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. Author manuscript; available in PMC: 2010 Sep 10.
Published in final edited form as: Structure. 2010 Mar 10;18(3):332–342. doi: 10.1016/j.str.2010.01.003

Molecular basis for shared cytokine recognition revealed in the structure of an unusually high affinity complex between IL-13 and IL-13Rα2

Patrick J Lupardus 1,1, Michael E Birnbaum 1,1, K Christopher Garcia 1,*
PMCID: PMC2850121  NIHMSID: NIHMS188925  PMID: 20223216

Summary

Interleukin-13 is a cytokine important for development of T-helper cell type 2 (Th2) responses and plays a critical role in asthma and allergy. The IL-13 Receptor α2 (IL-13Rα2) is a receptor for IL-13 lacking canonical Jak/STAT signaling functions. Here we present the crystal structure along with a mutational and biophysical analysis of the IL-13/IL-13Rα2 complex. While retaining a similar mode of IL-13 binding to its related signaling receptor IL-13Rα1, IL-13Rα2 utilizes peripheral receptor residues unused in the IL-13/IL-13Rα1 complex to generate a larger and more complementary interface for IL-13. This results in a four orders of magnitude increase in affinity, to the femtomolar level, compared to IL-13Rα1. Alanine scanning mutagenesis of the IL-13 interface reveals several common ‘hotspot’ residues important for binding to both receptors, but also identifies a prominent IL-13Rα2-specific contact. These results provide a framework for development of receptor subtype-selective IL-13 antagonists, and indicate a decoy function for IL-13Rα2.

Introduction

Cytokines are soluble proteins that mediate the growth, homeostasis, development, and effector functions of the immune response, as well as provide critical cross-talk between adaptive and innate immunity. Cytokines are key mediators of T-cell mediated immunity and drive the differentiation of activated CD4+ T-cells into the Th1, Th2, and Th17 T-helper subtypes by dimerization of cognate cytokine receptors and activation of Jak/STAT signaling (Leonard, 1999). The four-helix bundle cytokines IL-4 and IL-13 have important roles in the differentiation and effector functions of Th2-polarized T-cells, which are necessary for the antiparasitic immune response (Wynn, 2003). Largely produced by activated Th2 cells, IL-13 causes alternative activation of macrophages, induces class-switching of B-cells to the IgE and IgG4 antibody isotypes, stimulates mast cells and eosinophils, and can also induce changes in the respiratory and vascular epithelium (Hershey, 2003). Dysregulation of IL-13 mediated responses has been implicated in the pathology of asthma and allergic disease (Barnes, 2002; Wills-Karp, 2004).

IL-13 signaling is initiated through heterodimerization of the IL-13 receptor family members IL-13Rα1 and IL-4Rα in what is termed the Type II complex (Figure 1A) (Aman et al., 1996; Hilton et al., 1996). IL-13 is first recruited by IL-13Rα1, followed by binding of the binary IL-13/IL-13Rα1 complex to IL-4Rα to form the ternary signaling complex (LaPorte et al., 2008). Importantly, receptor dimerization is mediated by a composite IL-13/IL-13Rα1 interface, and IL-13 alone has no measurable affinity for IL-4Rα (Andrews et al., 2002). IL-4 can also signal through a type II receptor heterodimer, but assembles the receptor complex in the reverse order, which may partially explain the different signaling potencies of IL-4 versus 13 (Junttila et al., 2008; LaPorte et al., 2008). Upon dimerization, receptor-associated Jak kinases recruit and activate STAT6 to induce effector gene transcription (Kelly-Welch et al., 2003).

Figure 1. The overall structure of the IL-13/IL-13Rα2 complex.

Figure 1

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(A) Schematic representation of the IL-13 receptor family. S-Ig stands for s-type Ig domain, and CHR indicates the cytokine-binding homology region. Disulfide bonds in the receptors are represented by yellow lines. (B) The structure of the IL-13/IL-13Rα1/IL-4Rα signaling complex, (PDB ID 3BPO). (C–D) The structure of the IL-13/IL-13Rα2 complex. (C) Side and (D) face-on views of IL-13/IL-13Rα2. IL-13 helices A through D are colored yellow, and IL-13Rα2 domains 1 to 3 (D1–D3) are colored blue. The cytokine-receptor interaction sites II and III are highlighted in ovals. The single N-acetylglucosamine sugar residue attached to Asn215 is colored orange, and disulfide bonds are colored yellow.

IL-13 also has the ability to form a second complex with its high-affinity receptor, IL-13Rα2. Discovered contemporaneously with IL-13Rα1, IL-13Rα2 shares 21% sequence identity with IL-13Rα1 (Caput et al., 1996) and, like IL-13Rα1, contains an unusual top-mounted S-type Ig fold, followed by two fibronectin type-III like domains and a WSXWS box that make up the prototypical ‘cytokine-binding homology region’ (CHR) (Donaldson et al., 1998) (Figure 1A). The biological role of IL-13Rα2 has been a matter of debate, due to its high affinity for IL-13 (reported to be ~500 pM) and short intracellular domain (~20 amino acids) that lacks a canonical Box I/II Jak binding site and that does not activate STAT6 upon cytokine binding (Caput et al., 1996; Donaldson et al., 1998). IL-13Rα2 is upregulated during certain types of infections (Chiaramonte et al., 2003; Mentink-Kane et al., 2004; Morimoto et al., 2009), and is expressed on multiple cell types outside of the hematopoietic system (Donaldson et al., 1998). IL-13Rα2 has been found intracellularly, on the cell surface, and as a shed soluble receptor in mice (Daines and Hershey, 2002; Daines et al., 2006), although it has not been found as a soluble protein in human studies (O'Toole et al., 2008b). IL-13Rα2-knockout mice exhibit a phenotype corresponding to increased IL-13 production, including higher systemic concentrations of IL-13 and increased IgE and IgG4 production (Wood et al., 2003). This has led to a general consensus that the primary role of IL-13Rα2 is as a decoy receptor and dominant negative regulator of IL-13 (Hershey, 2003; O'Toole et al., 2008a). However, recent studies suggest that IL-13Rα2 may also have a role in signaling. It has been reported that IL-13 upregulates TGF-β in fibroblasts in an IL-13Rα2-dependent manner, and that this upregulation is STAT6-independent yet requires the intracellular domain of IL-13Rα2 (Fichtner-Feigl et al., 2006). These data have been corroborated in vivo, with a study showing that IL-13Rα2 and IL-13-dependent TGF-β production are involved in promoting immune evasion of tumors in mice (Fichtner-Feigl et al., 2008). While no dimeric signaling complex containing IL-13Rα2 has been defined, these data add to the potential roles of IL-13Rα2 in the immune response.

IL-13Rα2 has also attracted increasing interest for its potential role in cancer. IL-13Rα2 has been found to be significantly upregulated in a number of human cancers, including glioblastomas and ovarian carcinomas (Kioi et al., 2006; Mintz et al., 2002). The receptor has also attracted attention as a potential receptor for targeted, tumor-specific therapeutics (Kahlon et al., 2004).

With a number of different strategies to inhibit IL-13 as well as target IL-13Rα2 in therapeutic development, it is important to understand the molecular basis for shared cytokine recognition by the IL-13 receptor family. Molecular studies will also be important to help understand whether IL-13Rα2 is solely a decoy receptor, or whether it also has a function as a signaling receptor through homo- or heterodimerization. Here, we present the crystal structure of IL-13 in complex with IL-13Rα2 and a comparative biophysical analysis of the IL-13 interaction with IL-13Rα1 and IL-13Rα2 by alanine scanning and affinity measurement. We discover that while the IL-13 receptor-interacting residues are almost completely conserved, IL-13Rα2 has evolved an interlocking site II interface to bind IL-13 with extraordinarily high affinity rarely seen in nature, suggesting an important role for this receptor in blocking IL-13 sensitivity in IL-13Rα2 expressing cells.

Results

The structure of IL-13 bound to IL-13Rα2

To express the IL-13/IL-13Rα2 complex for crystallization, we co-infected insect HiFive cells with recombinant baculoviruses encoding IL-13 and the cytokine binding domains (D1–D3) of IL-13Rα2. As IL-13 and IL-13Rα2 are both heavily glycosylated (with four predicted Asn-linked glycosylation sites on IL-13 and three on IL-13Rα2), the insect cells were grown in the presence of kifunensine to render Asn-linked glycans endoglycosidase-H (Endo-H)-sensitive. IL-13 and IL-13Rα2 viruses were then co-infected with Endo-H virus to cleave the kifunensine-sensitized glycans in situ, leaving a single N-acetylglucosamine residue attached to the Asn sidechain (Lupardus and Garcia, 2008). After purification, surface lysines were reductively methylated (Walter et al., 2006) and the complex purified by size exclusion chromatography. Deglycosylated and methylated IL-13/IL-13Rα2 complex behaved identically to the unmodified complex, while crystal diffraction was improved from ~9 to 3.1 Å (Table 1).

Table 1.

Data collection and refinement statistics for the IL-13/IL-13Rα2 complex

IL-13/IL-13Rα2 native
Data collection
Space group I 2
Cell dimensions
    a, b, c (Å) 73.37, 86.57, 166.79
    α, β, γ (°) 90, 96.8, 90
Wavelength (Å) 1.0
Resolution (Å) 82.8-3.05 (3.21-3.05)
Rmerge 0.124 (0.581)
I / σI 7.6 (2.0)
Completeness (%) 100.0 (100.0)
Redundancy 3.8 (3.8)
Refinement
Resolution (Å) 82.8-3.05
No. reflections (total/test) 18755/954
Rwork / Rfree 21.9/26.9
No. atoms
    Protein 6027
    Sugar 28
    Calcium 2
    Water 31
B-factors
    Protein 55.9
    Sugar 75.5
    Calcium 77.4
    Water 38.7
R.m.s deviations
    Bond lengths (Å) 0.012
    Bond angles (°) 1.42
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Highest resolution shell is in parentheses

The overall architecture of the IL-13/IL-13Rα2 complex is similar to the structure of IL-13 in complex with IL-13Rα1 (Figure 1B–D) (LaPorte et al., 2008). Preservation of the general ‘two-site’ binding mode, in which IL-13 appears to be ‘pinched’ between the top and bottom domains of the receptor, is not surprising given IL-13Rα1 and IL-13Rα2 evolved from a common ancestor and have a similar domain structure (Arima et al., 2005; Boulay et al., 2003). Compared to the more common two-domain cytokine receptors, the cytokine-binding region of IL-13Rα2 consists of three domains: an N-terminal S-type Ig domain (D1) and two fibronectin III-like domains (D2 and D3) that make up the canonical cytokine binding homology region (CHR). The IL-13 receptor binding paradigm, established by the interaction of IL-4 and IL-13 with IL-13Rα1, is characterized by two binding sites, termed ‘site II’ and ‘site III’ (LaPorte et al., 2008). Site II is the canonical “Growth hormone-like” interaction of the D2-D3 ‘elbow’ region of the CHR by the A and D helices of IL-13 (Figure 1C and Figure 2B), while site III is comprised of the A–B and C–D loops of the cytokine contacting the D1 Ig-like domain through an extended anti-parallel β-sheet interaction (Figure 1C and Figure 2A). Both of these interactions are preserved in the IL-13/IL-13Rα2 complex, burying a total of approximately 1840 Å2 of surface area (1190 Å2 and 650 Å2 in site II and III, respectively).

Figure 2. The interactions of IL-13Rα2 with IL-13.

Figure 2

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The overall structure to the left is a ribbon depiction of IL-13 bound to a molecular surface of IL-13Rα2. Panel (A) View of the ‘site III’ interface showing the backbone β-sheet interactions between the c’ strand of IL-13Rα2 D1 and the CD loop strand of IL-13. Panel (B) View of the ‘site II’ interface, showing the interactions between the D2 and D3 loops of IL-13Rα2 and the A and D helices of IL-13. Interacting residues are shown as sticks, and polar interactions are drawn as dashed red lines.

The site II interface in the IL-13/IL-13Rα2 complex is formed by an unusual splayed, ‘spider-like’ arrangement of four side chains on the D-helix of IL-13 (K104, K105, F107, and R108) (Figure 2B). Looking top-down onto the D helix, each of these four residues are offset by approximately ~90°, with three of the four (K104, F107, and R108) inserting into depressions created by the D2–D3 CHR loops on IL-13Rα2. K104 fits in a groove created by the FG loop of D3 of IL-13Rα2, while R108 inserts into a canyon created by the BC loop of D3 and the AB and EF loops of D2. K104 and R108 also form polar bonds to the receptor at the periphery of the interface, with K104 forming a salt bridge with D318 and R108 participating in a hydrogen bond with the backbone carbonyl of P266. F107 occupies a pocket lined by the IL-13Rα2 residues Y315, the BC-FG loop disulfide (C269–C316), and R268, which also forms two hydrogen bonds with the peptide carbonyl of F107 and R108 in IL-13. The A-helix contributes R11, I14, and E15 to the interface, with R11 and E15 forming a pocket for and hydrogen bonding with the IL-13Rα2 FG loop residue Y315.

The defining feature of the site III interface is the extension of the antiparallel β-sheet of the IL-13Rα2 D1 Ig domain by two strands formed by the AB and CD loops of IL-13 (Figure 2A). The IL-13Rα2 interface is dominated by the backbone of the D1 c` β-strand along with the side chains of residues T83, I84, I85, and T86. These opposing side chains form a flat, nonpolar interface complementary to the IL-13 site III epitope, which is formed by the CD loop strand and a hydrophobic patch generated by M33, W35, and the methylene chain of K89. Each end of the c` strand of IL-13Rα2 is ‘capped’ with lysines (K82 and K89). Overall, the site III interaction is primarily driven by the backbone β-sheet hydrogen bonding with nonpolar van der Waals interactions between the side chains on each side.

IL-13 binding energetics

To biophysically characterize the interaction between IL-13 and IL-13Rα2 and compare the binding energetics to that of the IL-13/IL-13Rα1 complex, we utilized two methods to measure the affinity of the IL-13/IL-13Rα2 complex: thermodynamic analysis by isothermal titration calorimetry (ITC) and kinetic analysis by surface plasmon resonance (SPR). We performed ITC as described previously (LaPorte et al., 2008), utilizing fully glycosylated IL-13 and soluble extracellular domains for IL-13Rα1 and IL-13Rα2. When we measured the thermodynamics of IL-13 binding to IL-13Rα2 by ITC, we found the reaction to be strongly exothermic (ΔH= −20.0 kcal/mol), but the high affinity of the interaction produced a steep titration transition that could not be accurately fit to determine KD (Figure 3A). To accurately determine the dissociation constant, we performed displacement ITC, a method developed to reduce the apparent KD of an interaction by pre-complexing one of the binding partners with a ligand of lower affinity (Sigurskjold, 2000). We were able to take advantage of the relatively lower affinity of IL-13Rα1 for IL-13 (Figure 3B) and use it as a competitor for IL-13Rα2 binding. By this method, we were able to accurately fit the titration transition and measured a dissociation constant of 4.7 pM for the IL-13/IL-13Rα2 interaction (Figure 3C). As measured by ITC, IL-13Rα2 therefore has over 5,000-fold higher affinity for IL-13 than IL-13Rα1. This interaction is enthalpically driven and entropically unfavorable. Although one cannot unambiguously assign energetics simply by inspection of structure, the ITC results suggest that the interaction between IL-13 and IL-13Rα2 is, on balance, driven primarily by formation of polar bonds, which tend to have strong exothermic signatures, and to a lesser extent desolvation which tends to be entropically favorable, although the site II interface does contain several clearly hydrophobic interactions.

Figure 3. Biophysical characterization of the IL-13 interaction with IL-13Rα1 and IL-13Rα2.

Figure 3

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(A–C) Thermodynamic measurements of binding performed by isothermal titration calorimetry. Parameters measured for binding of (A) IL-13 to IL-13Rα2, (B) IL-13 to IL-13Rα1, and (C) displacement of IL-13Rα1 from IL-13 by IL-13Rα2 are summarized in the tables below each binding isotherm. Only DH could be calculated from the IL-13/IL-13Rα2 titration – the data could not be fit for KD due to the steepness of the curve. (D–E) Surface plasmon resonance measurement of binding kinetics for the IL-13/IL-13Rα1 and IL-13/IL-13Rα2 interactions.

We next measured the kinetics of binding for both the IL-13/IL-13Rα1 and IL-13/IL-13Rα2 interactions. C-terminally biotinylated IL-13Rα1 or IL-13Rα2 were immobilized on a streptavidin SPR surface, with a range of IL-13 concentrations passed over the surface to measure rates of association and dissociation. We found the affinity between IL-13 and IL-13Rα1 to be 1.7 nM by SPR (Figure 3D), consistent with previously reported values (Andrews et al., 2002), albeit tenfold higher affinity than observed via ITC. We then attempted to determine the affinity between IL-13 and IL-13Rα2 using SPR and found the complex had an exceptionally slow dissociation rate. Even when allowed to dissociate for 180 minutes (compared to 1 minute for IL-13/IL-13Rα1 dissociation measurements), there was essentially no dissociation of IL-13 from IL-13Rα2 observed (Figure 3E). Our measurements indicate a dissociation rate slower than 2×10−6 sec−1 and an interaction half-life of several days (t1/2 = ln2/koff). While an exact KD cannot be determined with high confidence due to limitations in accurately measuring the complex dissocation, these kinetic readings indicate IL-13Rα2 has sub-100 fM affinity for IL-13 by SPR. These results indicate the IL-13/IL-13Rα2 interaction is among the highest-affinity protein-protein complexes reported in the literature.

Comparison of the IL-13/receptor complexes

These extraordinary differences in affinity led us to carefully compare our structure to the structure of IL-13 in complex with IL-13Rα1 (PDB ID 3BPO) (LaPorte et al., 2008) in order to determine the molecular basis for the greater than 20,000-fold affinity differential observed between the complexes. Overlay of the IL-13 molecules indicates that the structure of the cytokine is nearly unchanged upon binding to its receptors (Figure S2), with a Cα backbone root mean square deviation (rmsd) of ~1.1 Å. Clearly, the cross-reactivity of IL-13 for IL-13Rα1 and IL-13Rα2 is not mediated by significant changes in the contact residues or side chain positions on the A and D helices of IL-13 (Figure S2). Comparison of rotamer positions of IL-13 for sites II and III show surprisingly few differences between the IL-13Rα1 and IL-13Rα2 complexes, with one major exception being the site II D-helix residue R108. Instead, the difference in affinity seems to be almost entirely mediated by sequence and structural differences of the contact surfaces presented by the two receptors (Figure 4A–F), and, in particular, the site II interface (Figure 4C–F). Perhaps most prominently, in site II the D2 EF loop of IL-13Rα2 protrudes towards the D helix of IL-13, and Y207 at the tip of this loop makes a number of important contacts with L101, K105 and R108 of IL-13 (Figures 4C, E). The D2 EF loop is disordered in the structure of IL-13/IL-13Rα1 (Figure 4D), and the absence of cytokine contacts leaves a large empty space between IL-13 helix D and IL-13Rα1 (Figure 4F); this empty space is occupied by the D2 EF loop in the IL-13Rα2 complex (compare Figure 4E to 4F). Due to the usage of the EF loop by IL-13Rα2, comparison of the site II interfaces show a significant increase in buried surface area (1190 Å2 versus 880 Å2) versus IL-13Rα1 (Figure 5). In fact, the IL-13Rα2 Y207 contact with K105 of IL-13 is distinct in the IL-13Rα2 complex (Figure 5) and denotes a clear site of specificity between the two complexes that we interrogated by mutagenesis (discussed below).

Figure 4. Comparison of the site II and site III interfaces for IL-13 in complex with IL-13Rα1 and IL-13Rα2.

Figure 4

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To make comparisons between the two IL-13/receptor complexes, the IL-13 molecules were structurally superimposed. (A–D) Comparison of residues that comprise the site III and site II interfaces for IL-13Rα2 (A and C, in blue) and IL-13Rα1 (B and D, in green). Cytokine-interacting residues are shown as sticks. (E–F) “Projection” view of IL-13 helices A and D depicted as ribbons and sticks interacting with semi-transparent molecular surfaces of (E) IL-13Rα2 and (F) IL-13Rα1. Interacting residues are shown as sticks, with the key ‘hotspot’ residues I14, K104, and F107 colored pink.

Figure 5. Shared and distinct contact surfaces in the IL-13/IL-13Rα2 and IL-13/IL-13Rα1 interfaces.

Figure 5

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Surface representation of IL-13 (middle panel), with residues interacting with IL-13Rα2 (pink), IL-13Rα1 (teal), or both receptors (yellow) highlighted. The IL-13-interacting surfaces of IL-13Rα2 and IL-13Rα1 are highlighted in pink and yellow, respectively (left and right panels).

The differences between the site III interfaces are less striking (Figure 4A–B), with 660 Å2 and 650 Å2 buried for IL-13Rα1 and IL-13Rα2 (Figure 5), respectively, and the interfaces dominated by backbone β-sheet hydrogen bonds and nonpolar van der Waals interactions. There is little sequence conservation between the IL-13 interacting residues in IL-13Rα1 and IL-13Rα2 (Figure 4A inset), suggesting that, as for site II, each complex has evolved a highly distinct set of receptor-ligand contacts.

Alanine-scanning mutagenesis of the IL-13 interface

Our discovery that IL-13 utilizes primarily the same residues to bind IL-13Rα1 and IL-13Rα2 with dramatically different affinities led us to investigate the relative energetic contributions that each side chain makes to the overall affinity of each complex. We identified the fifteen residues in IL-13 that made significant side chain interactions in either or both of the receptor-binding interfaces (Table S1 and Figure S3) and created a panel of alanine point mutants for SPR affinity measurements against IL-13Rα1 and IL-13Rα2. Due to the extremely slow dissociation rate between IL-13 and IL-13Rα2, we first performed a single-concentration binding screen by flowing 3 nM of each IL-13 mutant over immobilized IL-13Rα2. While there was some variation in observed association rate for several of the IL-13 mutants, only four mutants showed any appreciable dissociation within the 10 minute window used for the screen: I14A, K104A, K105A, and F107A (Figure S4). We performed complete kinetic analysis of these four mutants, and found the KD values for these mutants to be 1pM, 9 pM, 70 pM, and 289 pM for K105A, I14A, F107A, and K104A, respectively (Figure S4, Table 2). Each mutant showed an approximately 10-fold decrease in association rate (~1×108 M−1s−1 to ~1×107 M−1s−1), but a 10 to 10,000-fold increase in the dissociation rate (Table 2). ITC measurements for IL-13 K104A and F107A binding to IL-13Rα2 were also performed, and these interactions were found to have KD values of 2.8 and 1.3 nM, respectively (Figure S6). These results are between two and three orders of magnitude lower affinity than the wild-type IL-13 binding to IL-13Rα2 by ITC (4.7 pM), corresponding to the change in affinity seen in our SPR measurements.

Table 2.

Summary of kinetic and thermodynamic binding measurements for IL-13 mutants binding to IL-13Rα1 and IL-13Rα2.

SPR Kinetics/Equilibrium Affinity
IL-13
Mutant		 IL-13Rα1 immobilized IL-13Rα2 immobilized
kon(M−1s−1) koff (s−1) KD (M−1) kon(M−1s−1) koff (s−1) KD (M−1)
IL-13WT 2.35×106 3.97×10−3 1.69×10−9 1.22×108 1.93×10−6 1.58×10−14
IL-13 R11A 1.87×106 4.87×10−3 2.60×10−9
IL-13 I14A 3.17×10−7 1.79×107 1.52×10−4 8.47×10−12
IL-13 E15A 2.82×106 4.45×10−3 1.58×10−9
IL-13 V18A 3.26×106 4.43×10−3 1.36×10−9
IL-13 M33A 8.65×10−8
IL-13 D87A 2.74×106 7.51×10−3 2.74×10−9
IL-13 T88A 2.80×106 4.88×10−3 1.75×10−9
IL-13 K89A 1.28×10−8
IL-13 I90A 1.70×10−8
IL-13 E91A 1.35×10−8
IL-13 L101A 3.53×106 4.63×10−3 1.31×10−9
IL-13 K104A 3.93×10−7 9.64×106 2.71×10−3 2.89×10−10
IL-13 K105A 1.31×106 7.71×10−3 5.89×10−9 9.16×106 1.06×10−5 1.16×10−12
IL-13 F107A >1.0×10−7* 1.15×107 9.45×10−4 8.91×10−11
IL-13 R108A 2.11×10−7
ITC Thermodynamics
Titrand Titrant ΔH
(kcal/mol) 		ΔS
(cal/mol*K) 		ΔG
(kcal/mol) 		KD (M−1) n
IL-13 IL-13Rα1 −11.4 −2.8 −10.5 2.5×10−8 0.937
IL-13 IL-13Rα2 −20 N.D. N.D. N.D. 0.980
IL-13/
IL-13Rα1		 IL-R13α2 −20.1 −14.5 −15.7 4.7×10−12 0.943
IL-13 K104A IL-R13α2 −20.4 −28.2 −11.9 2.78×10−9 0.884
IL-13 F107A IL-R13α2 −17.1 −15.7 −12.3 1.27×10−9 0.949
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*

Accurate KD could not be determined due to non-specific binding of IL-13F107A to the SPR control channel at concentrations >1 µM

We next made affinity measurements via SPR for IL-13 binding to IL-13Rα1 to identify the energetically important interacting residues for this complex. We found that unlike IL-13Rα2, mutation of both site II and site III residues could disrupt the affinity of IL-13Rα1 for IL-13. In site III of IL-13, mutation of residues K89, I90, and E91 each decrease affinity for IL-13Rα1 by approximately 10-fold, while M33A has a more significant effect (~50 fold decrease) and D87A and T88A had no effect (Figure S5 and Table 2). In contrast, none of these IL-13 point mutants appreciably disrupted IL-13Rα2 binding. Each of these side chains contribute significant surface area to both site III binding interfaces (Figure 5 and S3), so it appears that the IL-13/IL-13Rα2 site III interface is more tolerant to point mutations, with the high affinity of the site II interface possibly able to compensate for site III disruption.

The site II mutants that most severely disrupt IL-13Rα2 binding (I14A, K104A, F107A) also significantly weaken the IL-13/IL-13Rα1 interaction, with binding being reduced ~200-fold for I14A and K104A (Table 2 and Figure S4). IL-13 F107A disrupts affinity for IL-13Rα1 by at least 50-fold; the actual extent of disruption is likely larger, as the IL-13 F107A mutant was poorly behaved at concentrations higher than 1 µM which caused the SPR sensorgrams to show severe perturbations at equilibrium and not approach the theoretical Rmax for the surface (Figure S4). In addition to those shared residues, each IL-13 complex also has distinct IL-13 D-helix residues that disrupt binding upon mutation to alanine. In the IL-13/IL-13Rα2 complex, mutation of IL-13 K105 to alanine reduces binding over 10-fold (Table 2), likely due to contacts the K105 sidechain makes with Y207 in the D2 E–F loop (Figure 4C, E). In the IL-13/IL-13Rα1 complex, IL-13 K105 does not contact the receptor due to the lack of involvement of the IL-13Rα1 EF loop in IL-13 binding; instead, IL-13 R108 packs into the space on IL-13Rα1 occupied by K105 and the D2 E–F loop in the IL-13Rα2 structure (Figures 4E, F). Predictably, mutation of K105 to alanine does not affect IL-13Rα1 binding due to its lack of contact in the structure, while changing R108 to alanine reduces affinity for IL-13Rα1 by 100-fold (Table 2). Interestingly, even though R108 appears to make extensive contacts with IL-13Rα2 (Figure 4E), mutation of this residue to alanine has no appreciable effect on complex affinity. Therefore, while there are slight differences in IL-13 residues energetically important for IL-13Rα1 and IL-13Rα2 complex formation that could serve as targets to produce IL-13 variants with more or less selectivity for IL-13Rα1 versus IL-13Rα2 (such as K105), the cluster of residues I14, K104, and F107 compose a mutually shared ‘hotspot’ surface and functional epitope essential for binding of IL-13 to both receptors.

Specific versus degenerate binding in the IL-13 receptor family

Prior studies have shown that while IL-13 binds to both IL-13Rα1 and IL-13Rα2, IL-4 can only bind IL-13Rα1 after first forming a complex with IL-4Rα and has no apparent affinity for IL-13Rα2 (Andrews et al., 2002; Andrews et al., 2006). An important question that we can now address is how IL-13Rα2 has evolved high affinity for IL-13 without also gaining additional affinity for IL-4. An alignment of the A and D helices of IL-4 bound to IL-13Rα1, IL-13 bound to IL-13Rα1, and IL-13 bound to IL-13Rα2 shows that the IL-13 ‘hotspot’ residues I14, K104, and F107 correspond to IL-4 residues I11, R121, and Y124, respectively (Figure 6). This high level of conservation, including similar rotamer conformations, indicates the core IL-13 receptor binding ‘hotspot’ on IL-4 and IL-13 is preserved among the complexes. Importantly, a number of peripheral IL-13 amino acids that participate in the IL-13Rα2 complex but neither of the IL-13Rα1 complexes are quite divergent, including two charge reversals (K105/E122 and E15/K12 on IL-13 and IL-4, respectively) (Figure 6). This leads us to conclude that IL-13Rα2 has evolved to specifically interact with IL-13 via increased shape complementarity and additional peripheral contacts with the cytokine, while IL-13Rα1 has remained promiscuous by primarily interacting with the shared core of IL-4/IL-13 hotspot residues and minimizing peripheral contacts.

Figure 6. An IL-4/IL-13 hotspot mediates binding to the IL-13 receptor family.

Figure 6

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The A and D helices of IL-13 molecules from our IL-13/IL-13Rα2 complex and the IL-13/IL-13Rα1 complex (PDB 3BPO) were aligned along with the A and D helices of IL-4 from the IL-4/IL-13Rα1 complex (PDB 3BPN). Side chains interacting with IL-13Rα1 or IL-13Rα2 are shown as stick models. The IL-13 receptor binding ‘hotspot’ consisting of the IL-13/IL-4 residues I14/I11, K104/R121, and F107/Y124 is highlighted.

Discussion

IL-13Rα2 is a high affinity IL-13-specific receptor thought to function as a ‘decoy’ and regulator of IL-13 signaling. Here we report the crystal structure of IL-13 in complex with IL-13Rα2, which reveals the molecular basis for the extraordinary affinity of the receptor for IL-13. An alanine scan of the IL-13 interface to determine the amino acids most important for binding to IL-13Rα1 and IL-13Rα2 indicates that both receptors utilize central set of site II ‘hotspot’ residues for binding to IL-13. Yet in contrast to IL-13Rα1, IL-13Rα2 makes a number of peripheral interface contacts to gain specificity and a four orders of magnitude increase in binding affinity.

Our structure and the accompanying biophysical analysis of the IL-13/IL-13Rα2 complex raise several important questions related to the in vivo function of IL-13Rα2. The femtomolar affinity of IL-13Rα2 for IL-13 is unprecedented in cytokine/receptor interactions, and ranks among the highest-affinity non-covalent protein-protein interactions as yet identified. Why is such tight binding necessary for IL-13 regulation? It has been previously suggested that IL-13Rα2 functions at least in part as a decoy receptor, reducing levels of IL-13 in the cellular microenvironment and downregulating IL-13 signaling on cells expressing the receptor. This has been suggested by experiments in which downregulation of IL-13 signaling after parasitic infection was dependent on IL-13Rα2 (Chiaramonte et al., 2003; Mentink-Kane et al., 2004) and in which an IL-13 overexpression phenotype is seen in an IL-13Rα2 mouse knockout model (Wood et al., 2003). A number of decoy receptors involved in cytokine regulation have been characterized, including IL-1 receptor II (IL-1RII), which neutralizes the proinflammatory cytokine IL-1 (Dinarello, 2009), and the DcR family of receptors (DcR1-3), which bind to the apoptosis-inducing TNF-related molecules TRAIL and FasL (Ashkenazi and Dixit, 1999). Additionally, a number of viruses utilize decoy receptors to modulate the activities of the TNF, IL-1, and IFN families of cytokines (Alcami, 2003). From our affinity measurements IL-13Rα2 has at least a 5,000 fold greater affinity for IL-13 than IL-13Rα1, so any expression of IL-13Rα2 on the surface of a cell would cause a substantial downregulation of IL-13 signaling. It is therefore very likely that a significant role for IL-13Rα2 in vivo is as a decoy receptor and brake on IL-13 signaling.

The high affinity of IL-13Rα2 for IL-13 would also seem to necessitate tight regulation of IL-13Rα2 expression and/or localization, due to the significant effect even small amounts of IL-13Rα2 could have on IL-13 signaling. It has been shown in U937 cells that IL-13Rα2 is only expressed at low levels on the cell surface but is rapidly upregulated in response to IFN-γ (Daines and Hershey, 2002). Additionally, strong IL-13Rα2 staining is seen in intracellular compartments (Daines and Hershey, 2002), suggesting the functional receptor may be held in intracellular stores and only trafficked to the cell surface in response to very specific cytokine-mediated signals. IL-13Rα2 is also internalized quickly upon IL-13 binding (Kawakami et al., 2001), suggesting that both the surface expression level and the amount of free receptor are kept under stringent control.

With regards to signaling, it has been shown that IL-13Rα2 does not signal through a canonical Jak/STAT signaling pathway due a lack of interaction with IL-4Rα as well as a short intracellular domain lacking Jak interaction motifs (Andrews et al., 2006). In the IL-13 type 2 signaling complex, the binary IL-13/IL-13Rα1 complex interacts with IL-4Rα through a composite cytokine-receptor and receptor-receptor binding surface (LaPorte et al., 2008). Comparison of the receptor–receptor dimerization interface of IL-13Rα1 with the same region on IL-13Rα2 shows significant structural differences that likely preclude an IL-4Rα/IL-13Rα2 interaction. IL-13Rα1 residues F297 and P300 (shown to be conserved also in common gamma chain) (LaPorte et al., 2008) correspond to tyrosine (Y293) and alanine (A296) in IL-13Rα2, with Y293 in particular pushed out into the interface and likely sterically restricting an interaction with IL-4Rα (Figure S7). Functional studies, SPR analysis, and native gel electrophoresis have all demonstrated no measurable affinity between IL-4Rα and the IL-13/IL-13Rα2 complex ((Andrews et al., 2002; Andrews et al., 2006), and data not shown). The unusually high affinity of IL-13Rα2 for IL-13 would also seem to be mutually exclusive with canonical Jak/STAT signaling, due to the need for ‘tuneability’, or a scaling up and down in signaling in response to differing levels of ligand. Alternate STAT-independent signaling modalities, as proposed previously (Fichtner-Feigl et al., 2006), may rely on undescribed mechanisms that do not require ‘tuneable’ affinity.

Experimental Procedures

Protein Expression and Purification

Human IL-13 (amino acids 21–132) and the IL-13Rα2 ectodomain (amino acids 29–331) were cloned into the pAcGP67-A vector (BD Biosciences) in frame with an N-terminal gp67 signal sequence and C-terminal hexahistidine tag and produced using the baculovirus expression system. Baculovirus stocks were prepared by transfection and amplification in Spodoptera frugiperda (Sf9) cells grown in SF900III media (Invitrogen), and protein expression was carried out in suspension Trichoplusia ni (Hi-Five) cells grown in InsectXpress media (Lonza). For biophysical experiments, IL-13, IL-13Rα1 (LaPorte et al., 2008) and IL-13Rα2 were individually expressed and captured from HiFive supernatants after 48 hours by Ni-agarose (Qiagen), concentrated and purified by size exclusion chromatography on a Superdex 200 column (GE Healthcare) equilibrated in 5 mM Hepes pH 7.0, 150 mM NaCl (Hepes-buffered saline). For crystallization, glycan-minimized IL-13/IL-13Rα2 complex was generated by co-infection of kifunensine-treated HiFive cells with IL-13, IL-13Rα2, and endoglycosidase-H viruses. After nickel purification, the complex was treated overnight with carboxypeptidase-A, diluted to ~1 mg/mL, and modified by reductive methylation (Walter et al., 2006). The endo-H trimmed and methylated complex was then concentrated and purified by size exclusion in Hepes-buffered saline. Peak fractions were pooled and concentrated to ~10–12 mg/mL for crystallization.

Crystallization and Data Collection

Initial crystallization trials yielded needle-like crystals from the PACT crystallization screen (Molecular Dimensions) using the sitting drop method at 25°C. Diffraction-quality crystals were ultimately grown in sitting drops at 25°C by mixing 0.1 mL protein (10 mg/mL in 5 mM Hepes-NaOH, pH 7.0, 150 mM NaCl) with an equal volume of 100 mM MES, pH 6.0, 200 mM CaCl2, 20% PEG-6000, and 4% v/v polypropylene glycol. Crystals grew to a maximum size of 100×100×100 mM in 2–3 weeks. Crystals were flash frozen in liquid nitrogen using mother liquor containing 25% glycerol as a cryoprotectant. A 3.1 Å data set was collected under cryo-cooled conditions at beamline 9-2 at the Stanford Synchotron Radiation Laboratory. Diffraction data were processed using MOSFLM (Leslie, 1992) and SCALA (Potterton et al., 2003). Data processing statistics can be found in Table 1.

Structure Determination and Refinement

Initial phase information was obtained by molecular replacement with the program PHASER (McCoy et al., 2007) using the coordinates of IL-13, followed by placement of the individual domains of IL-13Rα1 from the IL-13/IL-4Rα/IL-13Rα1 ternary complex structure (PDB ID 3BPO) (LaPorte et al., 2008). After the IL-13Rα1 domains were placed, the sequence was converted to poly-alanine and iterative rounds of simulated annealing refinement with CNS (Brunger, 2007) and model adjustment with COOT (Emsley and Cowtan, 2004) were carried out to build the IL-13Rα2 model. Once all amino acid changes and register problems were resolved, final rounds of positional, ADP, and TLS refinement were carried out using the PHENIX package (Afonine, 2005) resulting in final Rwork and Rfree values of 21.9% and 26.9%, respectively. Ramachandran analysis by MolProbity (http://molprobity.biochem.duke.edu) (Davis et al., 2007) indicates 90.4% of residues reside in the most favorable regions, with none in the disallowed regions. Contact maps and buried surface area values were calculated using the Protein Interfaces, Surfaces, and Assemblies (PISA) server (Krissinel and Henrick, 2007) (http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html). Overlay of coordinates was carried out using SUPERPOSE from the CCP4 suite (Potterton et al., 2003). All structural figures were prepared using PyMOL (http://www.pymol.org) (DeLano, 2002).

The final model consists of two IL-13/IL-13Rα2 complexes in the asymmetric unit, with IL-13 chain A and IL-13Rα2 chain C making up one complex and IL-13 chain B and IL-13Rα2 chain D making up the second copy. Several loops in between strands of the Ig-like domains of the IL-13Rα2 molecules were modeled as alanine or unmodeled due to a lack of electron density. A number of lysines throughout the structure were modeled as dimethyl-lysine when additional electron density was found around the terminal amine, and one calcium atom bound to each IL-13Rα2 was modeled into the structure, coordinated by Asp93 away from the IL-13/IL-13Rα2 binding interfaces. IL-13Rα2 chains C and D each have a single N-acetylglucosamine moiety attached to Asp215 in the model. The amino acid numbering scheme in the PDB file corresponds to the mature, signal peptide-cleaved chain of IL-13 and the non-cleaved sequence of IL-13Rα2 to facilitate comparison with the IL-13/IL-13Rα1 complex (PDB 3BPO). Chains B (IL-13) and D (IL-13Rα2) were used for all figures in the paper.

Surface Plamon Resonance

Surface plasmon resonance (SPR) experiments were conducted on a Biacore T100 instrument. IL-13 and all alanine mutants were expressed fully glycoslylated in HiFive cells and purified via gel filtration, and all mutants behaved identically to wild-type IL-13. IL-13Rα1 and IL-13Rα2 contained a C-terminal biotinylation peptide (LHHILDAQKMVWNHR) and were expressed and purified followed by biotinylation in vitro with GST-BirA ligase according to established protocols (O'Callaghan et al., 1999). After the biotinylation reaction was complete, receptors were re-purified by size exclusion chromatography to remove excess biotin. Protein concentrations were quantified by UV spectroscopy at 280 nm using a Nanodrop2000 spectrometer (Thermo Scientific), and readings were taken three times and averaged to determine protein concentration. All data was analyzed using the Biacore T100 evaluation software version 2.0 with a 1:1 Langmuir binding model.

Experiments were either conducted using a Biacore SA sensor chip or a Biacore Biotin CAPture Kit (GE Healthcare). Biotinylated receptors were captured at a low density (200–300 RU) and kinetic runs were conducted at 50 µL/min. An unrelated biotinylated protein was immobilized as a reference surface for the SA sensor chip with matching RU to the experimental surface. For the SA sensor chip, IL-13Rα1 was regenerated using 10 mM sodium acetate pH 4.0, 250 mM NaCl and IL-13Rα2 was regenerated using 10 mM glycine pH 2.5, 125 mM NaCl.

All measurements were made using three-fold serial dilution of IL-13 and IL-13 mutants as indicated. IL-13/IL-13Rα1 kinetics data was determined using 160 s association and 60 s dissociation. IL-13/IL-13Rα2 kinetics data was determined using 300 s association and 10,800 s (180 min) dissociation. The initial single-concentration binding screen to identify IL-13 mutants with altered binding to IL-13Rα2 used 3 nM IL-13, with 120 s association and 360 s dissociation. Kinetic experiments for IL-13 mutants binding to IL-13Rα1 were conducted identically to wild-type IL-13Rα1 measurements. When the data could not be fit for kinetics, equilibrium data was determined at 30 µL/min flow rate with 270 s association and 60 s dissociation. Kinetic data for IL-13 mutants binding to IL-13Rα2 was conducted with 300 s association and 1800 s (30 min) dissociation time for IL-13 I14A, K104A, and F107A, with 3600 s (60 min) dissociation for IL-13 K105A.

Isothermal Titration Calorimetry

Calorimetric titrations were carried out on a VP-ITC calorimeter (MicroCal, Northhampton, MA) at 30°C. Fully glycosylated proteins were expressed in HiFive cells and gel purified in HEPES-buffered saline (5 mM HEPES-NaOH, 150 mM NaCl). Proteins were quantified by UV spectroscopy as described above. Each experiment was conducted with 30 injections of 10 µL each of titrant. Injections were conducted over 20.5 s with 360 s between injections to allow the reaction to come to equilibration. For IL-13/IL-13Rα1 measurements, IL-13Rα1 was the titrant at a concentration of 40 µM and IL-13 was the titrand at 5 µM. For the wild type IL-13/IL-13Rα2 measurements, IL-13Rα2 was the titrant at 8 µM and IL-13 was the titrand at 1 µM. For measurement of the IL-13 mutants K104A and F107A with IL-13Rα2, IL-13Rα2 was the titrant in the syringe at a concentration of 40 µM and IL-13 was the titrand at 5 µM. For displacement ITC measurements (Sigurskjold, 2000), 5 µM IL-13 pre-incubated for 30 minutes with 100 µM IL-13Rα1 was used as the titrand and 40 mM IL-13Rα2 was used as the titrant. All data processing was performed using Origin 7.0. Stoichiometry measurements via ITC were all n = 0.88–1.0, indicating 1:1 molecular interactions.

Supplementary Material

01

Acknowledgements

We thank Natalia Goriatcheva for expert technical assistance, and members of the Garcia lab for helpful discussions. P.J.L. is a Damon Runyon Fellow, supported by the Damon Runyon Cancer Research Foundation (DRG-1928-06). M.E.B. is supported by a Regina Casper Stanford Graduate Fellowship and a National Science Foundation Fellowship. This work was funded by National Institutes of Health (AI51321 to K.C.G) and the Howard Hughes Medical Institute (K.C.G).

Footnotes

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Accession Numbers

The atomic coordinates and structure factors have been deposited in the Protein Data Bank with ID code 3LB6.

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