Functional epitope of common c chain for interleukin-4 binding Jin-Li Zhang, Manfred Buehner and Walter Sebald Theodor-Boveri-Institut fu ¨ r Biowissenschaften (Biozentrum), Physiologische Chemie II, Universita ¨ tWu ¨ rzburg, Germany Interleukin 4 (IL-4) can a ct on target cells through an IL-4 receptor c omplex consisting of the IL-4 receptor a chain and the c ommon c chain (c c ). A n IL-4 epitope for c c binding has previously been identified. In this study, the c c residues involved in IL-4 binding were defi ned by alanine-scanning mutational analysis. The epitope comprises c c residues I100, L102, and Y103 on loop EF1 together with L208 on loop FG2 as the major binding determinants. These predomin- antly h ydrophobic determinants i nteract with t he hydro- phobic IL-4 epitope composed of residues I11, N 15, a nd Y124. Double-mutant cycle a nalysis revealed co-operative interaction between c c and IL-4 side chains. Seve ral c c residues involved in IL-4 binding have b een previo usly shown to be mutated in X-linked severe combined immunodeficiency. The importance of these binding residues for c c function is discussed. These r esults provide a basis f or elucidating the molecular recognition mechanism in the IL-4 receptor system and a paradigm for other c c -dependent cytokine receptor systems. Keywords:commonc chain; interleukin 4; mutagenesis; protein–protein interaction; structure/function. Interleukin-4 ( IL-4) is a multifunctional cytokine that plays a critical role in t he regulation of immune responses [1,2]. It induces the generation o f Th2-dominated early immune response [3] and determines the immunoglobulin class switching t o I gE [4]. Dysregulation of IL-4 function is strongly correlated w ith type I hyper sensitivity reactions, such as allergies and asthma [5]. The IL-4 receptor complex is therefore a potential target f or the development o f antiallergic drugs. The central role of IL-4 in the develop - ment of Th2 cells sugge sts that it may be of benefit in the treatment of autoimmune disease characterized by an imbalance of Th cells [6]. Its ability to induce growth arrest and apoptosis in leukemic lymphoblasts in vitro [7] suggests that IL-4 is also a p romising cytokine for the treatment o f high-risk acute lymphoblastic leukemia. Understanding the molecular recognition m echanism in the IL-4 receptor system is a p rerequisite for the rational design of IL-4-like drugs. IL-4 is one of the s hort-chain four-helix bundle cytokines. Its effects depend on binding to and s ignaling t hrough a receptor complex consisting of a p rimary high-affinity binding subunit, the IL-4Ra, a nd a l ow-affinity receptor, depending on the cell type, the common c chain (c c ;typeI IL-4 receptor [8]) or IL-13Ra1 chain (type II IL-4 receptor [9]). All three receptors are members of the type I cytokine receptor superfamily, w hich is charac terized by the presence of at least one cytokine-binding homology r egion (CHR) composed of two fibronectin type III domains. The membrane distal domain contains a set of four conserved cysteines, and the membrane proximal domain contains a WSXWS motif [10]. T he fibronectin type III domain is comprised of s even b strands, t he sequences of which are conserved b etween members o f the family, while loop sequences connecting the b strands vary between family members and putatively contain r esidues that mediate distinct intermolecular c ontacts. These loop regions were therefore selected for this mutational analysis. A comprehensive mutational analysis of I L-4 i n w hich single residues were replaced by alanine or charged residues yielded high-resolution data on the binding epitopes f or the receptor chains. The IL-4 site 1 binding epitope for IL-4Ra consists of a mixed charge pair (E9, R88) as major determinants and five minor determinants l ocated on helices A, B, and C [11]. The importance of site 1-binding determinants and their partner residues o n I L-4Ra (D72, Y183 as key binding determinants) was subsequently confirmed and further d efined by d etermining the c rystal structure of the 1 : 1 IL-4/IL-4Ra ectodomain (IL-4- binding protein, I L-4BP) complex [12] and by mutational analysis of the IL-4BP binding epitope [13]. The results have already be en used for the rational design of IL-4 minipro- teins [14]. The IL-4 s ite 2 epitope for c c comprises residues I11 and N15 on helix A together with Y 124 on helix D as major b inding determinants and three minor determinants K12, R121, an d S125 o n helices A a nd D [ 15]. A double mutant of IL-4 that completely inhibits responses induced by IL-4 and IL-13 by disrupting the binding of the IL-4 site 2 epitope to c c or IL-13Ra1provedtobeaverypromising anti-asthma drug [ 16–18]. T wo f urther I L-4 m utants that selectively inhibit IL-4-induced activity on endothelial cells appeared to b e good candidate drugs for the treatment of certain autoimmune diseases [6] and high-risk acute lymphoblastic l eukemia [7]. However, t he residues on c c that contribute to IL-4 site 2 binding remain uncertain. Correspondence to W. Sebald, Theodor-Boveri-Institut fu ¨ rBiowis- senschaften (Biozentrum), Physiologische Chemie II, Universita ¨ t Wu ¨ rzburg, Am Hubland, D -97074 Wu ¨ rzburg, Germany. Fax: + 49 931 888 4113, Tel.: + 49 931 888 4111, E-mail: sebald@biozentrum.uni-wuerzburg.de Abbreviations: IL-4, interleukin-4; IL-4Ra, interleukin-4 receptor a chain; IL-4BP, IL-4 binding protein; c c , common c chain; IL-13Ra1, IL-13 receptor a1 chain; CHR, cytokine-binding homology region; Jak, Janus kinase; XSCID, X-linked severe combined immunodefi- ciency; hGHR, human growth hormone receptor; hEPOR, hum an erythropoietin receptor; b c ,commonb chain. (Received 14 November 2001, revised 16 January 2002, accepted 21 January 2002) Eur. J. Biochem. 269, 1490–1499 (2002) Ó FEBS 2002 c c is shared by several important cytokine receptor complexes, including those for IL-2, IL-4, IL-7, IL-9, IL-15 [8] and also for the recently described new member of the cytokine family, I L-21 [19]. c c alone binds ligands with very low affinity (K d % 150 l M for IL-4) [15]. Recruitment of c c into receptor complexes for the above cytokines increases receptor af finity for binding [20–22]. c c participates in cytokine signaling in several receptor complexes via JAK3 [23]. Mutations of either c c or JAK3 result in X-linked severe combined immunodeficiency (XSCID) which is characterized by a failure in T and NK cell d evelopment [24]. c c -knockout mice have been generated a nd their immune system successfully reconstituted by gene therapy [25,26]. Initial attempts a t gene t herapy for patients w ith XSCID had been successful for more than 10 m onths [27,28]. Thus, defining the IL-4-binding determinants on c c is important not only for elucidating molecular recognition and activation mechanisms in the IL-4 receptor system and possibly providing a paradigm for other c c -dependent cytokine receptor systems, but also for delineating the molecular pathology of XSCID. So far, the binding epitopes o f human and m urine c c for some c c -dependent cytokines have been studied. A molecular mapping study using the antagonistic monoclonal a ntibody PC.B8, which reacts with a discon- tinuous site on human c c , localized c c binding residues to four loops, but did not identify single specific residues for ligand binding [29]. Mutational analysis of murine c c employing heterodimeric IL-2R and IL-7R on whole cells suggests that c c epitopes for IL-2 and IL-7 binding overlap a nd comprise at least t hree distinct putative loop segments of the c c protein [ 30]. Here we report the effect of single amino-acid substitutions in the human c c ectodomain on IL-4 binding. Biosensor techniques employing s oluble r ecombinant I L-4, IL-4-BP and the wild type or mutant forms of human c c ectodomain revealed the c ontributions of c c residues to IL-4 binding. The possible co-operativity between some residues on t he c c epitope and t he IL-4 site 2 epitope was a nalyzed b y double-mutant cycle analysis. EXPERIMENTAL PROCEDURES Protein expression and purification The ectodomain of human c c comprising amino-acid residues 1–232 [20] was expressed with a C-terminal thrombin cleavage site (LVPRGS) plus a His 6 tag in SF9 insect cells according t o the manufacturer’s instructions (PharMingen). The protein was isolated from the culture medium of infected SF9 cells by standard procedures involving Ni 2+ /nitriloacetate/agarose (Qiagen), digested with thrombin (Sigma), and purified by ge l-filtration chromatography through a Superdex 200 HR 10/30 c ol- umn (Pharmacia). After exhaustive dialysis against water, the purified protein was freeze-dried and stored at )80 °C. ThecDNAforthemurinec c ectodomain comprising residues 1–233 [31] was cloned into the temperature- regulated e xpression vector pRpr9 fd [32], expressed i n Escherichia coli strain KS 474, and refolded as desc ribed [33]. The refolded protein was purified to homogeneity by gel-filtration chromatography through a Superdex 200 HR 10/30 column, and stored at )80 °C. The A 182, C207 IL-4BP variant was produced in SF9 cells, purified, and biotinylated at C207 as described [32]. IL-4 and IL-4 variants were expressed in E. coli, refolded, and pu rified to homogeneity as described [11,34]. Protein concentrations were determined by measuring A 280 ,using an absorption coefficient (e 280 ) ¼ 8860 M )1 Æcm )1 for IL-4, e 280 ¼ 7370 M )1 Æcm )1 for A124 IL-4, e 280 = 66 930 M )1 Æcm )1 for IL-4BP, e 280 ¼ 61 450 M )1 Æcm )1 for human c c , e 280 ¼ 60 170 M )1 Æcm )1 for A103 human c c , and e 280 ¼ 45 660 M )1 Æcm )1 for murine c c . Mutagenesis of the c c ectodomain cDNA for human c c ectodomain was submitted to in vitro cassette mutagenesis employing synthetic double-stranded oligonucleotides. The c c variants were expressed and purified as the wild-type human c c ectodomain. Biosensor interaction analysis The binding of c c variants to I L-4/IL-4BP was recorded on a BIAcore 2000 system ( Pharmacia B iosensor) as described [15]. Briefly, a CM5 biosensor chip was first loaded with streptavidin in flow cells 1 and 2. Subsequently biotinylated A182,C207 IL-4BP was immobilized at the streptavidin matrix of flow cell 2 at a density of % 200 resonance units. The following reaction cycle was applied using the c om- mand COINJECT: ( a) IL-4 at 0.1 l M in HBS buff er (1 0 m M Hepes, pH 7.4, 150 m M NaCl, 3 .4 m M EDTA, 0.005% surfactant P20) was perfused over flow cells 1 and 2 at a flow rate of 10 lLÆmin )1 at 25 °C f or 2 min; (b) 0.1 l M IL-4 plus c c ectodomain o r c c variants at 1–10 l M inthesamebuffer were perfused in the same way for 2 min; (c) H BS buffer alone was perfused f or 5 min; ( d) free receptors were regenerated by perfusion with 0.1 M acetic acid/1 M NaCl for 30 s. Sensograms were recorded at a data-sampling r ate of 2.5 Hz and evaluated as described [15]. Equilibrium binding of c c variants at 1, 2, 3, 5, 10 l M was measured for at least three times in duplicate. The mean standard deviation (mean r) was 13.8% ± 6 .5% for the K d values calculated from the fi ve variant concentrations. For the double mutant cycle analysis [35], the same procedure as a bove was used except that IL-4 variants [15,36] at 0.1 l M and c c variants at 2, 4, 6, 10, 20 l M were perfused (the mean r was 16.4% ± 7.4% for the K d values). The loss of binding free energy on mutatio n for IL-4 and c c wascalculatedasddG (kJÆmol )1 ) ¼ 5.69 log K d (mutant)/K d (wild-type). The interaction energy between two residues was calculated by the double-mutant cycle method as in Eqn. (1): ddG int ¼ ddG X-A þ ddG Y-B À ddG X-A;Y-B ð1Þ where ddG X-A and ddG Y-B are t he changes in binding energy on mutation of X to A and Y to B (mutation of IL-4 and c c in this experiment), respectively, and ddG X-A, Y-B the change on the simultaneous mutation of X to A and Y to B. ddG int is a measure of the co-operativity of the interaction of the two components mutated. ddG int ¼ 0indicatesthat the pair o f residues analyzed do n ot interact. A positive value of ddG int means that two residues interact favorably, and a negative value means that the two residues repel each other [37]. The individual errors (2 r, a ¼ 0.95) calculated from the mean for ddG int areshowninTable3. Ó FEBS 2002 Mutagenesis of human c c ectodomain (Eur. J. Biochem. 269) 1491 Molecular modeling of the IL-4–IL-4BP–c c ternary complex The present model i s based on the crystal structure of the complexofIL-4andIL-4BP(PDBentry1IAR[12]), augmented by the model of c c derived f rom human growth hormone receptor (hGHR), as obtained from an older model (T. Mueller, & W. Kammer, personal communica- tion, Universita ¨ tWu ¨ rzburg, Germany) 1 of the ternary complex of IL-4–IL-4BP–c c . This old model was based on the struc ture of f ree IL-4 (PDB entry 1 HIK [ 38]) a nd of models of the e xtracellular domains of IL-4Ra and c c obtained by analogy modeling following the structure of the hGHR complex (PDB entry 3HHR [39]). The 3 HHR data were o btained from the protein databa nk (PDB [40]). The old model was built in such a way that all cysteine residues formed proper d isulfide bonds, and all evidence from mutation experiments a vailable a t the time was used to adjust the binding epitope s of the recep tor chains. The resulting alignment r equired some nontr ivial rebuilding with insertions an d deletions, and, consequently, t he resulting model of the IL-4 receptor complex had to be extensively energy refined. The p rogram O [41] was used f or model building, and the program X - PLOR [42] for energy refinement. The differences between the experimentally determined binary complex and the corresponding components of the old model were significant in detail, but the gross changes were small en ough that t he binding topology of c c could be transferred to the new m odel without major problems. The local program DISDM 2 was used (H. J . Hecht, & M. Buehner, unpublished r esults) t o build and adjust the present model using the data of mutational a nalysis of IL-4 and c c . The program runs under Open-VMS and uses Datagraph VTC 8002 and VTC 8003 terminals for display. All model building was performed manually. For online refinement of conformational energy, the p rogram EREF was used [43], which is called from within DISDM 2. RESULTS Site-specific mutagenesis of amino acids in the c c ectodomain Alanine substitutions were targeted to residues in four putative i nterconnecting loops and the interdomain segment of the human c c ectodomain based on the published models [44–46], and sequence alignment performed between c c and several cytokine receptors, the major ligand-binding deter- minants of w hich were identified. These include the hGHR [39,47], the human erythropoietin receptor (hEPOR [48,49]), IL-4BP [12], and the human gp130 (hgp130 [50,51]). E ighteen c c variants were generated with amino- acid substitutions in the AB1, EF1, BC2, FG2 loops and t he interdomain segment (Fig. 1). A deletion m utant lacking residues 1–33 of the N-terminus of c c ,namedc c CHR, was also generated to find o ut whether this N-terminal region of c c is required for ligand bindin g. All human c c wild-type or variant proteins could b e purified to apparent homogeneity by Ni 2+ /nitrilotriacetate/ agarose and gel fi ltration. The wild-type human c c ectodo- main expressed in SF9 cells was recovered as monomeric and dimeric species [52,53]. The murine c c ectodomain expressed i n E. coli occurred as a monomer (Fig. 2). Initial biosensor studies showed that the different forms of human and m urine p roteins exhibited similar b inding affinity for the IL-4–IL-4BP complex. The mixture of monomeric and dimeric human c c interacts with the complex with a K d of Fig. 1. Amino-acid substitutions in the ectodomain of the human com- mon c chain (c c ). The amino-acid sequence of c c is shown with boxed portions i ndicating predicted b-strands which are designated by the letter below the box. R esidues substituted in this study are indicated by asterisks. Fig. 2. Gel-filtration analysis of the human c c ectodomain expr essed in SF9 cells and the m urine c c ectodomain expressed in E. coli. The samples were applied to a Superdex 200 HR 10/30 c olumn and eluted w ith the same buffer. The two peaks of human c c represent dimer (A) and monomer (B). 1492 J L. Zhang et al.(Eur. J. Biochem. 269) Ó FEBS 2002 4 l M , a nd murine c c with a K d of 1.6 l M (Fig. 3 and Table 1). In addition, different preparations of wild-type human c c ectodomain consistently showed a K d of 4 l M irrespective of the monomer t o d imer ratio (data not shown). Therefore, the mixtures o f dimeric and monomeric human c c protein w ere u sed for all b iosensor measurements. The c c epitope for IL-4 binding The method of measuring the binding of c c to IL-4–IL-4BP by biosensor was established previously [15]. The d issocia- tion constant K d evaluated from the concentration dependence of e quilibrium binding proved to be very reliable for measuring the interaction of the c c ectodomain with IL-4–IL-4BP. The measured K d for interaction of c c ectodomain variants with IL-4–IL-4BP are compiled in Table 1. Eight c c variants including c c CHR exhibited unchanged binding characteristics. Changes in binding affinity were observed in 11 c c variants. The K d of six variants was too high to be r eliably determined. A r ough estimate yields K d values of about 200–300 l M for I100A, L102A, Y103A and L208A, and K d values of about 500– 1000 l M for C160A and C209A. The K d values of five variants, N128A, H15 9A, L161A, E162A, and G210A, were found to be increased threefold to fourfold compared with the K d of wild-type c c , suggesting that these residues are part of the c c binding interface, but do not play a key role in binding. The loss of binding affinity of the four variants I100A, L102A, Y103A a nd L208A i s not likely to be c aused by extended structural alterations, a s I 100A, L102A, and L208AwerereportedtobindtoIL-2andIL-7withthe same affinity a s wild-type c c , and the Y 103A mutation resulted in only twofold to thre efold reduced IL-2 and IL-7 binding [30]. Thus, the four residues I100, L 102, Y103 and L208 are hot spots on c c , contributing > 9 kJÆmol )1 each. The five minor residues investigated contribute only 2.9– 3.5 kJÆmol )1 . The two cysteine variants C160A and C209A exhibited a largely r educed binding affinity ( K d > 500 l M ). Thismaybecausedbystructuralperturbationofthe protein. A direct role in binding, however, cannot be excluded for these residues. Double-mutant cycle analysis of the IL-4–c c interface The c o-operativity of the interaction of some residues on the IL-4 site 2 epitope and the c c ectodomain was determined in this experiment. Double-mutant cycles were con structed only for the mutants with minimal effects on b inding (Tables 2 and 3), because the K d values for the interaction between variants o f the main binding residues I100, L102, Y103, a nd L2 08 of c c andIL-4variantsaswellasthe interaction betwee n variant I 11A o f I L-4 a nd c c variants were too high to b e reliably d etermined. Interaction be tween residues on IL-4 and c c can be grouped according t o their coupling energies (Table 3). The main binding determinant of IL-4 Y124 failed to e xhibit positive coupling energies with any of the c c residues analyzed. IL-4 Y124 probably interacts w ith the main functional side chains of the receptor located on loop EF1 (I100, L102, Y103), the binding of the alanine variants of which was too weak to be analyzed by this approach. Remarkably, IL-4 S125 neighboring Y124 does show coupling to receptor N128 in addition to that to Fig. 3. Sensograms rec ording the binding of h uman and m urine c c ectodomains to the IL-4–IL-4BP complex. IL-4BP was i mmobiliz ed on the biosensor matrix. At time zero, perfusion with 100 n M IL-4 was initiated. The saturation binding of IL-4 was arbitrarily set as zero. After 120 s, perfusion was continued with 1 00 n M IL-4 plus c c ecto- domain. In different cycles, 5 l M human (a) or murine (b ) c c ecto- domains w e re applied. Perfusion with buff er alone starte d at time 240 s. The ruler indicates resonance units (RU) corresponding to 1–10 l M murine c c ectodomain. Th e resonance u nit f or 5 l M human c c corresponded to that for 1.6–2 l M murine c c . Table 1. Equilibrium binding between c c ectodomain mutants and IL-4–IL-4BP. The dissociation constants K d were evaluated from equilibrium binding between wild-type (wt) or mutants (mut) of the c c ectodomain and immobilized IL-4BP saturated with IL-4. The l oss of free energy of binding on mutation was calculated as ddG (kJÆmol )1 ) ¼ 5.69 log K d (mut)/K d (wt). Alanine variant Equilibrium binding ddG (kJÆmol )1 ) K d (l M ) K d (mut)/K d (wt) Murine c c (wt) 1.6 Human c c (wt) 4.0 1.0 0.0 Human c c CHR 4.5 1.1 0.2 Loop 1 (AB1) N44A 2.8 0.7 )0.9 V45A 3.3 0.8 )0.5 Loop 3 (EF1) E99A 5.5 1.4 0.8 I100A >320 >80 >11 L102A >240 >60 >10 Y103A >300 >80 >11 Q104A 5.6 1.4 0.8 Loop 4 (ID) Q127A 2.4 0.6 )1.3 N128A 15 3.7 3.2 Loop 5 (BC2) N158A 1.4 0.4 )2.3 H159A 17 4.1 3.5 C160A >900 >230 >13 L161A 13 3.2 2.9 E162A 13 3.2 2.9 Loop 6 (FG2) P207A 3.4 0.9 )0.4 L208A >166 >40 >9 C209A >490 >120 >12 G210A 15 3.7 3.2 Ó FEBS 2002 Mutagenesis of human c c ectodomain (Eur. J. Biochem. 269) 1493 G210. The IL-4 side c hain of N15 functionally interacts with the central receptor s ide chain N128, a nd also with H159 located at the periphery of the functional c c epitope. The relative positions of the c oupling side c hains as proposed by our theoretical model of the ternary complex (see below) are presented i n t he open-book view in Fig. 4A,B. T he two receptor side chains N1128 and H159 are 12 A ˚ apart in t he c c model. This could indicate that our c c model is inaccurate, because this model does not completely fit the results of t he double-mutant cycle analysis. Alternatively, the interaction of IL-4 side chain N15 with H159 (coupling energy only 0.8 k JÆmol )1 ) m ay be indirect. Of p articular interest is the I L-4 side chain of R121, which, after being substituted with D or E, leads t o a selective IL-4 agonist specifically impaired in IL-13R a1 binding [6,7,16,17,34]. The IL-4 R 121 was distinct in showing positive coupling during interaction with the c c side chain L161. Model of the structure of the IL-4–IL-4BP–c c ternary complex In a series of steps, c c was adapted to achieve a good fit to the core structure (the binary complex; Fig. 5). The procedure s tarted with moving the whole chain (Ôrigid bodyÕ). Then domains and subdomains were moved indi- vidually. The binary core complex was changed as little as possible, being an experimentally determined structure and thus the most reliable p art of the model, but some minor changes in side chain o rientation could not be avoided for proper a daptation. An important point was to keep t he C-terminal domains of the receptor c hains c lose together, as this was expected to be essential for dimer formation and thereby signaling through the membrane. The structures of c c and the ternary complex were modeled so that residues that exhibit positive coupling energies during double- mutant cycle analysis w ere placed c lose to each other. Occasionally, however, t here was a Ôconflict of interestÕ between the requirements of interaction and those of dimerization. DISCUSSION This mutational analysis defines human c c residues involved in IL-4 binding. T he residues are located in the EF1, BC2, and FG2 loops and the interdomain segment of c c .The functional b inding epitope of c c includes r esidues I100, L102, Y103, and L208 as major binding dete rminants and five residues, N128, H159, L161, E162, and G210, as minor determinants. O ur results a lso show that the truncated c c CHR has the same binding affinity as the complete c c ectodomain, indicating that the short N-terminal region of c c is not required for ligand binding. This is true for most type I cytokine receptors, except for hgp130 [51] and granulocyte colony-stimulating factor receptor [54]. There- fore, c c CHR, the short form of the c c ectodomain, may be Table 2. Double mutant cycle analysis of interaction between c c and IL-4. The d issoc iation constants K d were evaluated from equilibrium binding between wild-type (wt) o r mutants (mut) o f the c c ectodomain and im mobilized IL-4BP saturated w ith w ild-t ype o r mutants of IL-4. The loss of free e nergy o f binding on mutation was calculated a s ddG ¼ 5.69 log K d (mut)/K d (wt). ddG sum is the sum of th e l osses of f ree energy of binding upon m ut ation for IL-4 and c c separately. ND, Sensogram could not be evaluated because of weak binding. IL-4 variants c c chain variants K d (l M ) ddG (kJÆmol )1 ) ddG sum (kJÆmol )1 ) wt wt 4.0 N15A wt 20 4.0 R121A wt 12 2.8 Y124F wt 6.9 1.4 S125A wt 8.5 1.9 wt N128A 15 3.2 wt H159A 17 3.5 wt L161A 13 2.9 wt E162A 13 2.9 wt G210A 15 3.2 N15A N128A 34 5.3 7.2 N15A H159A 61 6.7 7.5 N15A L161A 75 7.2 6.9 N15A E162A ND – 6.9 N15A G210A 59 6.7 7.2 R121A N128A 50 6.2 6.0 R121A H159A 75 7.2 6.3 R121A L161A 27 4.7 5.7 R121A E162A 127 8.6 5.7 R121A G210A 81 7.4 6.0 Y124F N128A 22 4.2 4.6 Y124F H159A 33 5.2 4.9 Y124F L161A 24 4.5 4.3 Y124F E162A 83 7.5 4.3 Y124F G210A 29 4.9 4.6 S125A N128A 19 3.9 5.1 S125A H159A 57 6.6 5.4 S125A L161A 53 6.4 4.8 S125A E162A 37 5.5 4.8 S125A G210A 22 4.2 5.1 Table 3. Co-operativity between r esidue pairs in the i nteraction interface o f c c and IL -4. The couplin g e nergy between a p a ir of residues was c alc ulated as d dG int ¼ ddG sum ) ddG (data from Table 2) ac cording to eqn ( 1). T he underlined values indicate f avorable i nteraction. The numbers in parentheses are the calculated errors (2 r, a ¼ 0.95). ND, Sensogram could not be evaluated because of weak binding. c c chain variants ddG of IL-4 variants (kJÆmol )1 ) N15A R121A Y124F S125A N128A 1.9 (0.62) )0.2 (0.66) 0.4 (0.80) 1.2 (0.90) H159A 0.8 (0.64) )0.9 (1.10) )0.3 (0.83) )1.2 (0.86) L161A )0.3 (0.99) 1.0 (0.80) )0.2 (0.64) )1.6 (0.72) E162A ND )2.9 (0.90) )3.2 (0.76) )0.7 (0.68) G210A 0.4 (0.66) )1.4 (0.87) )0.3 (0.48) 0.9 (0.67) 1494 J L. Zhang et al.(Eur. J. Biochem. 269) Ó FEBS 2002 Fig. 5. M odel of I L-4–IL-4BP–c c ternary complex. The s tructures o f I L-4, IL-4BP, and CHR of c c are depicted as ribbons and colored blue, red, and green, respectively. The major binding residues on c c and IL-4 site II epitopes are represented by s ticks. The figure was g enerated using MOLSCRIPT and RASTER 3 D. Fig. 4. O pen-book view o f complementary functional IL -4 (site 2 ) (A) and c c (B) binding epitopes, and missense mutations in the putative loops of c c implicated in patients with XSCID (62) (C) . The structures of IL-4 and c c from our model are depicted as ribbons. The mutated residues are represented b y space-filling m odels. The colors o f residues i n IL-4 and c c binding s ites indicate the loss o f binding free energy [ddG (kcalÆmo l )1 ) ¼ 1.36 log (K d variant/K d wild-type)] d ue to alanine substitutio n (see Tab les 1 and 2; 1 kcalÆmol )1 ¼ 4.18 kJÆmol )1 ). The data for I11, K12, an d Y124 wer e taken from Letzeler 2 et a l. [15]. The letters in parentheses in (C) indic ate the other mutations found in the s ame p osition . The figure was produced with MOLSCRIPT and RASTER 3 D . Ó FEBS 2002 Mutagenesis of human c c ectodomain (Eur. J. Biochem. 269) 1495 better suited to form crystals of IL-4–IL-4BP–c c than the complete c c ectodomain for solving the structure of t he low- affinity complex by X-ray diffraction. It appears that binding of c c to IL-4 is sustained predominantly by h ydrophobic i nteractions. O f t he nine residues involved in IL-4 binding, five, in particular all four major d eterminants, are hydrophobic. We propose that residues I100, L102, and Y103 of loop EF1, a nd L208 of FG2 form a hydrophobic c luster to interact with the hydrophobic epitope composed of residues I11, N15, a nd Y124 on helices A and D of IL-4 ( [12,15]; Figs 4 and 5). Similar hydrophobic determinants have b een found in several type I cytokine receptors, including hGHR [39], hEPOR [48], hgp130 [50], and the human common b chain (hb c [55–57]). Two of the three loops EF1, BC2 a nd FG2 of these receptors appear to establish two major f unctional interfaces with the ligands, and the binding is dominated by one or two h ydrophobic aromatic r esidues. For example, W104 and W 169 in loops EF1 and BC2 o f hGHR, F 93 and F205 in loops EF1 and FG2 of h EPOR, F169 in loop EF1 of hgp130, and Y365 and Y421 in loops BC2 and FG2 of b c are all key residues in binding interactions (Fig. 6). In terms of c c , Y103 is homologous to W104 of hGHR, to F93 of hEPOR and to F169 of hgp130, and the FG2 loop containing L208 may have a similar function to the loop containing W169 in hGHR. In this regard, Y103 and L208 may h ave t he most im portant r ole in the hydrophobic cluster for binding to IL-4. The two c c variants, C160A and C209A, exhibited very high K d values (> 490 l M and > 90 0 l M , respectively). The two cysteines may form a disulfide bond between loops BC2 and FG2. This prediction is consistent with our model and one of the published models [46] of c c . The contribution of the two residues to binding could not be directly determined. The disulfide bond may be only important for maintaining the structural integrity of c c . However, i t cannot be ruled out that the disulfide group participates directly in binding. These questions may be answered when the structures of both free c c and the IL-4–IL-4BP–c c ternary complex are solved. Double-mutant cycle analysis could identify co-operativ- ity between two side chains [ 35], and predict a more d etailed map of interacting residues without knowledge of t he structures of the two proteins analyzed. Unfortunately, the coupling e nergies between the m ajor determinants on c c and IL-4 site 2 cannot be measured because of the low b inding affinity of the alanine variants. Nevertheless, our experiment revealed favorable interactions between several pairs of c c and IL-4 side chains. The results support our prediction of hydrophobic interaction between the functional c c epitope and I L-4 site 2 reasonably w ell. Accordingly, the b inding epitope of c c can be divided into two functional interfaces (Figs 4A,B and 5): ( a) I100, L 102, and Y 103 on t he EF1 loop interact mainly with IL-4 Y124 and S125; (b) L208 and other residues on the BC2 a nd FG2 loops interact mainly with IL-4 N15, and probably I11 (coupling w ith c c residues could not be determined). The most i mportant is the interface on the EF1 loop of c c , because the partner residue IL-4 Y124 is a k ey determinant f or binding ( contributing 10.9 kJÆmol )1 ) [ 15], a nd the Y124D mutant exhibits a complete antagonist activity [36]. The IL-4 R121 which is more important for IL-13Ra1 binding [6,7,16,17] was found to interact with L161 on the BC2 and FG2 interface of c c . Its interaction with the binding residues on t he EF1 loop of c c could not be exclud ed. Therefore, it would be interesting to determine t he IL-1 3Ra1 epitope for IL-4 binding and compare it with the c c epitope defined in this experiment. It is unfortunate for our modeling process that the most effective mutations did not yield interaction data. Therefore, we had to rely on the residues of the weaker (but measurable) interaction which, although they are expected to work over larger distances and t hus provide less stringent constraints than d esirable, nevertheless l ead to a quite reasonable model as far as the gross features are concerned. For all the details on a t ruly atomic scale, howeve r, we have to await the crystal structure of the ternary complex. This study focu ses o n t he molecular description of the mechanism of recognition between human IL-4 and c c . Nevertheless, it will be important to understand how the c c mutations and the associated changes in IL-4 binding affect the biological a ctivity of c c during IL-4 signaling or t he signaling of the cytok ines that d epend on c c . Previous experiments with IL-4 mutant proteins [15] revealed that substitutions in the c c binding e pitope l ead to partial agonists and IL-4 antagonists. The binding affinity of such mutants t o t he receptor on whole cells was a t m ost threefold reduced compared with wild-type IL-4 (see, e.g [6]), indicating that c c binding contributes only m arginally to IL-4 binding affinity with the whole receptor complex (see also [21]). Remarkably, only a l owering of t he c c binding affinity of more than 100-fold, measured b y Biosensor in certain IL-4 mutants, produced partial agonist activities of less than 20%. It could be p redicted that the c c mutant proteins with reduced IL-4 bindin g affinity will exhibit the same alterations in biological activity as the c omplementary IL-4 proteins. Furthermore, some point mutations have been reported t o a brogate or diminish t he high affinity of IL-4 (and also IL- 2 and IL-7) for Epstein-Barr virus-transformed B cell lines or cells transfected with Fig. 6. Alignment of loops of different cytokine receptors involved in ligand binding. The struct ure-based sequence alignment of hIL-4Ra, hGHR, hEPOR, and hgp130 was taken from Hage et al.[12].The sequence s o f h c c and hb c were aligned manually. The c entral part of the EF1, BC2, and FG2 l oops of receptors are selectively shown. Key residues fo r b inding are underlined. The d eletions are marked Ô–Õ.The interaction of the receptor with the respective ligand is classified as follows: a AD or AC helix interface involved in receptor binding; b polarity of the interface; c affinity of ligand–receptor interaction. 1496 J L. Zhang et al.(Eur. J. Biochem. 269) Ó FEBS 2002 mutant sequences derived from patients with XSCID [58–61]. Two c c mutants (A134V and R202C) were found to produce t wofold and fourfold reduced IL-4 and IL-2 binding, and to be less effective in modulating Jak3 activation stimulated by IL-4 and IL-2, respectively [60,61]. A134 is located at the periphery of the c c epitope identified in this study and has not been included in the present experiment. Some of the residues in the c c epitope for IL-4 binding as identified in this study (Y103, L161, L208 and G210) have been found to be mutated in patients with XSCID (Fig 4B,C [62]). The XSCID phenotype seems to be caused predominantly by the dis ruption of IL-7 and/or IL-15 signaling [28,63]. Thus, the c c epitopes for binding of IL-4 and of IL-7 and/or IL-15 most likely share Y103, L161, L208 and G 210 as binding det erminants. Remarkably, L161 and G210 of c c are only minor determinants for IL-4 binding. T he severe deficiency produced in XSCID m ay result from the particular substitutions (G210R and L161S; Fig. 4C [62]); this could be more disruptive than an alanine substitution. Alternatively, L161 and G210 may be major determinants for IL-7 and/or IL-15 binding. More d etailed molecular information is needed on how far the c c epitopes for binding of IL-4, IL-2, IL-7, I L-9, IL-15, and IL-21 differ or coincide. Then t he severity of the clinical manifestation in patients with XSCID c an possibly be correlated w ith c c mutations in major or minor binding determinants. Thecommonnatureofc c raises the possibility that common residues for binding different ligands may exist in this receptor. Indeed, some common residues contributing to binding of different ligands have been found in hb c [55,56] and hgp130 [51]. Our result and the mutagenesis analysis of the b inding of the m urine c c chain t o I L-2 a nd IL-7 [30] show that Y103 of c c is a key ligand-interacting residue for IL-2, IL-4, and IL-7. Y103 is probably a common critical residue for all c c -dependent receptor systems. In a ddition, in that study [30], the counterpart of three dominated residues I100, L 102 a nd L208 of human c c for I L-4 binding were reported not to be important for IL-2 a nd IL-7 binding. These residues are probably unique to IL-4 binding, a s suggested by the fact that c c binding sites for different cytokines overlap but are not identical [29,64]. However, it cannot be ruled out that some of the binding residues of c c defined in our study also participate in IL-2 and IL-7 binding, as, in the aforementioned s tudy, only one re sidue (Y103) was shown to be directly involved in IL-2 and IL-7 binding. Y 103A or Y103R mutations resulted in only slightly (twofold to threefold) reduced IL-2 and IL-7 binding [30]. The difference between these results and our own may partly originate f rom the different methods applied. Therefore, further studies will be required to determine w hether Y103 and other residues i dentified in the present study are also involved in binding of other c c -dependent cytokines. The co-ordinate of the model of the IL-4–IL-4BP–c c ternary complex is available from the authors. ACKNOWLEDGEMENTS The authors are grateful to Dr J . Nickel for h elpful discussion, Dr Siddiqi for drawing the figures, and W. Ha ¨ delt and C. So ¨ der for excellent technical assistance. 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IL-4, interleukin-4; IL-4Ra, interleukin-4 receptor a chain; IL-4BP, IL-4 binding protein; c c , common c chain; IL-13Ra1, IL-13 receptor a1 chain; CHR, cytokine -binding. There- fore, c c CHR, the short form of the c c ectodomain, may be Table 2. Double mutant cycle analysis of interaction between c c and IL-4. The d issoc iation constants