Structural Analysis and Modeling of a Synthetic Interleukin-2 Mimetic and Its Interleukin-2R{beta}2 Receptor*

Thierry Rose {ddagger}, Jean-Louis Moreau §, Ralph Eckenberg § and Jacques Thèze § 

From the {ddagger}Département de Biologie Structurale et Chimie, §Unité d'Immuno-Génétique Cellulaire, Département de Médecine Moléculaire, Institut Pasteur, 28 rue du Dr. Roux, 75724 Paris Cedex 15, France

Received for publication, February 19, 2003 , and in revised form, April 2, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Peptide p1–30, which is composed of the 30 amino-terminal residues ({alpha}-helix A) of human interleukin-2 (IL-2), binds as a tetramer to the dimeric IL-2R{beta}2 receptor, whereas the entire IL-2 recognizes the tricomponent receptor IL-2R{alpha}{beta}{gamma}. p1–30 is an IL-2 mimetic that activates CD8 low lymphocytes and natural killer cells, because these cells produce IL-2R{beta} constitutively. It also induces a strong lymphokine-activated killer cell response. A series of truncated peptides were analyzed by circular dichroism and analytical centrifugation to elucidate the role of p1–30 residues. We propose a model where residues 10–30 of the p1–30 peptide form an {alpha}-helix with eight hydrophobic side chains on the same surface buried in a hydrophobic core when four anti-parallel helices combine to form a bundle. IL-2R{beta} dimerization was further studied, and three-dimensional models of the free IL-2R{beta}2 receptor and the p1–304·IL-2R{beta}2 complex were built by comparative modeling based on the crystal structure of the erythropoietin receptor complex, because this belongs to the same hematopoietin family as IL-2. These models suggest that binding of the p1–30 tetramer rotates the COOH-terminal domains and brings both transmembrane regions 50 Å closer together, driving the association of the two intracytoplasmic domains that would transduce the signal into the cytoplasm.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Human interleukin-2 (IL-2)1 is a cytokine 133 residues in length with a molecular mass of 15–18 kDa depending on its degree of glycosylation. It is involved in inflammatory reactions and immune responses (13). IL-2 is made up of a compact bundle of four anti-parallel {alpha}-helices connected by three loops (4). The effects of IL-2 on its target cells are mediated by specific cell surface receptors (IL-2R{alpha}, IL-2R{beta}, and IL-2R{gamma}) (5) encoded by three different genes (6, 7). IL-2R{alpha}, the first component to be identified, is a 55-kDa protein that binds to IL-2 with a Kd of ~10 nM (8, 9). The second IL-2R component, IL-2R{beta}, is a 75-kDa protein with a large intracytoplasmic domain (286 amino acids) (1012). The last component to be identified, IL-2R{gamma}, is a 64-kDa protein (13). IL-2R{beta} and IL-2R{gamma} belong to the hematopoietin receptor family (14), whereas IL-2R{alpha} belongs to a different and unrelated family (15). Two receptors are functional in the human system. The association of human IL-2R{beta} and IL-2R{gamma} forms a receptor of intermediate affinity with a Kd of ~1 nM, whereas the expression of all three chains leads to the formation of a high affinity IL-2R (Kd ~10 pM). A structural model of the IL-2·IL-2R complex derived from the three-dimensional structure of growth hormone-GH receptor co-crystals has been discussed (5, 16). {beta} and {gamma} Subunits are related and appear to fit in an immunoglobulin scaffold common to the cytokine receptor family. Interactions among IL-2 and the IL-2R{beta} and IL-2R{gamma} subunits have already been analyzed (1720), and the experimental data appear to support the model.

We and others (21) have demonstrated that {alpha}-helix A in IL-2 (residues 8–28) is essential for binding to the IL-2R{beta} chain, the most important component of the IL-2R system critically involved in signal transduction. More recently, we showed that peptide p1–30, which includes {alpha}-helix A, tetramerizes and forms bundles of four helices, thus mimicking the structure shown by a cytokine of the hematopoietin family. This neocytokine behaves as an IL-2 partial mimetic after binding to its receptor, which is composed of IL-2R{beta} dimers (22). At the cellular level, p1–30 activates intracellular Shc and p56lck but fails to activate the Janus kinases (Jak1 and Jak3) or the signal transducer and activator of transcription 5 (STAT5). By contrast, p1–30 activates Tyk2, another Janus-like kinase. At the immunological level, p1–30 induces lymphokine-activated killer (LAK) cells and preferentially activates CD8 lymphocytes and NK cells, which constitutively express IL-2R{beta} chains. Significant production of interferon {gamma} is also detected after p1–30 stimulation. IL-2 is used in cancer therapy, but its therapeutic potential is limited by various side effects including a significant "vascular leak syndrome." Because p1–30 is specifically targeted to cytotoxic cells, it may have selective anti-tumor activity. Furthermore, our data suggest that active analogs of p1–30 could be developed with a higher therapeutic index.

Because p1–30 has therapeutic potential and is a unique tool to assess the role played by the different IL-2R chains in the mechanism of signal transduction, we investigated the structural features of this neocytokine and the nature of its interactions with its receptor. We have already reported a preliminary analysis of the molecular basis of p1–304 tetramerization. Furthermore, we showed that extracellular NH2-terminal domain (fragments 31–200) of the IL-2R{beta} µ{beta} molecule forms a dimer in solution prior to the binding of p1–30 (22).

In this paper, we further examine the role played by p1–30 residues in the structure of active molecular species based on a structural study of peptide fragments covering different parts of the peptide. The correlation between the structural properties of these peptides and their effects on cell activation is analyzed. We clarify the importance of p1–30 tetramerization in receptor recognition. We confirm that IL-2R{beta} forms dimers in solution prior to the binding of p1–30. We present theoretical three-dimensional models of free p1–30 and IL-2R{beta} as well as a model of the p1–304·IL-2R{beta}2 complex built by comparative modeling using as templates the free and bound crystallographic structures of the erythropoietin receptor (23) and human growth hormone receptor (24). Finally, we propose a mechanism for signal transduction through a conformational change of IL-2R{beta}2 triggered by p1–30 binding.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines and Proliferation Assays—The T cell line TS1{beta} was derived from the IL-9-dependent mouse T cell line TS1 transfected with human IL-2R{beta} and grown in IL-2 (25). All of the cultures used complete medium supplemented with recombinant human IL-2 (Chiron-Europe, Amsterdam, The Netherlands).

Peptide synergy with IL-2 was tested by an indirect assay of proliferation. Cells were stimulated by various concentrations of IL-2 in the presence of 60 µM peptides in 96-well flat-bottomed microtiter plates (104 cells/well in a final volume of 0.2 ml, 105 cells/well). After 36 h of stimulation, cultures were pulsed for 15 h with 0.5 µCi/well [3H]thymidine. All of the measurements were made in duplicate (averages are shown), and all of the experiments were carried out at least three times. Representative experiments are shown.

IL-2 Peptides and IL-2R{beta}Peptides were synthesized and purified as described previously (21). They were then verified by mass spectrometry and amino acid analysis after total hydrolysis. The following IL-2 peptides were used here (numbers indicate the position in the IL-2 sequence): p1–30, p1–22, p10–30, p1–31, p1–10, p5–15, p15–25, and p20–30. In p1–31Lys-20, Asp-20 was replaced by Lys. In p1–30Cys-31, a Cys was added at the COOH terminus of the p1–30 sequence. Molar extinction coefficients were calculated for the peptides from their UV absorptions at 230 and 242 nm (12,133 and 2047 M 1 cm1, respectively, for p1–30) scaled on their amino acid analysis.

p1–30Cys31 was labeled on the COOH-terminal Cys-31 with a fluorescein group. 6.107 mol of lyophilized peptide was mixed with 5.106 mol of fluorescein 5-maleimide (Molecular Probes) in 200 µl of 100 mM sodium phosphate and 150 mM NaCl buffer at pH 7.2 and gently spun for 16 h at 20 °C. Excess dye was removed by two successive exclusion chromatography runs. The purified labeled peptide p1–31CysFlc31 (a molecular mass of 3805 Da, {epsilon}242 nm 2104 M 1 cm1, {epsilon}490 nm 83,000 M–1 cm1, n = 0.732 ml/mg) with a fluorescein group (Flc) branched on the COOH-terminal Cys-31 accounted for >92% peptide.

The 31–230 residues of the IL-2R{beta} chain (IL-2R{beta}31–230, a molecular mass of 23,352 Da) were produced and purified as a solubilized fragment as described by Eckenberg et al. (22). The molar extinction coefficient of this fragment was calculated from its UV absorption at 280 nm (59,450 M1 cm1) scaled on its amino acid analysis.

Circular Dichroism (CD) and Molecular Weight Analysis—CD measurements were acquired in the far-UV on a spectropolarimeter (Jobin-Yvon CD6, Paris). 5–20 scans were recorded from 180 to 260 nm at 20 °C in 20 mM sodium phosphate buffer, pH 7.2. The helix content was estimated from CD spectra (26).

Diffusion-sedimentation equilibrium studies were performed at 20 °C on peptide solutions (10 and 150 µM) or on IL-2R{beta}31–230 (1 and 14 µM) in 20 mM sodium phosphate, pH 7.2, using a Beckman XL-A analytical ultracentrifuging system at 25,000 and 42,000 rpm and standard double sector cells with 1.2-mm thick aluminum centerpieces. Absorbances were recorded at 230, 242, and 360 nm for the peptides and 280 and 360 nm for IL-2R{beta}31–230. Lyophilized peptides were dissolved in 20 mM sodium phosphate, pH 7.2, and diluted to the desired concentration with the same buffer. 150 µl of peptide or protein sample solution were introduced into the sample sector of the centrifugation cell onto 50 µl of fluorocarbon FC43. The corresponding equilibration buffer (210 µl) was placed in the reference sector. Absorbances were recorded as a function of radial distance at different wavelengths depending on initial peptide or protein concentration and centrifugation speed. After reaching the selected centrifugation speed, radial scans were recorded at 2-h intervals for at least 18 h to check that an equilibrium had been reached. 10–99 successive scans were recorded and averaged. It was checked at 360 nm that no optical artifacts were affecting the base line. Protein distribution at equilibrium was analyzed by Origin-based Optima XL-A data analysis software (Beckman). The different fitting models (single ideal component, two ideal components, and association of identical ideal components) for single data sets (XLA-Single program) were systematically tested, and the best fit was retained on the basis of both the {chi}2 value and the lack of systematic deviation of the residuals. In all of the cases, the base line was set at zero absorbance and not allowed to drift during the fitting procedure. Partial specific volume values (n) used in the molecular weight calculations were determined from the amino acid composition of the peptides (p1–10, n = 0.710 ml/mg; p5–15, n = 0.722 ml/mg; p10–20, n = 0.759 ml/mg; p1–22, n = 0.720 ml/mg; p10–30, n = 0.752 ml/mg; p20–30, n = 0.729 ml/mg; p1–30, n = 0.710 ml/mg; p1–31Cys31, n = 0.710 ml/mg; and p1–31CysFlc31, n = 0.732 ml/mg) and IL-2R{beta}31–230 (n = 0.719 ml/mg). Solvent density was taken as 1.001g/ml at 20 °C.

The binding of p1–30, p1–31Cys-31, and p1–31CysFlc31 to IL-2R{beta}31–230 was tested by analytical ultracentrifugation in 20 mM sodium phosphate, pH 7.1, at 20 °C. We used a 3-cell rotor for each experiment: one cell was filled with peptide, one was filled with IL-2R{beta}31–230, and the last was filled with a mixture of peptide and IL-2R{beta}31–230. Peptide and IL-2R{beta}31–230 concentrations were 50 and 14 µM, respectively. Absorbances were recorded at 242, 280, and 490 nm after reaching equilibrium at 22, 25, 28, and 42 x 103 rpm.

P1–30 Models—Several models of four helical p1–30 peptides were built using the crystal structures of IL-2 (3INK [PDB] ) (4), erythropoietin (1BUY [PDB] ) (27), and the bacterial ColE1 operon repressor ROP (1ROP [PDB] ) (28) as templates downloaded from the Protein Data Bank (29). Peptides p1–302 were threaded along the two longest helices of IL-2 (residues 8–28 and 83–96 from 3INK [PDB] ), respecting the hydrophobic side chain periodicity. The final tetramer was then built by symmetry operations. Every interacting helix pair is anti-parallel and annotated 1-1-1-1 (up-down-up-down) by counting the number of parallel-oriented neighbors. The helices were arranged in other configurations along the same four longitudinal axes using different symmetry operators to build tetramers with four parallel helices annotated 4 (up-up-up-up), three parallel helices, and one antiparallel helix annotated 3-1 (up-up-up-down) and 2x two parallel helices annotated 2-2 (up-up-down-down).

Residues were substituted, and hydrogens were added by Insight II software (Accelrys, San Diego,CA). The conformation of the peptide bundle was optimized by simulated annealing during molecular dynamic runs and then minimized (200 steepest descent and then 300 conjugate gradient steps) by Discover software (Accelrys). We used the CFF91 force field, set the dielectric constant {epsilon} to 1*r (r is the distance between charges), and fixed the non-bond cut off to 14 Å during the dynamics and to infinity during the minimization processes.

Models of the IL-2R Receptor and the p1–304·IL-2R Complex—IL-2R {alpha}-, {beta}-, and {gamma}-chain sequences were downloaded from the Swiss Protein data base as IL2A_HUMAN, IL2B_HUMAN, and CYRG_HUMAN (www.expasy.ch). The IL-2R {beta}- and {gamma}-chains were aligned by ClustalX software (30) using the secondary structure frame from erythropoietin and growth hormone receptors as constraints. We built three structures for IL-2R{beta}2 by comparative modeling using Modeler 4 software (31). We folded two sequences of {beta}-chain fragments 31–200 over the homodimeric erythropoietin receptor (1ERN [PDB] ) (23) in its free form and over the erythropoietin-bound form (1EER [PDB] ) (32). We then folded the same sequences over the homodimeric human growth hormone receptor (3HHR [PDB] ) (24). Four p1–304 helix bundle arrangements were then docked between the two {beta}-chains of the two models super-imposed over templates composed of EPO·EPOR (1EER [PDB] ) and GH·GHR (3HHR [PDB] ), yielding eight models depicting the complexes.

The free and bound forms were optimized by molecular dynamics and minimization as noted earlier for the helix bundles. 50 models for each complex were ranked according to their stereochemical quality, secondary structure contents were evaluated by the Procheck 3.0 software (33), the compatibility of folds with sequences were scored by Verify 3D (34), and the side chain contact statistics were processed by the What-Check (35).

Solvent-accessible surface areas (ASA) were computed using the Connolly algorithm rolling a spherical probe 1.4 Å in radius around van der Waals spheres of atoms. The interface between two molecules ({Delta}ASA) was calculated as the difference between the complex surface area and the sum of the two separated component surface areas.

Binding Free Energy Estimation—The relative binding free energies between two molecules were estimated from the three-dimensional models using a method based on the areas of polar (oxygen and nitrogen atoms) and non-polar surfaces (carbon and sulfur atoms) exposed to solvent after component dissociation (36) as shown in Equation 1.

(Eq. 1)

Changes in heat capacity {Delta}Cp,calc were calculated from the expression shown in Equation 2.

(Eq. 2)

Changes in enthalpy {Delta}H0 were evaluated at 60 °C using the expression shown in Equation 3,

(Eq. 3)
and they were extrapolated at 25 °C as shown in Equations 4 and 5.

(Eq. 4)

(Eq. 5)

Changes in entropy were calculated as the sum of the conformational entropy , the association entropy , and the solvation entropy defined in Equation 6,

(Eq. 6)
where {Delta}ASASC,i and {Delta}ASABB,i are the ASA changes in the side chain or the backbone of residue i on binding. ASAAXA-SC,i and ASAAXA-BB,i are the ASA of the side chain or the backbone of residue i in an extended Ala-X-Ala tripeptide. Values for backbone entropy and were drawn from d'Aquino et al. (37), and the values for chain entropy () were from Lee et al. (38) as shown in Equations 7 and 8.

(Eq. 7)

(Eq. 8)

Finally, the binding free energy was computed from {Delta}G0 ={Delta}H0T{Delta}S0.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Structure-Function Analysis of IL-2 Peptides—The biological effects of various IL-2 peptides were investigated on the IL-2 proliferation of TS1{beta} cells. Synergy was measured for various concentrations of IL-2 in the presence of different peptide sizes at 60 µM (Fig. 1). This experiment showed that p1–30, p1–31, and p1–31Lys-20 induced the same level of cell proliferation, whereas p10–30 induced 25% less and p20–30 induced 50% less. P1–10, p5–15, p10–20, and p1–22 did not stimulate cell proliferation at all.



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FIG. 1.
p1–30 induces cell proliferation and acts in synergy with IL-2 on human IL-2 receptor {beta}-chain. TS1{beta} cells (104/well) were incubated with a fixed concentration (60 µM) of specific IL-2 peptides and stimulated with various concentrations of IL-2 (0.005–10 nM). After 36 h, cells were pulsed for 15 h with 0.5 µCi of [3H]thymidine before harvesting. Proliferative responses were then assayed.

 

The secondary structures of p1–10, p5–15, p1–22, p10–20, p10–30, and p1–30 were studied by circular dichroism. Fig. 2A shows the spectra obtained in the far-UV range with 150 µM peptides p1–10, p10–20, p20–30, p5–15, p10–30, p1–22, and p1–30. Only two peptides showed >5% helical conformation under these conditions: p10–30 (35 ± 5%) and p1–30 (50 ± 5%) (Table I). The concentration dependence of the dichroic signal was also evaluated between 3 and 150 µM for each peptide (Fig. 2B). The helix content of p1–30 and p10–30 depended on the peptide concentration. The maximum helix content was obtained for p1–30 above 30 µM, and the half-maximum was obtained at 6 µM. For p10–30, the maximum was above 100 µM, and the half-maximum was obtained at 60 µM. The dichroism signal at 198 nm was weakly concentration-dependent for peptides p1–22 and p20–30 (Fig. 2B). Both peptides have very low helix contents but a propensity to form {beta}-strands (Fig. 2A) independent of the peptide concentration above 50 µM and dependent on the peptide concentration below 50 µM (Fig. 2B). p1–10, p5–15, and p10–20 did not form any periodic secondary structures under the test conditions.



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FIG. 2.
Structural analysis of peptide p1–30. A, circular dichroism spectra ({Delta}{epsilon}) from 180 to 260 nm are shown for p1–10, p5–15, p1–22, p10–20, p10–30, p20–30, and p1–30 at 150 µM in 20 µM sodium phosphate buffer, pH 7.2, at 20 °C. B, peptide concentration dependence (3–150 µM) of the CD signal ({Delta}{epsilon}) at maximum peaks.

 

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TABLE I
Secondary and quaternary structure of peptides in solution

 

The quaternary structure of p1–30 was analyzed by sedimentation-diffusion equilibrium experiments performed at various peptide concentrations, spanning its impotent function to potent function. The results are illustrated in Fig. 3 and are reported in Table I. p1–30 behaved mainly as a tetramer below 10 µM and as an octamer at 150 µM. The tetramer-octamer Kd is approximately 60 µM. Below 10 µM, the molecular masses obtained for the lightest species ranged between 10,340 and 16,480 Da, corresponding to 4.0 ± 0.9 peptides/oligomer (Fig. 3I). These results are in close agreement with those obtained by size-exclusion chromatography (22) and therefore confirm that the p1–30 tetramer has a compact conformation. As shown in Fig. 3H and reported in Table I, the p10–30 peptide forms a tetramer at 150 µM by associating two dimers with a Kd of 88 µM, and this may explain is biological activity. p1–10, p5–15, p1–22, and p20–30 are dimers at 150 µM with Kd of 50, 113, 65, and 42 µM, respectively, and they remain inactive.



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FIG. 3.
Quaternary structure of the peptides and IL-2R{beta}31–230. Absorbance profiles versus radius from the center of rotation (C, F, I, L, O, and R) were obtained by sedimentation-diffusion equilibrium at 25,000 rpm in 20 mM sodium phosphate, pH 7.2, at 20 °C for peptides p1–10 (J–L), p10–20 (M–O), p1–22 (A–C), p10–30 (D–F), p20–30 (P–R), and p1–30 (G–I) (initial concentration was 150 µM). Residual errors as a function of radial distance from the center of rotation (A, B, D, E, G, H, J, K, M, N, P, and Q) show the data quality of fit to the selected models as described under "Materials and Methods." Results are reported in Table I.

 

The 10–30 sequence of p1–30 could be responsible for the association of four monomers folded to form helix bundles as proposed by the analysis of circular dichroism experiments, whereas the 1–10 sequence might be responsible for the tetramer dimerization that forms the octameric species. The concentration dependence of helix formation and tetramerization is strongly correlated. The association of four p1–30 peptides stabilizes their helices probably within the 10–30 residue range. It is noteworthy that p20–30 and p1–22, which form {beta}-strands, sediment as dimers (Fig. 3, B and Q, and Table II), but only p20–30 exhibits biological activity.


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TABLE II
Secondary and quaternary structure of IL-2R{beta} in solution

Details of the major species in solution, weight, and dissociation constant were tested over the appropriate range of concentrations.

 

Analysis of IL2-R{beta} Dimerization—The weight of the IL-2R{beta}31–230 molecular species was further studied by analytical ultracentrifugation. At 1 µM protein, sedimentation-diffusion experiments at equilibrium showed an association of two monomers with a Kd of 3 µM. At 14 µM, the molecular mass obtained was very close to the dimer mass (Fig. 4). The mixing of p1–30 (150 µM) with IL-2R{beta}31–230 (1 µM) did not affect the absorbance profile at 295 nm (p1–30 does not absorb at this wavelength) after the equilibrium had been reached at 25,000 rpm. P1–30 did not induce IL-2R{beta}31–230 dimerization in solution at its active concentration. We also verified whether p1–30 binds IL-2R{beta}31–230 dimers in a concentration range from 1 to 14 µM {beta}-chain. We labeled the peptide by placing a fluorescein group on a carboxyl-terminal cysteine and recorded its absorbance profile at 490 nm under the condition described above for p1–30. This peptide, p1–30CysFlc31, showed a concentration-dependent helix conformation and formed octamers and tetramers with Kd values close to those of p1–30 (Table I). Fig. 4 shows 295-nm absorbance profiles for p1–30CysFlc31 (150 µM) and IL-2R{beta}31–230 (14 µM) in separate compartments and mixed in the same compartment (the results are reported in Table II). Clearly, absorbance profiles at 490 nm show that p1–30CysFlc31 gains weight in the presence of IL-2R{beta}31–230. However, because a large number of different molecular species at equilibrium were present in the solution, it was impossible to determine an accurate Kd value. The computer simulation of the sedimentation-diffusion profile suggests that the p1–304 tetramer binds the IL-2R dimer (ideal monospecies) with a Kd greater than the highest peptide concentration we used (Kd >150 µM) between 200 and 300 µM under p1–304-p1–308 equilibrium conditions (octamer dissociation of ~50 µM).



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FIG. 4.
Quaternary structure of IL-2R. A–C, sedimentation equilibrium of IL-2R{beta}31–200; initial concentration was 14 µM. Absorbances were recorded at 295 nm. A model of a dimer (A) shows a closer fit than a monomer-dimer equilibrium (B). D–I, closed circles, sedimentation equilibrium of p1–31CysFl31 and IL-2R{beta}31–200; initial concentrations were 150 and 14 µM, respectively. D–F, profiles at 295 nm (aromatic residue absorbance of IL-2R{beta}31–230) of the peptide-protein mixture was corrected for the absorbance profile of p1–31CysFlc31 at this wavelength. Open circle, IL-2R{beta}31–230 14 µM alone. G–I, profiles at 490 nm; fluorescein absorbance from the mixture of p1–31CysFlc31 and IL-2R{beta}31–200. Open circle, p1–31CysFlc31 150 µM alone.

 

Three-dimensional Models of p1–304Three-dimensional models of p1–304 were built from diverse four-helix bundles (IL-2, erythropoietin, and ROP) by different tuning alignments of the helix sequences for the different hydrophobicity profiles. Obviously, the 8–28 helix built from p1–30 is amphipathic. The bundle arrangements buried 6 of the 10 aliphatic hydrophobic side chains when the helix axes were parallel, and bundle arrangements buried 8 of 10 aliphatic hydrophobic side chains when the axes were right-tilted. Binding free energies for the transition from monomer to tetramer were 0–3 kcal/mol lower for the 1-1-1-1 bundle than the other configurations using the polar/nonpolar solvent accessible surface-based method. The 1-1-1-1 configuration seemed even more favorable when we included a term for potential energy from the CFF91 force field that accounts for electrostatic contributions made by monopolar-monopolar interactions that decreased the bundle energy by 3–8 kcal/mol compared with other helix arrangements. Attractive contributions from electrostatic dipoles formed by two anti-parallel helices were approximately–0.5 kcal/mol/pair. However, the total contribution made by dipolar-dipolar interactions was approximately–4 kcal/mol smaller than the entropy earned by grouping hydrophobic residues together in the same core away from the water. The 1-1-1-1 arrangement is certainly the most favorable with helix axes tilted relative to the bundle axis (5–11 kcal/mol lower) (Fig. 5). This configuration mimics the actual arrangement of the four-helix bundle family of cytokines such as IL-2 and erythropoietin.



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FIG. 5.
p1–304 helix bundle. Helices are juxtaposed in an anti-parallel arrangement (1–1–1–1) viewed perpendicularly to the bundle axis (A) or along the axis (B). Hydrophobic side chains are displayed as sticks with each chain a different color.

 

Three-dimensional Models of IL-2R{beta}2 and p1–304·IL-2R{beta}2 Fig. 6 shows three-dimensional models of IL-2R and the p1–304·IL-2R complex. Table III gives their interface areas and the values in the corresponding templates. The three-dimensional models of free IL-2R (Fig. 6, A and B) based on the crystal structure of the erythropoietin receptor (1ERN [PDB] ) give {beta}-chain contact areas of approximately 1760 Å2, larger than the erythropoietin receptor homodimeric chain interface (1430 Å2) used as a template. The distance between the two IL-2R{beta}31–230 COOH termini is 83 Å, i.e. approximately the same as for EPOR in the template model (1ERN [PDB] ).



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FIG. 6.
Three-dimensional models of p1–304·IL-2R{beta}2 complex and of free IL-2R{beta}2. Ribbon graphs of the IL-2R peptide-free dimer (A, front view; B, top view) built by comparative modeling using EPOR as template (1ERN [PDB] ) and of the p1–304·IL-2R complex (C and D) based on EPO·EPOR (1EER [PDB] ).

 

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TABLE III
Interface areas

 

The 31–200 fragment of the IL-2R{beta} chain interacts with two p1–30 peptides in the p1–304·IL-2R complex. Anti-parallel arrangements consistently gave the lowest binding free energies for the p1–30 peptides orientated in the same way as proposed by Bamborough et al. (16) for IL-2 helix A in the model put forward for the IL-2·IL-2R{alpha}{beta}{gamma} complex. The model of the p1–304(1-1-1-1)·IL-2R complex (Fig. 6, C and D) shows a 2-fold symmetry that is not present in any of the templates employed. The axis of the helix bundle (50 x 25 x 25 Å) is perpendicular to the 2-fold symmetry axis of the two IL-2R{beta}31–230 subunits (100 x 70 x 35 Å) that are in the same plane. The distance between the two COOH termini is approximately 28 Å (39 Å for the template). In the three-dimensional model of the p1–304(1-1-1-1)·IL-2R complex (Fig. 6, C and D) derived from the EPO·EPOR complex (1EER [PDB] ), the interface between the two IL-2R{beta}31–230 chains is ~300 Å2 in the model (76 Å2 for the template), and between p1–304 and IL-2R, the interface is ~4300 Å2 (3300 Å2 for the template). In the three-dimensional model of the p1–304(1-1-1-1)·IL-2R{beta}2 complex derived from the GH·GHR receptor (3HHR [PDB] ), the interface between the two IL-2R{beta} chains is approximately 1100 Å2 in the model (952 Å2 for the template), and between p1–304 and IL-2R{beta}2, the interface is approximately 4684 Å2 (4118 Å2 for the template). By way of a comparison, in the IL-2·IL-2R{alpha}{beta}{gamma} three-dimensional model, the interface between the two IL-2R{beta} chains and IL-2R{gamma} chains is approximately 1920 Å2, and between IL-2 and IL-2R{beta}{gamma}, the interface is 3800 Å2. The reduction in the {beta}:{beta} interface (–1460 Å2) when passing from the peptide-free IL-2R receptor (1760 Å2) to the peptide-bound receptor p1–304·IL-2R (300 Å2) is compensated by the gain of peptide-receptor interaction (4300 Å2).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We prepared a range of synthesized peptides, which covered various lengths of the amino-terminal sequence of IL-2, and tested their effects on TS1{beta} cell proliferation. p1–30, p1–31, p1–31Lys-20, and p10–30 acted in synergy with IL-2, enhancing the basic cytokine-induced cell stimulation, whereas peptides p1–10, p5–15, p1–22, p10–20, and random peptides did not. Peptide activity was inhibited by a neutralizing monoclonal antibody directed against IL-2R{beta}. Human IL-2R{beta} expression was an absolute requirement for the p1–30-proliferative response. The possible implications of IL-2R{alpha} and {gamma} subunits in p1–30 binding were rejected because p1–30 was active in the absence of IL-2R{alpha} expression in TS1{beta} cells, and its activity was not significantly affected by a neutralizing monoclonal antibody directed against IL-2R{gamma}. We concluded that p1–30 activity is dependent only on interactions with human IL-2R{beta}. p10–30 is active on TS1{beta} cells, indicating that the COOH-terminal part of the p1–30 peptide is more important than its NH2 terminus. We then sought to discover any specific structural features associated with this activity. Circular dichroism studies in the far-UV indicated that the active peptides contained a high proportion of {alpha}-helix at their active concentration, whereas inactive peptides did not. Remarkably, the helix contents of p1–30 and p10–30 depended on their concentration, suggesting that peptide oligomerization stabilizes their folding. At a concentration below 5 µM, p1–30 was inactive and half the optimal helix content was lost. p1–30 and p10–30 are mainly homotetramers in the concentration range where their biological activities are observed.

Our investigation has shed light on mechanisms that explain previously reported observations. p1–30 shows some propensity to form octamers with a dissociation constant to tetramers of approximately 50 µM. The optimal peptide concentration for IL-2 synergy assays was also around 50 µM, i.e. conditions where the concentration of tetramer peptide is the highest. At higher peptide concentrations, p1–30 activity decreases (22). This finding suggests that competition occurs between the assembly of two p1–304 forming a peptide octamer or the binding of p1–304 with the IL-2R{beta}2 receptor. From our models, octamers would not bind to IL-2R{beta} and would therefore act as inhibitors of p1–30 activity. The sedimentation of p1–10 and p5–15 was suggestive of dimer formation. The p1–30 NH2 terminus could be involved in the association of two tetramers. However, because no peptide aggregation was detected up to 50 µM, we assumed that only two tetramers were pooled together head to head or as an eight-helix bundle. The p20–30 peptide was described as active. Circular dichroism analysis showed that it forms {beta}-strands. Analytical ultracentrifugation experiments revealed that it acted as a dimer. The same phenomenon has also been described for erythropoietin receptor binding agonist peptides such as EMP1 (39, 40). The binding of p20–302 dimers organized in {beta}-strands was obviously different in its features from p1–30 assembled in {alpha}-helix bundle. The decrease in cell activation at high concentrations of p20–30 was interpreted as resulting from {beta}-strand peptide aggregation.

The p1–30 amino acid sequence includes seven leucines (positions 12, 14, 17, 18, 19, 21, and 25), two isoleucines (positions 24 and 28), and one methionine (position 23) among the 18 carboxyl-terminal residues. The periodicity of these amino acids suggests that most of these hydrophobic side chains are grouped on the same face and form an amphiphilic helix. By similarity with many known structures of four-helix bundles, we propose an arrangement where the four hydrophobic faces are pooled one in front of each other, forming a hydrophobic core that stabilizes the homotetramer. This assembly mimics a cytokine of the hemopoietin family. However, the arrangement of the {alpha}-helices in tetrameric p1–30 has not yet been determined experimentally, but we predict a 1-1-1-1 configuration (up-down-up-down) as most favorable from the binding energies of the four helices in the three-dimensional model bundle (Fig. 5).

The biological effects of most cytokines are exerted after heterodimerization or homodimerization of their receptors. The p1–304 tetramer has potentially four recognition sites for IL-2R{beta}. Analytical ultracentrifugation suggests that IL-2R dimers are formed in the absence of the peptide. It also proposes the stoichiometry of the p1–304·IL-2R assembly as four peptides for two receptor fragments. However, IL-2R{beta}31– 230 is monomeric in solution below 1 µM, and p1–30 concentrations up to 150 µM do not promote its dimerization. The Kd of IL-2R{beta} dimerization may be lower than that for the 31–200 fragment because of its membrane anchoring, which allows bidimensional diffusion in a preoriented manner (in and out). Thus, we propose that IL-2R{beta} dimers are formed prior to the binding of p1–30 tetramer, which is responsible for the signaling. Therefore, we do not expect transmembrane and internal IL-2R{beta} domains to contribute to the binding.

The models based on EPO·EPOR (23) suggest that IL-2R{beta}31–230 COOH termini are >80 Å apart in the peptide-free state and closer than 30 Å when the peptides are bound. The interface area between two IL-2R{beta}31–230 chains in the dimers appears to decrease 10-fold upon binding p1–304. This loss is compensated by the large interface formed between p1–30 and IL-2R{beta}31–230. P1–304 does not contribute to IL-2R{beta}31–230 association, dimers of IL-2R{beta} chain preexists in solution, and the binding of the peptide bundle to the dimer receptor requires a costly conformational receptor change. Thus, these predictions suggest that peptide association and dissociation to and from the receptor follow slow kinetics. The two {beta}-chains do not interact as tightly as in the peptide-free IL-2R{beta}2 model or as observed for the {beta}- and {gamma}-chains in the IL-2R{alpha}{beta}{gamma} model (16). The mechanism proposed for erythropoietin should be applicable (23, 41). The binding of p1–304 partially disrupts the {beta}:{beta} interface, rotates the COOH-terminal domains clockwise by 30o, and brings them closer together. These extremities are attached to the transmembrane region by extension in the full-length of the {beta}-chain. This arrangement is suggestive of an interaction between the intracytoplasmic domains responsible for signal transduction, whereas these domains are held far from each other in the peptide-free receptor. The NH2-terminal domain of IL-2R{beta}, which forms a six-strand {beta}-barrel, would also be closer to the membrane surface when the peptide is bound. The reduction in the {beta}:{beta} interface area is compensated by peptide-receptor interactions from the peptide-free to peptide-bound model, and the sum of interface areas is increased by 2840 Å2. This is in the same order of magnitude as for EPO·EPOR (1946 Å2, Table III). These models suggest that the binding of the p1–30 tetramer rotates the COOH-terminal domains and brings both transmembrane regions 50 Å closer together, driving the association of the two intracytoplasmic domains that transduces the signal into the cytoplasm.

This model explains how p1–30 induces signals through IL-2R{beta}2 different from IL-2, because IL-2 signals are generated by IL-2R{alpha}{beta}{gamma} complexes. In the same manner as IL-2 activation, Shc and p56lck were activated, but unlike IL-2 activation, Jak-1 and Jak-3 were not phosphorylated and the STAT pathway was not induced. In the IL-2·IL-2R model, Shc and p56lck are dependent on IL-2R{beta}, whereas the STAT pathway is dependent on IL-2R{beta}·IL-2R{gamma} heterodimerization. Therefore, p1–30 signals appear to be dependent only on IL-2R{beta}, and this further supports the p1–304·IL-2R model described above. Surprisingly, it was also shown that p1–30, similar to IL-2, induced Tyk-2 phosphorylation.

Peptide p1–30 has therapeutic potential. Because NK cells express large amounts of IL-2R{beta}, whereas monocytes express IL-2R{gamma} (42), NK cells could constitute a major target of p1–30. IL-2 increases the cytotoxic response of NK cells, inducing the expression of granzyme B and perforine. The corresponding signaling pathways are unknown, and induction of these effects by p1–30 is not excluded. IL-2 also stimulates monocytes/macrophages and induces tumor necrosis factor-{alpha} and nitric oxide production, which may be responsible for the side effects observed in patients injected with IL-2. In this context, the p1–30 peptide, which is useful to further investigate the IL-2·IL-2R system, could also have practical applications if proven to be active in vivo and induce fewer side effects in patients. Furthermore, p1–30 derived peptide may induce less vascular leak syndrome (VLS). VLS involves damage to vascular endothelial cells leading to vascular leak, edema, and organ failure. A recent study (43) provides evidence that a (X)D(Y)-conserved motif in the plant toxin ricin A chain, IL-2, and other VLS-inducing proteins may be responsible for binding to endothelial cells and initiating VLS. This motif is located in {alpha}-helix A of IL-2 centered on Asp-20. Short peptides containing this motif induce VLS, whereas mutated peptides do not. Because the p1–30 peptide also exhibits this motif, we generated a mutated peptide (p1–30Lys-20) abrogating this motif and tested its activity. We firstly studied its capacity to induce TS1{beta} cell proliferation in comparison with p1–30. TS1{beta} cells were stimulated by various concentrations of IL-2 (102 to 5.101 nM) in the presence of peptide p1–31 or p1–31Lys-20 (60 µM) (peptides p1–31 and p1–30 have a similar activity). No differences were detected with p1–30. This result demonstrated that Asp-20 is not implicated in p1–30 activity. If further studies confirm that VLS is strictly dependent on this motif, this will open the way for the possible production of mutated peptides of therapeutic interest.

In conclusion, p1–30-derived peptides may prove to be useful for immunotherapy. In this context structural modeling may help in the design of new molecules with higher therapeutic indices.


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

To whom correspondence should be addressed: Dépt. de Médecine Moléculaire, Unité d'Immuno-Génétique, Institut Pasteur, 28 rue du Dr. Roux, 75724 Paris Cedex 15, France. Tel.: 33-01-45-68-86-00; Fax: 33-01-45-68-88-38; E-mail: jtheze{at}pasteur.fr.

1 The abbreviations used are: IL, interleukin; CD, circular dichroism; EPO, erythropoietin; Flc, fluorescein; Jak, Janus kinase; LAK, lymphokine-activated killer cell; PTK, protein tyrosine kinase; STAT, signal transducers and activators of transcription; VLS, vascular leak syndrome; Flc, fluorescein group; n, partial specific volume; ASA, accessible surface areas. Back


    ACKNOWLEDGMENTS
 
All of the peptides used in this study were synthesized by Dr. Marc Bossus in the Unit of Prof. André Tartar (Institut Pasteur de Lille). Their cooperation during the present work has been very critical. We are also grateful to Dr. Pedro Alzari (Unité de Biochimie Structurale, Institut Pasteur), Dr. Michael Nilges (Unité de Bio-Informatique Sructurale, Institut Pasteur), and Dr. Françoise Baleux (Unité de Chimie Organique, Institut Pasteur) for valuable discussion and critical review of the paper.



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