISCUSSION

Previous studies have suggested that two regions on huIFN- 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).

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 ICvalue 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-.

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-. 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

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.



FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed.

The abbreviations used are: HuIFN-, 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.

The numbering of huIFN- 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).

The numbering of huIFN-R peptides and protein was derived from the cDNA sequence described by Aguet et al. (1988) including the 14-amino-acid signal sequence.

A scrambled version of the 120-141(Tyr)acm peptide IADPIC(acm)RELKKIPEGRYDQGKK which was synthesized.

Initial NMR studies were performed with the modified form of the receptor peptide 120-141(Tyr)acm. Subsequently, select NMR experiments were repeated with the native 120-141acm peptide in order to verify with the native peptide, the observations that had been obtained for the modified receptor peptide 120-141(Tyr)acm. For consistency, all figures and tables represent the data obtained with the modified peptide 120-141(Tyr)acm.

The abbreviations for the sequential H-H distances in the peptide follow the notation outlined in Wuthrich (1986).


ACKNOWLEDGEMENTS

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- 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.


REFERENCES
  1. Aguet, M., Dembic, Z., and Merlin, G. (1988) Cell 55, 273-280 [Medline] [Order article via Infotrieve]
  2. Bax, A., and Davis, D. G. (1985) J. Magn. Res. 65, 355-360
  3. Bazan, J. F. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 6934-6938 [Abstract]
  4. Bradford, M. (1976) Anal. Biochem. 72, 248-254 [CrossRef][Medline] [Order article via Infotrieve]
  5. Bushweller, J. H., and Bartlett, P. A. (1991) Biochemistry 30, 8144-8151 [Medline] [Order article via Infotrieve]
  6. Campbell, P. A., and Sykes, B. D. (1991) J. Magn. Res. 93, 77-92
  7. Clore, G. M., and Gronenborn, A. M. (1982) J. Magn. Res. 48, 402-417
  8. Cook, J. R., Jung, V., Schwartz, B., Wang, P., and Pestka, S. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 11317-11321 [Abstract]
  9. deVos, A. M., Ultsch, M., and Kossiakoff, A. A. (1992) Science 255, 306-312 [Medline] [Order article via Infotrieve]
  10. Dighe, A. S., Farrar, M. A., and Schreiber, R. D. (1993) J. Biol. Chem. 268, 10645-10653 [Abstract/Free Full Text]
  11. Dratz, E. A., Furstenau, J. E., Lambert, C. G., Thireault, D. L., Rarick, H., Schepers, T., Pakhlevaniants, T., and Hamm, H. E. (1993) Nature 363, 276-281 [CrossRef][Medline] [Order article via Infotrieve]
  12. Dyson, H. J., Rance, M., Houghten, R. A., Lerner, R. A., and Wright, P. E. (1988) J. Mol. Biol. 201, 161-200 [Medline] [Order article via Infotrieve]
  13. Ealick, S. E., Cook, W. J., Vijay-Kumar, S., Carson, M., Nagabhushan, T. L., Trotta, P. P., and Bugg, C. E. (1991) Science 252, 698-702 [Medline] [Order article via Infotrieve]
  14. Farrar, M. A., and Schreiber, R. D. (1993) Annu. Rev. Immunol. 11, 571-611 [CrossRef][Medline] [Order article via Infotrieve]
  15. Farrar, M. A., Fernandez-Luna, J., and Schreiber, R. D. (1991) J. Biol. Chem. 266, 19626-19635 [Abstract/Free Full Text]
  16. Farrar, M. A., Campbell, J. D., and Schreiber, R. D. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 11706-11710 [Abstract]
  17. Fountoulakis, M., Lahm, H. W., Maris, A., Friedlein, A., Manneberg, M., Stueber, D., and Garotta, G. (1991) J. Biol. Chem. 266, 14970-14977 [Abstract/Free Full Text]
  18. Fountoulakis, M., Zulauf, M., Lustig, A., and Garotta, G. (1992) Eur. J. Biochem. 208, 781-787 [Abstract]
  19. Garcia, K. C., Ronco, P. M., Verroust, P. J., Brunger, A. T., and Amzel, L. M. (1992) Science 257, 502-507 [Medline] [Order article via Infotrieve]
  20. Garotta, G., Ozmen, L., Fountoulakis, M., Dembic, Z., van Loon, A. P. G. M., and Stuber, D. (1990) J. Biol Chem. 265, 6908-6915 [Abstract/Free Full Text]
  21. Georgiades, J. A., Baron, S., Fleischmann, W. R., Jr., Langford, M., Weigent, D. A., and Stanton, G. J. (1984) in Interferons and Their Applications: Handbook of Experimental Pharmacology (Cane, P. E., and Carter, W. A., eds) Vol. 71, pp. 305-337, Springer-Verlag, Berlin
  22. Geysen, H. M., Rodda, S. J., Mason, T. J., Tribbick, G., and Schoofs, P. G. (1987) J. Immunol. Methods 102, 259-274
  23. Gibbs, V. C., Williams, S. R., Gray, P. W., Schreiber, R. D., Pennica, D., Rice, G., and Goeddel, D. V. (1992) Mol. Cell. Biol. 11, 5860-5866
  24. Gibson, U. E. M., and Kramer, S. M. (1989) J. Immunol. Methods 125, 105-113
  25. Gray, P. W., and Goeddel, D. V. (1983) in Proceedings of a Symposium on the Biological Basis of New Developments in Biotechnology (Hollaender, A., Laskin, A. I., and Rodgers, P., eds) pp. 35-61, Plenum Press, New York
  26. Gray, P. W., Leong, S., Fennie, E. H., Farrar, M. A., Pingel, J. T., Fernandez-Luna, J., and Schreiber, R. D. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 8497-8501 [Abstract]
  27. Greenlund, A. C., Schreiber, R. D., Goeddel, D. V., and Pennica, D. (1993) J. Biol. Chem. 268, 18103-18110 [Abstract/Free Full Text]
  28. Hemmi, S., Bohni, R., Stark, G., DiMarco, F., and Aguet, M. (1994) Cell 76, 803-810 [Medline] [Order article via Infotrieve]
  29. Hibino, Y., Kumar, C. S., Mariano, T. M., Lai, D., and Pestka, S. (1992) J. Biol. Chem. 267, 3741-3749 [Abstract/Free Full Text]
  30. Landry, S. J., Jordan, R., McMacken, R., and Gierasch, L. M. (1992) Nature 355, 455-457 [CrossRef][Medline] [Order article via Infotrieve]
  31. Langer, J. A., and Pestka, S. (1988) Immunol. Today 9, 393-400 [Medline] [Order article via Infotrieve]
  32. Lowry, O. H., Rosenbrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275 [Free Full Text]
  33. Lunn, C. A., Davies, L., Dalgarno, D., Narula, S. K., Zavodny, P. J., and Lundell, D. (1992) J. Biol. Chem. 267, 17920-17924 [Abstract/Free Full Text]
  34. Merrifield, R. B. (1963) J. Am. Chem. Soc. 85, 2149-2154
  35. Nagabhushan, T. L., and Leibowitz, P. J. (1985) in Interferon Alpha-2: Preclinical and Clinical Evaluation (Kisner, D. L., and Smyth, J. F., eds) pp. 1-12, Martinus Nijhof Publishers, Boston
  36. Nagabhushan, T. L., Trotta, P. P., Le, H. V., Seelig, G. F., and Kosecki, R. A. (June 14, 1988) U.S. Patent4,751,078
  37. Neuhaus, D., and Williamson, M. (1989) The Nuclear Overhauser Effect in Structural and Conformational Analysis, pp. 175-181, VCH Publishers, New York
  38. Noggle, J. H., and Schirmer, R. E. (1971) The Nuclear Overhauser Effect: Chemical Applications, pp. 4-75, Academic Press, New York
  39. Otting, G. (1993) Curr. Opin. Struct. Biol. 3, 760-768 [CrossRef]
  40. Rance, M., Sorensen, M., Bodenhausen, G., Wagner, G., Ernst, R. R., and Würthrich, K. (1983) Biochem. Biophys. Res. Commun. 117, 479-485 [Medline] [Order article via Infotrieve]
  41. Rini, J. M., Schulze-Gahmen, U., and Wilson, I. A., (1992) Science 255, 959-965 [Medline] [Order article via Infotrieve]
  42. Rizo, J., and Gierasch, L. M. (1992) Annu. Rev. Biochem. 61, 387-418 [CrossRef][Medline] [Order article via Infotrieve]
  43. Scherf, T., Hiller, R., Naider, F., Levitt, M., and Anglister, J. (1992) Biochemistry 31, 6884-6897 [Medline] [Order article via Infotrieve]
  44. Seelig, G. F., and Prosise, W. W. (1992) J. Cell. Biochem. 16C, 101
  45. Seelig, G. F., White, G., Prosise, W. W., Pennington, M. W., Windsor, W. T., Nagabhushan, T. L., and Trotta, P. P. (1989) J. Interferon Res. 4, 184
  46. Seelig, G. F., Prosise, W. W., and Taremi, S. S. (1994) J. Biol. Chem. 269, 358-363 [Abstract/Free Full Text]
  47. Soh, J., Donnelly, R. J., Kotenko, S., Mariano, T. M., Cook, J. R., Wang, N., Emanuel, S., Schwartz, B., Miki, T., and Pestka, S. (1994) Cell 76, 793-802 [Medline] [Order article via Infotrieve]
  48. Sprang, S. R., and Bazan, J. F. (1993) Curr. Opin. Struct. Biol. 3, 815-817
  49. Stanfield, R. L., Fieser, T. M., Lerner, R. A., and Wilson, I. A. (1990) Science 248, 712-719 [Medline] [Order article via Infotrieve]
  50. States, D. J., Haberkorn, R. A., and Ruben, D. J. (1982) J. Magn. Res. 48, 286-292
  51. Stradley, S. J., Rizo, J., and Gierasch, L. M. (1993) Biochemistry 32, 12586-12590 [Medline] [Order article via Infotrieve]
  52. Stuber, D., Friedlein, A., Fountoulakis, M., Lahm, H-W., and Garrota, G. (1993) Biochemistry 32, 2432-2430
  53. Sykes, B., and Campbell, P. A. (1991) J. Magn. Res. 93, 77-92
  54. Szente, B. E., Soos, J. M., and Johnson, H. M. (1994) Biochem. Biophys. Res. Commun. 203, 1645-1654 [CrossRef][Medline] [Order article via Infotrieve]
  55. Trinchieri, G., and Perussia, B. (1985) Immunol. Today 6, 131-136
  56. Trotta, P. P., Seelig, G. F., Le, H. V., and Nagabhushan, T. L. (1986) in Interferons as Cell Growth Inhibitors and Antitumor Factors, (Friedman, R., Merigan, T., and Sveevalson, T., eds) pp. 497-507, A. R. Liss Inc., New York
  57. Trotta, P. P., Seelig, G. F., Kosecki, R. A., and Reichert, P. (July 16, 1987) European Patent Application 88306421.4
  58. Van Volkenburg, M. A., Griggs, N. D., Jarpe, M. A., Pace, J. L., Russell, S. W., and Johnson, H. M. (1993) J. Immunol. 151, 6206-6213 [Abstract/Free Full Text]
  59. Wüthrich, K. (1986) NMR of Proteins and Nucleic Acids, pp. 162-166, John Wiley and Sons, New York

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