Báo cáo Y học: Functional epitope of common c chain for interleukin-4 binding

Eur. J. Biochem. 269, 1490–1499 (2002) (cid:211) FEBS 2002

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¨t Wu¨rzburg, Germany

interaction between cc and IL-4 side chains. Several cc residues involved in IL-4 binding have been previously shown to be mutated in X-linked severe combined immunodeficiency. The importance of these binding residues for cc function is discussed. These results provide a basis for elucidating the molecular recognition mechanism in the IL-4 receptor system and a paradigm for other cc-dependent cytokine receptor systems.

Keywords: common c chain; interleukin 4; mutagenesis; protein–protein interaction; structure/function.

Interleukin 4 (IL-4) can act on target cells through an IL-4 receptor complex consisting of the IL-4 receptor a chain and the common c chain (cc). An IL-4 epitope for cc binding has previously been identified. In this study, the cc residues involved in IL-4 binding were defined by alanine-scanning mutational analysis. The epitope comprises cc residues I100, L102, and Y103 on loop EF1 together with L208 on loop FG2 as the major binding determinants. These predomin- antly hydrophobic determinants interact with the hydro- phobic IL-4 epitope composed of residues I11, N15, and Y124. Double-mutant cycle analysis revealed co-operative

[9]). All three receptors are members of the type I cytokine receptor superfamily, which is characterized by the presence of at least one cytokine-binding homology region (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]. The fibronectin type III domain is comprised of seven b strands, the sequences of which are conserved between members of the family, while loop sequences connecting the b strands vary between family members and putatively contain residues that mediate distinct intermolecular contacts. These loop regions were therefore selected for this mutational analysis.

Interleukin-4 (IL-4) is a multifunctional cytokine that plays a critical role in the regulation of immune responses [1,2]. It induces the generation of Th2-dominated early immune response [3] and determines the immunoglobulin class switching to IgE [4]. Dysregulation of IL-4 function is strongly correlated with type I hypersensitivity reactions, such as allergies and asthma [5]. The IL-4 receptor complex is therefore a potential target for the development of antiallergic drugs. The central role of IL-4 in the develop- ment of Th2 cells suggests 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 promising cytokine for the treatment of high-risk acute lymphoblastic leukemia. Understanding the molecular recognition mechanism in the IL-4 receptor system is a prerequisite for the rational design of IL-4-like drugs.

IL-4 is one of the short-chain four-helix bundle cytokines. Its effects depend on binding to and signaling through a receptor complex consisting of a primary high-affinity binding subunit, the IL-4Ra, and a low-affinity receptor, depending on the cell type, the common c chain (cc; type I IL-4 receptor [8]) or IL-13Ra1 chain (type II IL-4 receptor

A comprehensive mutational analysis of IL-4 in which single residues were replaced by alanine or charged residues yielded high-resolution data on the binding epitopes for 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 located on helices A, B, and C [11]. The importance of site 1-binding determinants and their partner residues on IL-4Ra (D72, Y183 as key binding determinants) was subsequently confirmed and further defined by determining the crystal structure of the 1 : 1 IL-4/IL-4Ra ectodomain (IL-4- binding protein, IL-4BP) complex [12] and by mutational analysis of the IL-4BP binding epitope [13]. The results have already been used for the rational design of IL-4 minipro- teins [14]. The IL-4 site 2 epitope for cc comprises residues I11 and N15 on helix A together with Y124 on helix D as major binding determinants and three minor determinants K12, R121, and S125 on helices A and 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 cc or IL-13Ra1 proved to be a very promising anti-asthma drug [16–18]. Two further IL-4 mutants that selectively inhibit IL-4-induced activity on endothelial cells appeared to be good candidate drugs for the treatment of certain autoimmune diseases [6] and high-risk acute lymphoblastic leukemia [7]. However, the residues on cc that contribute to IL-4 site 2 binding remain uncertain.

Correspondence to W. Sebald, Theodor-Boveri-Institut fu¨ r Biowis- 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; cc, 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, human erythropoietin receptor; bc, common b chain. (Received 14 November 2001, revised 16 January 2002, accepted 21 January 2002)

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Mutagenesis of human cc ectodomain (Eur. J. Biochem. 269) 1491

cc is shared by several

)1Æcm)1

(e280) (cid:136) 8860 M

)1Æcm)1

)1Æcm)1 for IL-4BP, e280 (cid:136) 61 450 M

The A182, 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 purified to homogeneity as described [11,34]. Protein concentrations were determined by measuring A280, using for an absorption coefficient for A124 IL-4, e280 = IL-4, e280 (cid:136) 7370 M )1Æcm)1 for 66 930 M )1Æcm)1 for A103 human cc, human cc, e280 (cid:136) 60 170 M and e280 (cid:136) 45 660 M

)1Æcm)1 for murine cc.

Mutagenesis of the cc ectodomain

cDNA for human cc ectodomain was submitted to in vitro cassette mutagenesis employing synthetic double-stranded oligonucleotides. The cc variants were expressed and purified as the wild-type human cc ectodomain.

Biosensor interaction analysis

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, IL-21 [19]. cc alone binds ligands with very low affinity (Kd (cid:25) 150 lM for IL-4) [15]. Recruitment of cc into receptor complexes for the above cytokines increases receptor affinity for binding [20–22]. cc participates in cytokine signaling in several receptor complexes via JAK3 [23]. Mutations of either cc or JAK3 result in X-linked severe combined immunodeficiency (XSCID) which is characterized by a failure in T and NK cell development [24]. cc-knockout mice have been generated and their immune system successfully reconstituted by gene therapy [25,26]. Initial attempts at gene therapy for patients with XSCID had been successful for more than 10 months [27,28]. Thus, defining the IL-4-binding determinants on cc is important not only for elucidating molecular recognition and activation mechanisms in the IL-4 receptor system and possibly providing a paradigm for other cc-dependent cytokine receptor systems, but also for delineating the molecular pathology of XSCID.

So far, the binding epitopes of human and murine cc for some cc-dependent cytokines have been studied. A molecular mapping study using the antagonistic monoclonal antibody PC.B8, which reacts with a discon- tinuous site on human cc, localized cc binding residues to four loops, but did not identify single specific residues for ligand binding [29]. Mutational analysis of murine cc employing heterodimeric IL-2R and IL-7R on whole cells suggests that cc epitopes for IL-2 and IL-7 binding overlap and comprise at least three distinct putative loop segments of the cc protein [30]. Here we report the effect in the human cc of single amino-acid substitutions ectodomain on IL-4 binding. Biosensor techniques employing soluble recombinant IL-4, IL-4-BP and the wild type or mutant forms of human cc ectodomain revealed the contributions of cc residues to IL-4 binding. The possible co-operativity between some residues on the cc epitope and the IL-4 site 2 epitope was analyzed by double-mutant cycle analysis.

E X P E R I M E N T A L P R O C E D U R E S

Protein expression and purification

The binding of cc variants to IL-4/IL-4BP was recorded on a BIAcore 2000 system (Pharmacia Biosensor) 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 (cid:25) 200 resonance units. The following reaction cycle was applied using the com- mand COINJECT: (a) IL-4 at 0.1 lM in HBS buffer (10 mM Hepes, pH 7.4, 150 mM NaCl, 3.4 mM EDTA, 0.005% surfactant P20) was perfused over flow cells 1 and 2 at a flow rate of 10 lLÆmin)1 at 25 (cid:176)C for 2 min; (b) 0.1 lM IL-4 plus cc ectodomain or cc variants at 1–10 lM in the same buffer were perfused in the same way for 2 min; (c) HBS buffer alone was perfused for 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 rate of 2.5 Hz and evaluated as described [15]. Equilibrium binding of cc variants at 1, 2, 3, 5, 10 lM was measured for at least three times in duplicate. The mean standard deviation (mean r) was 13.8% (cid:139) 6.5% for the Kd values calculated from the five variant concentrations. For the double mutant cycle analysis [35], the same procedure as above was used except that IL-4 variants [15,36] at 0.1 lM and cc variants at 2, 4, 6, 10, 20 lM were perfused (the mean r was 16.4% (cid:139) 7.4% for the Kd values). The loss of binding free energy on mutation for IL-4 and cc was calculated as ddG (kJÆmol)1) (cid:136) 5.69 log Kd (mutant)/Kd (wild-type). The interaction energy between two residues was calculated by the double-mutant cycle method as in Eqn. (1):

(cid:133)1(cid:134)

ddGint (cid:136) ddGX-A (cid:135) ddGY-B (cid:255) ddGX-A;Y-B

The ectodomain of human cc comprising amino-acid residues 1–232 [20] was expressed with a C-terminal thrombin cleavage site (LVPRGS) plus a His6 tag in SF9 insect cells according to the manufacturer’s instructions (PharMingen). The protein was isolated from the culture medium of infected SF9 cells by standard procedures involving Ni2+/nitriloacetate/agarose (Qiagen), digested with thrombin (Sigma), and purified by gel-filtration chromatography through a Superdex 200 HR 10/30 col- umn (Pharmacia). After exhaustive dialysis against water, the purified protein was freeze-dried and stored at )80 (cid:176)C. The cDNA for the murine cc ectodomain comprising residues 1–233 [31] was cloned into the temperature- regulated expression vector pRpr9 fd [32], expressed in Escherichia coli strain KS 474, and refolded as described [33]. The refolded protein was purified to homogeneity by gel-filtration chromatography through a Superdex 200 HR 10/30 column, and stored at )80 (cid:176)C.

where ddGX-A and ddGY-B are the changes in binding energy on mutation of X to A and Y to B (mutation of IL-4 and cc in this experiment), respectively, and ddGX-A, Y-B the change on the simultaneous mutation of X to A and Y to B. ddGint is a measure of the co-operativity of the interaction of the two components mutated. ddGint (cid:136) 0 indicates that the pair of residues analyzed do not interact. A positive value of ddGint 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 (cid:136) 0.95) calculated from the mean for ddGint are shown in Table 3.

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Molecular modeling of the IL-4–IL-4BP–cc ternary complex

1

The present model is based on the crystal structure of the complex of IL-4 and IL-4BP (PDB entry 1IAR [12]), augmented by the model of cc derived from human growth hormone receptor (hGHR), as obtained from an older model (T. Mueller, & W. Kammer, personal communica- tion, Universita¨ t Wu¨ rzburg, Germany) of the ternary complex of IL-4–IL-4BP–cc. This old model was based on the structure of free IL-4 (PDB entry 1HIK [38]) and of models of the extracellular domains of IL-4Ra and cc obtained by analogy modeling following the structure of the hGHR complex (PDB entry 3HHR [39]). The 3HHR data were obtained from the protein databank (PDB [40]). The old model was built in such a way that all cysteine residues formed proper disulfide bonds, and all evidence from mutation experiments available at the time was used to adjust the binding epitopes of the receptor chains. The resulting alignment required some nontrivial rebuilding with insertions and deletions, and, consequently, the resulting model of the IL-4 receptor complex had to be extensively energy refined. The program O [41] was used for 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 enough that the binding topology of cc could be transferred to the new model without major problems. The local program DISDM2 was used (H. J. Hecht, & M. Buehner, unpublished results) to build and adjust the present model using the data of mutational analysis of IL-4 and cc. 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 program EREF was used [43], which is called from within DISDM2.

Fig. 1. Amino-acid substitutions in the ectodomain of the human com- mon c chain (cc). The amino-acid sequence of cc is shown with boxed portions indicating predicted b-strands which are designated by the letter below the box. Residues substituted in this study are indicated by asterisks.

R E S U L T S

Site-specific mutagenesis of amino acids in the cc ectodomain

Alanine substitutions were targeted to residues in four putative interconnecting loops and the interdomain segment of the human cc ectodomain based on the published models [44–46], and sequence alignment performed between cc and several cytokine receptors, the major ligand-binding deter- minants of which were identified. These include the hGHR [39,47], (hEPOR the human erythropoietin receptor [48,49]), IL-4BP [12], and the human gp130 (hgp130 [50,51]). Eighteen cc variants were generated with amino- acid substitutions in the AB1, EF1, BC2, FG2 loops and the interdomain segment (Fig. 1). A deletion mutant lacking residues 1–33 of the N-terminus of cc, named cc CHR, was also generated to find out whether this N-terminal region of cc is required for ligand binding.

All human cc wild-type or variant proteins could be purified to apparent homogeneity by Ni2+/nitrilotriacetate/ agarose and gel filtration. The wild-type human cc ectodo- main expressed in SF9 cells was recovered as monomeric and dimeric species [52,53]. The murine cc ectodomain expressed in E. coli occurred as a monomer (Fig. 2). Initial

biosensor studies showed that the different forms of human and murine proteins exhibited similar binding affinity for the IL-4–IL-4BP complex. The mixture of monomeric and dimeric human cc interacts with the complex with a Kd of

Fig. 2. Gel-filtration analysis of the human cc ectodomain expressed in SF9 cells and the murine cc ectodomain expressed in E. coli. The samples were applied to a Superdex 200 HR 10/30 column and eluted with the same buffer. The two peaks of human cc represent dimer (A) and monomer (B).

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Mutagenesis of human cc ectodomain (Eur. J. Biochem. 269) 1493

Table 1. Equilibrium binding between cc ectodomain mutants and IL-4–IL-4BP. The dissociation constants Kd were evaluated from equilibrium binding between wild-type (wt) or mutants (mut) of the cc ectodomain and immobilized IL-4BP saturated with IL-4. The loss of free energy of binding on mutation was calculated as ddG (kJÆmol)1) (cid:136) 5.69 log Kd(mut)/Kd(wt).

Equilibrium binding

Alanine variant ddG (kJÆmol)1) Kd (lM) Kd(mut)/Kd(wt)

1.6 4.0 4.5 1.0 1.1 0.0 0.2

2.8 3.3 0.7 0.8 )0.9 )0.5

5.5 >320 >240 >300 5.6 1.4 >80 >60 >80 1.4 0.8 >11 >10 >11 0.8

Fig. 3. Sensograms recording the binding of human and murine cc ectodomains to the IL-4–IL-4BP complex. IL-4BP was immobilized on the biosensor matrix. At time zero, perfusion with 100 nM IL-4 was initiated. The saturation binding of IL-4 was arbitrarily set as zero. After 120 s, perfusion was continued with 100 nM IL-4 plus cc ecto- domain. In different cycles, 5 lM human (a) or murine (b) cc ecto- domains were applied. Perfusion with buffer alone started at time 240 s. The ruler indicates resonance units (RU) corresponding to 1–10 lM murine cc ectodomain. The resonance unit for 5 lM human cc corresponded to that for 1.6–2 lM murine cc. 2.4 15 0.6 3.7 )1.3 3.2

4 lM, and murine cc with a Kd of 1.6 lM (Fig. 3 and Table 1). In addition, different preparations of wild-type human cc ectodomain consistently showed a Kd of 4 lM irrespective of the monomer to dimer ratio (data not shown). Therefore, the mixtures of dimeric and monomeric human cc protein were used for all biosensor measurements.

The cc epitope for IL-4 binding

1.4 17 >900 13 13 0.4 4.1 >230 3.2 3.2 )2.3 3.5 >13 2.9 2.9

exhibited a largely reduced binding affinity (Kd > 500 lM). This may be caused by structural perturbation of the protein. A direct role in binding, however, cannot be excluded for these residues.

Double-mutant cycle analysis of the IL-4–cc interface

The method of measuring the binding of cc to IL-4–IL-4BP by biosensor was established previously [15]. The dissocia- tion constant Kd evaluated from the concentration dependence of equilibrium binding proved to be very reliable for measuring the interaction of the cc ectodomain with IL-4–IL-4BP. The measured Kd for interaction of cc ectodomain variants with IL-4–IL-4BP are compiled in Table 1. Eight cc variants including cc CHR exhibited unchanged binding characteristics. Changes in binding affinity were observed in 11 cc variants. The Kd of six variants was too high to be reliably determined. A rough estimate yields Kd values of about 200–300 lM for I100A, L102A, Y103A and L208A, and Kd values of about 500– 1000 lM for C160A and C209A. The Kd values of five variants, N128A, H159A, L161A, E162A, and G210A, were found to be increased threefold to fourfold compared with the Kd of wild-type cc, suggesting that these residues are part of the cc binding interface, but do not play a key role in binding. The loss of binding affinity of the four variants I100A, L102A, Y103A and L208A is not likely to be caused by extended structural alterations, as I100A, L102A, and L208A were reported to bind to IL-2 and IL-7 with the same affinity as wild-type cc, and the Y103A mutation resulted in only twofold to threefold reduced IL-2 and IL-7 binding [30]. Thus, the four residues I100, L102, Y103 and L208 are hot spots on cc, 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

The co-operativity of the interaction of some residues on the IL-4 site 2 epitope and the cc ectodomain was determined in this experiment. Double-mutant cycles were constructed only for the mutants with minimal effects on binding (Tables 2 and 3), because the Kd values for the interaction between variants of the main binding residues I100, L102, Y103, and L208 of cc and IL-4 variants as well as the interaction between variant I11A of IL-4 and cc variants were too high to be reliably determined. Interaction between residues on IL-4 and cc can be grouped according to their coupling energies (Table 3). The main binding determinant of IL-4 Y124 failed to exhibit positive coupling energies with any of the cc residues analyzed. IL-4 Y124 probably interacts with 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

Murine cc (wt) Human cc (wt) Human cc CHR Loop 1 (AB1) N44A V45A Loop 3 (EF1) E99A I100A L102A Y103A Q104A Loop 4 (ID) Q127A N128A Loop 5 (BC2) N158A H159A C160A L161A E162A Loop 6 (FG2) P207A L208A C209A G210A 3.4 >166 >490 15 0.9 >40 >120 3.7 )0.4 >9 >12 3.2

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Table 2. Double mutant cycle analysis of interaction between cc and IL-4. The dissociation constants Kd were evaluated from equilibrium binding between wild-type (wt) or mutants (mut) of the cc ectodomain and immobilized IL-4BP saturated with wild-type or mutants of IL-4. The loss of free energy of binding on mutation was calculated as ddG (cid:136) 5.69 log Kd(mut)/Kd(wt). ddGsum is the sum of the losses of free energy of binding upon mutation for IL-4 and cc separately. ND, Sensogram could not be evaluated because of weak binding.

receptor side chains N1128 and H159 are 12 A˚ apart in the cc model. This could indicate that our cc model is inaccurate, because this model does not completely fit the results of the double-mutant cycle analysis. Alternatively, the interaction of IL-4 side chain N15 with H159 (coupling energy only 0.8 kJÆmol)1) may be indirect. Of particular interest is the IL-4 side chain of R121, which, after being substituted with D or E, leads to a selective IL-4 agonist specifically impaired in IL-13Ra1 binding [6,7,16,17,34]. The IL-4 R121 was distinct in showing positive coupling during interaction with the cc side chain L161.

Model of the structure of the IL-4–IL-4BP–cc ternary complex

In a series of steps, cc was adapted to achieve a good fit to the core structure (the binary complex; Fig. 5). The procedure started with moving the whole chain ((cid:212)rigid body(cid:213)). 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 part of the model, but some minor changes in side chain orientation could not be avoided for proper adaptation. An important point was to keep the C-terminal domains of the receptor chains close together, as this was expected to be essential for dimer formation and thereby signaling through the membrane. The structures of cc and the ternary complex were modeled so that residues that exhibit positive coupling energies during double- mutant cycle analysis were placed close to each other. Occasionally, however, there was a (cid:212)conflict of interest(cid:213) between the requirements of interaction and those of dimerization.

IL-4 variants ddG (kJÆmol)1) ddGsum (kJÆmol)1) Kd (lM) cc chain variants

D I S C U S S I O N

G210. The IL-4 side chain of N15 functionally interacts with the central receptor side chain N128, and also with H159 located at the periphery of the functional cc epitope. The relative positions of the coupling side chains as proposed by our theoretical model of the ternary complex (see below) are presented in the open-book view in Fig. 4A,B. The two

This mutational analysis defines human cc residues involved in IL-4 binding. The residues are located in the EF1, BC2, and FG2 loops and the interdomain segment of cc. The functional binding epitope of cc includes residues I100, L102, Y103, and L208 as major binding determinants and five residues, N128, H159, L161, E162, and G210, as minor determinants. Our results also show that the truncated cc CHR has the same binding affinity as the complete cc ectodomain, indicating that the short N-terminal region of cc 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, cc CHR, the short form of the cc ectodomain, may be

4.0 20 12 6.9 8.5 15 17 13 13 15 34 61 75 ND 59 50 75 27 127 81 22 33 24 83 29 19 57 53 37 22 4.0 2.8 1.4 1.9 3.2 3.5 2.9 2.9 3.2 5.3 6.7 7.2 – 6.7 6.2 7.2 4.7 8.6 7.4 4.2 5.2 4.5 7.5 4.9 3.9 6.6 6.4 5.5 4.2 wt wt wt wt wt N128A H159A L161A E162A G210A N128A H159A L161A E162A G210A N128A H159A L161A E162A G210A N128A H159A L161A E162A G210A N128A H159A L161A E162A G210A 7.2 7.5 6.9 6.9 7.2 6.0 6.3 5.7 5.7 6.0 4.6 4.9 4.3 4.3 4.6 5.1 5.4 4.8 4.8 5.1 wt N15A R121A Y124F S125A wt wt wt wt wt N15A N15A N15A N15A N15A R121A R121A R121A R121A R121A Y124F Y124F Y124F Y124F Y124F S125A S125A S125A S125A S125A

Table 3. Co-operativity between residue pairs in the interaction interface of cc and IL-4. The coupling energy between a pair of residues was calculated as ddGint (cid:136) ddGsum ) ddG (data from Table 2) according to eqn (1). The underlined values indicate favorable interaction. The numbers in parentheses are the calculated errors (2 r, a (cid:136) 0.95). ND, Sensogram could not be evaluated because of weak binding.

ddG of IL-4 variants (kJÆmol)1)

N15A R121A Y124F S125A cc chain variants

1.9 (0.62) 0.8 (0.64) )0.3 (0.99) ND 0.4 (0.66) )0.2 (0.66) )0.9 (1.10) 1.0 (0.80) )2.9 (0.90) )1.4 (0.87) 0.4 (0.80) )0.3 (0.83) )0.2 (0.64) )3.2 (0.76) )0.3 (0.48) N128A H159A L161A E162A G210A 1.2 (0.90) )1.2 (0.86) )1.6 (0.72) )0.7 (0.68) 0.9 (0.67)

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Mutagenesis of human cc ectodomain (Eur. J. Biochem. 269) 1495

2 Fig. 4. Open-book view of complementary functional IL-4 (site 2) (A) and cc (B) binding epitopes, and missense mutations in the putative loops of cc implicated in patients with XSCID (62) (C). The structures of IL-4 and cc from our model are depicted as ribbons. The mutated residues are represented by space-filling models. The colors of residues in IL-4 and cc binding sites indicate the loss of binding free energy [ddG (kcalÆmol)1) (cid:136) 1.36 log (Kd variant/Kd wild-type)] due to alanine substitution (see Tables 1 and 2; 1 kcalÆmol )1 (cid:136) 4.18 kJÆmol)1). The data for et al. [15]. The letters in parentheses in (C) indicate the other mutations found in the same position. I11, K12, and Y124 were taken from Letzeler The figure was produced with MOLSCRIPT and RASTER3D.

Fig. 5. Model of IL-4–IL-4BP–cc ternary complex. The structures of IL-4, IL-4BP, and CHR of cc are depicted as ribbons and colored blue, red, and green, respectively. The major binding residues on cc and IL-4 site II epitopes are represented by sticks. The figure was generated using MOLSCRIPT and RASTER3D.

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better suited to form crystals of IL-4–IL-4BP–cc than the complete cc ectodomain for solving the structure of the low- affinity complex by X-ray diffraction.

cannot be ruled out that the disulfide group participates directly in binding. These questions may be answered when the structures of both free cc and the IL-4–IL-4BP–cc ternary complex are solved.

It appears that binding of cc to IL-4 is sustained predominantly by hydrophobic interactions. Of the nine residues involved in IL-4 binding, five, in particular all four major determinants, are hydrophobic. We propose that residues I100, L102, and Y103 of loop EF1, and L208 of FG2 form a hydrophobic cluster to interact with the hydrophobic epitope composed of residues I11, N15, and Y124 on helices A and D of IL-4 ([12,15]; Figs 4 and 5). Similar hydrophobic determinants have been found in several type I cytokine receptors, including hGHR [39], hEPOR [48], hgp130 [50], and the human common b chain (hbc [55–57]). Two of the three loops EF1, BC2 and FG2 of these receptors appear to establish two major functional interfaces with the ligands, and the binding is dominated by one or two hydrophobic aromatic residues. For example, W104 and W169 in loops EF1 and BC2 of hGHR, F93 and F205 in loops EF1 and FG2 of hEPOR, F169 in loop EF1 of hgp130, and Y365 and Y421 in loops BC2 and FG2 of bc are all key residues in binding interactions (Fig. 6). In terms of cc, 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 have the most important role in the hydrophobic cluster for binding to IL-4.

Double-mutant cycle analysis could identify co-operativ- ity between two side chains [35], and predict a more detailed map of interacting residues without knowledge of the structures of the two proteins analyzed. Unfortunately, the coupling energies between the major determinants on cc and IL-4 site 2 cannot be measured because of the low binding affinity of the alanine variants. Nevertheless, our experiment revealed favorable interactions between several pairs of cc and IL-4 side chains. The results support our prediction of hydrophobic interaction between the functional cc epitope and IL-4 site 2 reasonably well. Accordingly, the binding epitope of cc can be divided into two functional interfaces (Figs 4A,B and 5): (a) I100, L102, and Y103 on the EF1 loop interact mainly with IL-4 Y124 and S125; (b) L208 and other residues on the BC2 and FG2 loops interact mainly with IL-4 N15, and probably I11 (coupling with cc residues could not be determined). The most important is the interface on the EF1 loop of cc, because the partner residue IL-4 Y124 is a key determinant for binding (contributing 10.9 kJÆmol)1) [15], and 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 cc. Its interaction with the binding residues on the EF1 loop of cc could not be excluded. Therefore, it would be interesting to determine the IL-13Ra1 epitope for IL-4 binding and compare it with the cc epitope defined in this experiment.

The two cc variants, C160A and C209A, exhibited very high Kd values (> 490 lM and > 900 lM, 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 cc. The contribution of the two residues to binding could not be directly determined. The disulfide bond may be only important for it maintaining the structural

integrity of cc. However,

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 thus provide less stringent constraints than desirable, nevertheless lead to a quite reasonable model as far as the gross features are concerned. For all the details on a truly atomic scale, however, we have to await the crystal structure of the ternary complex.

This study focuses on the molecular description of the mechanism of recognition between human IL-4 and cc. Nevertheless, it will be important to understand how the cc mutations and the associated changes in IL-4 binding affect the biological activity of cc during IL-4 signaling or the signaling of the cytokines that depend on cc. Previous experiments with IL-4 mutant proteins [15] revealed that substitutions in the cc binding epitope lead to partial agonists and IL-4 antagonists. The binding affinity of such mutants to the receptor on whole cells was at most threefold reduced compared with wild-type IL-4 (see, e.g [6]), indicating that cc binding contributes only marginally to IL-4 binding affinity with the whole receptor complex (see also [21]). Remarkably, only a lowering of the cc binding affinity of more than 100-fold, measured by Biosensor in certain IL-4 mutants, produced partial agonist activities of less than 20%. It could be predicted that the cc mutant proteins with reduced IL-4 binding affinity will exhibit the same alterations in biological activity as the complementary IL-4 proteins. Furthermore, some point mutations have been reported to abrogate or diminish the high affinity of IL-4 (and also IL-2 and IL-7) for Epstein-Barr lines or cells transfected with virus-transformed B cell

Fig. 6. Alignment of loops of different cytokine receptors involved in ligand binding. The structure-based sequence alignment of hIL-4Ra, hGHR, hEPOR, and hgp130 was taken from Hage et al. [12]. The sequences of hcc and hbc were aligned manually. The central part of the EF1, BC2, and FG2 loops of receptors are selectively shown. Key residues for binding are underlined. The deletions are marked (cid:212)–(cid:213). The interaction of the receptor with the respective ligand is classified as follows: aAD or AC helix interface involved in receptor binding; bpolarity of the interface; ca(cid:129)nity of ligand–receptor interaction.

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R E F E R E N C E S

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mutant sequences derived from patients with XSCID [58–61]. Two cc mutants (A134V and R202C) were found to produce twofold 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 cc epitope identified in this study and has not been included in the present experiment.

2. Nelms, K., Keegan, A.D., Zamorano, J., Ryan, J.J. & Paul, W.E. (1999) The IL-4 receptor: signaling mechanisms and biologic functions. Annu. Rev. Immunol. 17, 701–738. 3. Paul, W.E. & Seder, R.A. (1994) Lymphocyte responses and cytokines. Cell 76, 241–251.

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Some of the residues in the cc 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 disruption of IL-7 and/or IL-15 signaling [28,63]. Thus, the cc epitopes for binding of IL-4 and of IL-7 and/or IL-15 most likely share Y103, L161, L208 and G210 as binding determinants. Remarkably, L161 and G210 of cc are only minor determinants for IL-4 binding. The severe deficiency produced in XSCID may 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 detailed molecular information is needed on how far the cc epitopes for binding of IL-4, IL-2, IL-7, IL-9, IL-15, and IL-21 differ or coincide. Then the severity of the clinical manifestation in patients with XSCID can possibly be correlated with cc mutations in major or minor binding determinants.

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The common nature of cc 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 hbc [55,56] and hgp130 [51]. Our result and the mutagenesis analysis of the binding of the murine cc chain to IL-2 and IL-7 [30] show that Y103 of cc is a key ligand-interacting residue for IL-2, IL-4, and IL-7. Y103 is probably a common critical residue for all cc-dependent receptor systems. In addition, in that study [30], the counterpart of three dominated residues I100, L102 and L208 of human cc for IL-4 binding were reported not to be important for IL-2 and IL-7 binding. These residues are probably unique to IL-4 binding, as suggested by the fact that cc 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 cc defined in our study also participate in IL-2 and IL-7 binding, as, in the aforementioned study, only one residue (Y103) was shown to be directly involved in IL-2 and IL-7 binding. Y103A 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 from the different methods applied. Therefore, further studies will be required to determine whether Y103 and other residues identified in the present study are also involved in binding of other cc-dependent cytokines.

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The co-ordinate of the model of the IL-4–IL-4BP–cc

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