Structural reorganization of the interleukin-7 signaling complex

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Feb 14, 2012 - structure of the unliganded form of the interleukin-7 alpha receptor ... the activating receptor for IL-2, IL-4, IL-7, IL-9, IL-15, and IL-. 21 (9).
Structural reorganization of the interleukin-7 signaling complex Craig A. McElroya, Paul J. Hollandb, Peng Zhaoc, Jae-Min Limc, Lance Wellsc, Edward Eisensteinb,d, and Scott T. R. Walshb,e,1 a Battelle Biomedical Research Center, Columbus, OH 43201; bInstitute for Bioscience and Biotechnology Research, W. M. Keck Laboratory for Structural Biology, dFischell Department of Bioengineering, and eDepartment of Cell Biology and Molecular Genetics, University of Maryland, College Park, Rockville, MD 20850; and cComplex Carbohydrate Research Center, Departments of Chemistry and Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602

We report here an unliganded receptor structure in the common gamma-chain (γc) family of receptors and cytokines. The crystal structure of the unliganded form of the interleukin-7 alpha receptor (IL-7Rα) extracellular domain (ECD) at 2.15 Å resolution reveals a homodimer forming an “X” geometry looking down onto the cell surface with the C termini of the two chains separated by 110 Å and the dimer interface comprising residues critical for IL-7 binding. Further biophysical studies indicate a weak association of the IL-7Rα ECDs but a stronger association between the γc/IL-7Rα ECDs, similar to previous studies of the full-length receptors on CD4+ T cells. Based on these and previous results, we propose a molecular mechanism detailing the progression from the inactive IL-7Rα homodimer and IL-7Rα–γc heterodimer to the active IL-7–IL-7Rα–γc ternary complex whereby the two receptors undergo at least a 90° rotation away from the cell surface, moving the C termini of IL-7Rα and γc from a distance of 110 Å to less than 30 Å at the cell surface. This molecular mechanism can be used to explain recently discovered IL-7– and γc-independent gain-of-function mutations in IL-7Rα from B- and Tcell acute lymphoblastic leukemia patients. The mechanism may also be applicable to other γc receptors that form inactive homodimers and heterodimers independent of their cytokines. X-ray crystallography

| biophysics | homodimerization | cancer mutations

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nterleukin (IL)-7Rα (CD127) is an essential pleiotropic receptor in immunology. IL-7Rα functions as a receptor for two signaling cascades: IL-7 and thymic stromal lymphopoietin (TSLP). In the IL-7 pathway, IL-7 interacts with IL-7Rα and the gamma chain (γc) (CD132), forming the signaling complex. This pathway effects extracellular matrix remodeling and the development and homeostasis of B and T cells (reviewed in ref. 1). Similarly, TSLP interacts with its receptor (TSLPR) and IL-7Rα, forming the signaling complex. Activation of the TSLP signaling pathway induces dendritic cell activation and B-cell development (reviewed in ref. 2). These pathways using IL-7Rα have been implicated in several diseases including severe combined immunodeficiency (reviewed in ref. 3), autoimmune conditions (colitis and multiple sclerosis) (4–6), and cancers (7, 8). Thus, it is imperative to dissect the molecular mechanisms of IL-7Rα assembly with its cognate ligands. IL-7Rα is a transmembrane receptor belonging to the cytokine receptor homology class 1 (CRH1) family. IL-7Rα is a heterotypic cytokine receptor belonging to the γc family. γc functions as the activating receptor for IL-2, IL-4, IL-7, IL-9, IL-15, and IL21 (9). Full-length human IL-7Rα consists of a 219-residue extracellular domain (ECD), a 25-residue transmembrane domain (TMD), and a 195-residue intracellular domain (ICD) (SI Appendix, Fig. 1) (10). IL-7 signaling is thought to function via the classical stepwise cytokine-induced receptor heterodimerization paradigm (reviewed in ref. 11). In this mechanism, IL-7 interacts with the IL-7Rα ECD, forming a 1:1 assembly, which subsequently recruits γc, producing the signaling complex. The association of the two receptors by IL-7 activates intracellular www.pnas.org/cgi/doi/10.1073/pnas.1116582109

phosphorylation events through the JAK/Stat, PI3/Akt, and SRC signaling cascades, ultimately leading to enhanced gene transcription and cellular responses (reviewed in ref. 1). The stepwise cytokine-induced heterodimerization mechanism of IL-7Rα has been called into question. Coimmunoprecipitation and fluorescence correlation spectroscopy studies demonstrate an IL-7–independent preassembly of IL-7Rα homodimers and IL-7Rα–γc heterodimers on T cells (12, 13). Neither the preassembly of IL-7Rα homodimers nor IL-7Rα–γc heterodimers induces signaling (12, 13). Similarly, preformed receptor homoand heterodimers have been reported for IL-2Rβ (14), IL-4Rα (15), and IL-9Rα (16). The preassembly model of cytokine receptors, first reported for the homotypic erythropoietin receptor (EPOR) (17), represents a new cytokine receptor signaling paradigm (reviewed in ref. 18). It remains an open question as to whether the two binding mechanisms and/or other undiscovered mechanisms function independently or synergistically. Ultimately, the structural details describing preassembly of preformed receptors of short-chain heterotypic receptors like the γc family have remained unanswered until now. We report the crystal structure of the unliganded IL-7Rα ECD. The structure reveals an IL-7Rα homodimer in the asymmetric unit, with the dimer interface comprising many of the same residues involved in IL-7 binding. We compare the unliganded IL-7Rα structure with the structures of IL-7Rα with IL-7 (19), other γc receptors (20–23), and the EPOR homodimer structure (21). Biophysical studies indicate weak self-association of the IL-7Rα ECDs but a stronger association between the IL7Rα and γc ECDs. These results provide the basis for structural models of normal and aberrant IL-7 signaling. Finally, the proposed structural mechanism may be applicable to the γc family. Results Architecture of the Unliganded IL-7Rα Structure. The IL-7Rα ECD (residues 1–219) was expressed and purified as described previously (24). The crystal structure of IL-7Rα was solved using molecular replacement to a resolution of 2.15 Å. The structure was refined with Rcryst and Rfree values of 0.214 and 0.258, respectively (SI Appendix, Table 1). Surprisingly, the asymmetric unit consists of two IL-7Rα molecules packed against each other

Author contributions: L.W., E.E., and S.T.R.W. designed research; C.A.M., P.J.H., P.Z., J.-M.L., E.E., and S.T.R.W. performed research; C.A.M., P.J.H., P.Z., J.-M.L., L.W., E.E., and S.T.R.W. analyzed data; and C.A.M., P.J.H., P.Z., J.-M.L., L.W., E.E., and S.T.R.W. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. K.C.G. is a guest editor invited by the Editorial Board. Data deposition: The atomic coordinates and structure factors reported in this paper have been deposited in the Protein Data Bank, www.pdb.org (PDB ID code 3up1). 1

To whom correspondence should be addressed. E-mail: [email protected].

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1116582109/-/DCSupplemental.

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Edited by K. Christopher Garcia, Stanford University School of Medicine, Stanford, CA, and accepted by the Editorial Board January 5, 2012 (received for review October 11, 2011)

with pseudotwofold symmetry (Fig. 1). The receptor chains are similar, with a Cα backbone root-mean-square deviation (rmsd) of 0.93 Å for residues 18–209 (SI Appendix, Figs. 2–5). An O-linked glycan attached to S133 of chain B (S133B) causes backbone changes on the order of 1.4–7.7 Å relative to chain A. This conformational change also slightly shifts the main chain of the two preceding β-strands, A2 and B2. Other than the small differences of the interfacial region, the remaining differences correspond to loop regions and are likely a result of crystal-packing differences. IL-7Rα consists of two fibronectin type-3 (FN3) domains (D1 and D2) connected by a 310-helical linker with an overall L-shaped architecture similar to structures of IL-7Rα in complex with IL-7 (19) and other CRH1 members (25). Chains A and B have elbow angles of 77° and 79°, respectively. IL-7Rα contains three disulfide bonds in the D1 domain connecting β-strands A1-B1 (C22-C3), F1-G1 (C88-C98), and C1-C′1 (C54-C62). Electron density was absent for the N- (1A-11A and 1B-15B) and C-terminal (213A-219A and 212B-219B) regions of IL-7Rα, suggesting dynamic behavior. Two N-acetyl glucosamines (GlcNAc) and a fucose are attached

to N131 of both chains of IL-7Rα—even after treatment with PNGase F. PNGase F cleaves the whole N-glycan, but cannot remove the N-glycan if an α(1→3) fucose is attached to the first GlcNAc. No other N-glycans were visualized for the other Asn sites of IL-7Rα (N29, N45, N162, N212, and N213). Interestingly, the region surrounding the side chain of S133B contained significant difference density, suggesting an O-glycan of this Ser (SI Appendix, Fig. 3). The O-glycan of S133B forms numerous contacts with the receptor residues. Chain A of IL-7Rα displayed no evidence of this modification, indicating that it was either not O-glycosylated or is dynamic. Mass spectrometry confirmed S133 to be glycosylated with an N-acetyl galactosamine and further identified an O-linked glycan of T132 (SI Appendix, Figs. 6–8 and Table 2). There was no crystallographic evidence of an O-glycan attached to T132 of either chain. Future studies will explore the importance of O-glycosylation of IL-7Rα. IL-7Rα Dimer Interface. A surprising feature of the unliganded IL7Rα structure is the “X” geometry of the two receptors when

Fig. 1. Crystal structure of an unliganded IL-7Rα homodimer. (A) Ribbon diagram of the IL-7Rα homodimer oriented looking down onto the cell surface. Chains A and B are colored purple and green, respectively. The six loop regions involved in the interface and ligand binding are labeled L1–L6. The disulfide bonds and the N- and O-glycans are represented as sticks and labeled. (B) Edge view of what the IL-7Rα homodimer structure would look like on a cell surface. The residues that can be N-glycosylated on IL-7Rα are displayed. Blacks lines represent unmodeled C-terminal residues. (C) Expansion of the binding interface formed in the IL-7Rα homodimer depicted as sticks or spheres. For clarity, only residues are labeled for the stick representations for the individual chains in the two views. The two insets are related by a 180° rotation around the y axis.

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Biophysical Measurements of IL-7Rα Associations. The self-association of the IL-7Rα ECD was assayed using size-exclusion chromatography (SEC), analytical ultracentrifugation (AUC), and surface plasmon resonance (SPR). Both unglycosylated (5 μM) and glycosylated (10 μM) IL-7Rα eluted as monomers by SEC (24). More quantitative assessment of potential self-association of the IL-7Rα ECD involved sedimentation equilibrium experiments using AUC. Sedimentation equilibrium curves of IL-7Rα at multiple concentrations and rotor speeds yielded a single, homogeneous species with a buoyant molecular weight (Mb) of 9,277 ± 120 (SI Appendix, Fig. 9). IL-7Rα showed no tendency to aggregate into higher-order oligomers at loading concentrations of 23 μM, and the confidence values for the molecular weight were reasonable for a glycoprotein. The molecular weight (Mw) of IL-7Rα was estimated by comparing the Mw based on composition with that obtained from the computed partial specific volume (ν) and from Mb determined by sedimentation equilibrium. This approach converged on a value of ∼20% (wt/wt) glycosylation, and indicates reasonable values for a ν of 0.7145 mL·g−1 and a Mw of 32,850 ± 415 Da. SPR experiments further probed the association of the IL-7Rα and γc ECDs. A very weak self-association estimate of 610 ± 140 μM (Kd) was observed for the IL-7Rα ECDs using steady-state SPR analysis (Fig. 2A). A stronger self-association of 3 μM was reported for the homodimerization of the IL-2Rβ ECDs, the receptor homologous to IL-7Rα (26). Fig. 2B displays the binding kinetics of the γc–IL-7Rα interaction. Analysis of these curves fit best to a two-step conformational exchange binding model (SI Appendix, Fig. 10) with k1, k−1, k2, and k−2 rates of 2.6 × 103 M−1·s−1, 2.6 × 10−2 s−1, 4.3 × 10−3 s−1, and 1.4 × 10−3 s−1, respectively, yielding a Kd of 3.1 ± 1.2 μM. A stronger binding affinity was also observed for the interaction of full-length IL-7Rα with γc than IL7Rα self-association on the cell surface of T cells (13). Likewise, a higher binding affinity was observed for the interaction of full-length IL-2Rβ with γc than IL-2Rβ self-association on cells (14, 27). Further structural studies of IL-7Rα and γc will shed light on the individual contributions of the ECD and TMD to IL-7–independent association. Structural Comparison of Unliganded and Liganded States of IL-7Rα.

The unliganded IL-7Rα structure is similar to the liganded IL7Rα structure bound to IL-7. There are liganded structures of IL7 bound to unglycosylated (two complexes) and glycosylated (one McElroy et al.

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looking down onto the cell surface: Their N and C termini are located on opposite ends, and the binding interface consists of similar elbow loop residues that were previously shown to be critical for IL-7 interaction (19) (Fig. 1). The IL-7Rα binding interface uses 19 and 20 residues and buries 568 and 600 Å2 of surface area for chain A and B, respectively (average of 584 Å2). There is one main-chain hydrogen bond (h-bond) between K138B O and E59A N (2.7 Å). The interface comprises 28% of charged residues, 17% of polar residues, and 56% of apolar residues, and has a shape complementarity (Sc) of 0.60. The asymmetry of the binding interface involves different degrees of packing interactions of the loop residues. For the six loop regions, contacts are formed only from loops L2, L3, L5, and L6 of chain A and loops L2–L6 of chain B (SI Appendix, Fig. 4). The asymmetry of the dimer interface primarily results from sidechain orientation differences, but there are two notable mainchain conformational changes between the two chains. The first main-chain difference is the conformation of loop L5. Not contacting the binding interface directly, the O-glycan of S133B alters the L5 loop conformation of chain B, adopting a single turn of α-helix, which does not occur in chain A (SI Appendix, Figs. 2 and 5). The second main-chain difference is the conformation of loop L6. The overall geometry of loop L6 for the two chains is similar, but there is a 2.2-Å displacement of the H191 Cα atoms, resulting in side-chain rotamer changes of H191 and Y192.

Fig. 2. SPR binding studies of IL-7Rα and γc at 25 °C. (A) Steady-state binding curve for the weak self-association of the IL-7Rα homodimer by plotting Rmax versus IL-7Rα concentration. (B) Binding kinetic sensorgrams of the association of γc with IL-7Rα are indicated with black lines. The sensorgrams were globally fit to a two-step conformational exchange, with two on- and off-rate constants depicted by red lines. RU, resonance unit.

complex) forms of IL-7Rα (19). Chain A of the unliganded IL7Rα is more closely related to the structures of IL-7Rα in complex with IL-7 than it is to chain B of the unliganded IL-7Rα. Backbone superimpositions of residues 18–209 gives an average rmsd of 0.55 Å for chain A to the liganded IL-7Rα structures (SI Appendix, Figs. 2, 4, and 5). When chain B is superimposed onto the liganded IL-7Rα structures, the average backbone rmsd increases to 1.06 Å. Not surprisingly, the largest differences between chain B and the complex structures are in the same regions seen in comparison with chain A. These results suggest that either there are no large conformational changes upon IL-7 binding to IL-7Rα or that the dimerization of IL-7Rα somehow mimics IL-7 binding, thereby triggering a similar conformational change. The unliganded binding interface of the IL-7Rα homodimer buries a smaller amount of surface area, has a fewer number of hbonds, and is less specific than the interface of IL-7Rα bound to IL-7. Depending on the chain, the unliganded IL-7Rα homodimer structure does not use all of the six loop regions L1–L6, whereas in the liganded IL-7Rα structures all loop regions engage IL-7 (SI Appendix, Fig. 4). Thus, the amount of buried surface area for the unliganded IL-7Rα homodimer is reduced by 134 Å2 compared with the liganded state of IL-7Rα (584 vs. 718 Å2; we list the average buried surface area of the two proteins). Finally, the unliganded IL-7Rα interface has only one h-bond versus five h-bonds for the liganded IL-7Rα structures and a poorer shape complementarity of 0.60 versus 0.68 for the liganded IL-7Rα PNAS | February 14, 2012 | vol. 109 | no. 7 | 2505

structures [Sc values varied by 0.04 for the liganded IL-7Rα structures, 0.65–0.69 (19)]. Discussion Structural Comparisons to CRH1 Members. Comparison of the IL7Rα homodimer structure to other CRH1 members reveals significant angular changes of the two FN3 domains. The FN3 domain geometries were analyzed by three angles: elbow (ε; angle between the D1 and D2 domains), twist (τ; angle between the x axis of the D1 and D2 domains), and swivel (σ; spin of the D2 domain in the x-z plane) (SI Appendix, Table 3) (28). The unliganded IL-7Rα structure displays more obtuse ε and τ angles (Δε = 3°, Δτ = 4°) but a more acute σ angle (Δσ = 5°) to the liganded IL-7Rα structures (19). In comparison with IL-2Rβ from IL-2–activating structures (20, 29), the unliganded IL-7Rα structure displays more obtuse ε and σ angles (Δε = 3°, Δσ = 20°) but a more acute τ angle (Δτ = 9°) (SI Appendix, Fig. 11). The unliganded IL-7Rα structure shows more acute ε and σ angles (Δε = 6°, Δσ = 9°) but a more obtuse τ angle (Δτ = 3°) to IL-4Rα from an IL-4–activating structure (23). There are binary and ternary complex structures of IL-4Rα, and this receptor displays similar ε and τ angles, but the σ angle increases by 39° when IL-4Rα/IL-4 binds γc (22, 23). Future structural studies of γc family members (e.g., IL-7 ternary complex) will be essential in understanding nonactivating and activating receptor geometries and also receptor pleiotropy from a structural perspective. The only definitive unliganded homodimer structure of the CRH1 family is EPOR (21). [Although the unliganded growth hormone receptor structure revealed a homodimer assembly, homodimerization was triggered through the transmembrane region, not the ECD (30).] The binding interfaces between IL7Rα and EPOR display significant variations (SI Appendix, Fig. 12 and Table 3). The EPOR dimer interface has a larger amount of buried surface area, 722 Å2, and more h-bonds, 9 (21), than the IL-7Rα dimer interface (584 Å2 and one h-bond). Although largely apolar (EPOR = 43%, IL-7Rα = 56%), the binding interfaces do vary in their polarity, with polar and charged residues comprising 33% and 24% of the EPOR dimer interface and 17% and 28% of the IL-7Rα dimer interface, respectively. Additionally, the EPOR dimer interface is almost perfectly symmetric, whereas the IL-7Rα dimer interface is highly asymmetric. Significant structural changes exist between the IL-7Rα homodimer and the EPOR homodimer. If chain A of IL-7Rα is superimposed onto chain A of EPOR using secondary structural elements (SI Appendix, Fig. 13), there is a 47° rotation and a 12-Å

translation (using the conserved disulfide bond) of chain B of IL7Rα to chain B of EPOR. Large differences are also observed for the FN3 geometries of IL-7Rα compared with EPOR. IL-7Rα displays more obtuse ε and σ angles (Δε = 10°, Δσ = 4°) but similar τ angles. These large conformational changes of the homodimeric structures reveal a level of structural complexity that the receptors undergo before and after binding their ligands that allows us to speculate on IL-7Rα signaling mechanisms. IL-7Rα Signaling Model. The IL-7Rα dimer structure explains why

homodimerization of IL-7Rα does not activate the IL-7 or TSLP pathways. Although there is currently no structure of the IL-7 ternary complex, there are activating structures of other γc members, namely IL-2 and IL-4 (20, 23, 29). The average distance between the C-terminal domains of these structures between the α/β receptors and γc is 27 Å. We predict a similar distance for the C-terminal domains of IL-7Rα and γc in an activating complex; however, the distance between the C-terminal domains of the homodimeric IL-7Rα structure is 110 Å. Thus, the JAK1 molecules bound to the IL-7Rα ICDs are kept physically apart, inhibiting self-activation. A similar mechanism was presented for the EPOR homodimer (21, 31). In this case, the C-terminal domains of the EPOR homodimer are separated by a smaller distance of 73 Å relative to the IL-7Rα homodimer. Upon EPO binding, the distance between the two C-terminal domains reduces to 39 Å, bringing the two ICDs in close proximity, leading to activation. Based on the IL-7Rα homodimer structure, we present a structural mechanism for signal inactivation in the ligand-independent association of IL-7Rα and γc. We demonstrate a stronger binding affinity for heterodimerization of the IL-7Rα/γc ECDs than homodimerization of the IL-7Rα ECDs (Kds of 3.1 μM vs. >610 μM at 25 °C). Previous studies also reported stronger binding affinity for full-length IL7Rα and γc than the IL-7Rα homodimer on T cells at 37 °C (13). The IL-2– and IL-4–activating structures revealed extensive binding surfaces between the C-terminal D2 domains of the α/β receptors with γc (20, 23, 29); the results presented here disfavor the ligand-independent association of IL-7Rα and γc through their C-terminal D2 domains. Rather, the loop residues of IL7Rα that are used to bind IL-7 and itself would likely be involved, leading us to propose the structural mechanism of the IL-7 signaling pathway highlighted in Fig. 3. The IL-7Rα homodimer keeps the JAK1 molecules bound to the ICDs separated by 110 Å. Likewise, the IL-7Rα–γc heterodimer [we modeled this complex by superimposing the secondary structural

Fig. 3. Proposed signaling mechanism of the IL-7 pathway. Signaling model of IL-7Rα–IL-7–γc interactions indicating preassembly of the IL-7Rα homodimer and IL-7Rα–γc heterodimer from cell biological studies (12, 13). There are no structures of the IL-7Rα–γc heterodimer or the IL-7Rα–IL-7–γc ternary complex. The distance between the D2 domains of IL-7Rα and γc was an average of the γc structures with IL-2 [PDB ID code 2bfi (20); PDB ID code 2erj (29)] and IL-4 [PDB ID code 3bpl (23)].

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IL-7Rα Signaling in Gain-of-Function Muteins. Gain-of-function mutations in IL-7Rα have been identified in patients with B- and T-cell acute lymphoblastic leukemias (B-ALL and T-ALL) (8, 32). In one study, B-ALL patients contained insertions and deletions of residues in the N-terminal region of the IL-7Rα TMD, with all of the sequences containing an extra Cys residue (32). In other B-ALL patients, the IL-7Rα ECD contains an S165-to-C165 mutation (32). The same study also reported TALL patients containing 30 different sequence insertions and deletions in the N-terminal region of the IL-7Rα TMD, 27 of which have an extra Cys (32). The mutations in the TMD of IL7Rα were different among the B-ALL and T-ALL patients. In a different study, T-ALL patients contained 17 different insertions and deletions in the N-terminal region of the IL-7Rα TMD, with 13 of them containing an additional Cys (8). These T-ALL mutations were different between the two studies (8, 32). Both studies tested several of the B- and T-ALL muteins in IL-7Rα and demonstrated IL-7– and γc-independent activation of the signaling pathway (8, 32). The structural mechanism presented above can be further extended to speculate on the molecular basis of the ALL muteins. We posit the B- and T-ALL muteins of IL-7Rα function independent of ligands by positioning at least two copies of IL-7Rα in geometries such that the C-terminal ends of their ECDs or TMDs are less than 30 Å apart. To provide structural support for this hypothesis, we constructed energy-minimized structural models of the S165C disulfide-linked homodimer of the B-ALL mutein of the IL-7Rα ECD and one of the T-ALL insertion sequences (JPSP7) in the IL-7Rα TMD (Fig. 4). The S165C BALL mutein of IL-7Rα forms an additional disulfide bond between two IL-7Rα molecules on the cell surface (32). S165 is positioned in a solvent-exposed loop region of the D2 domain, allowing for easy disulfide-bond formation between two IL-7Rα molecules (Fig. 4A). The structural model of the S165C mutein reveals that the C-terminal ends of the D2 domains of the IL7Rα ECDs would be 29 Å apart, well within the distance predicted to activate the pathway. The JPSP7 T-ALL mutein contains a three-residue insertion highlighted in bold into the IL-7Rα TMD sequence: PILLTCPTISILSFFSVALLVILACVLW (8). The IL-7Rα TMD is predicted to adopt a single α-helix spanning the lipid bilayer. The peptide sequences were fed into an algorithm developed by DeGrado and coworkers that optimizes the depth and angular geometry of transmembrane α-helical segments in a lipid bilayer based on known membrane protein structures (33). The algorithm predicts the first transmembrane residue of the T-ALL mutein to be I228, with residues PILLTCPT solvent-exposed. Results led to the conclusion that this T-ALL mutein forms an IL-7Rα homodimer/oligomer at the cell surface (8). A structural model of the C225 disulfide-linked IL-7Rα homodimer was constructed (Fig. 4B). The C-terminal residues from 209 to 219 of the wild-type juxtamembrane region are highly mobile. The distance between the Cαs of W247 of the two TMDs is 11 Å, well within the distance required to bring the JAK1s bound to the ICDs together, leading to activation. The linking of two IL-7Rαs through a disulfide bond in the juxtamembrane region is not a unique mechanism. Similar mechanisms have been reported for TSLPR (34), EPOR (35, 36), and epidermal growth factor receptors (37). However, not all of the T-ALL muteins contained an unpaired cysteine in this region of IL-7Rα. Therefore, we posit that the insertions and/or McElroy et al.

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elements of the γc ECD onto chain B (rmsd = 1.9 Å)] would keep JAK1 and JAK3 separated by a large distance preventing activation. When IL-7 binds both IL-7Rα and γc, the receptors undergo an ∼90° rotation away from the cell surface, bringing the two C termini of IL-7Rα and γc to within less than 30 Å and activating signal transduction.

Fig. 4. Structural models of two IL-7Rα mutations found from patients with either B-ALL or T-ALL. (A) Ribbon diagram of the disulfide-linked homodimer of the IL-7Rα ECDs through an S165-to-C165 mutation from a B-ALL patient. (B) Ribbon diagram of IL-7Rα including the juxtamembrane and transmembrane regions. For clarity, only one of the IL-7Rα ECDs is shown. Insertion of three amino acids by the T-ALL mutation from patient JPSP7 likely causes extension of the transmembrane region into the extracellular face, resulting in a disulfide-linked homodimer of two receptor molecules by C225.

deletions of the T-ALL muteins alter the orientation of the IL7Rα TMD, enabling higher-order oligomeric states capable of self-activating the IL-7 pathway. Future studies of the T-ALL muteins will delineate the molecular mechanisms of IL-7Rα and aid in developing new diagnostic and treatment strategies for these cancers. There are many reasons why the IL-7Rα homodimer structure likely represents a physiologically relevant conformation on a cell surface. First, self-association of the full-length IL-7Rα has been reported on the T-cell surface (13). Second, the structure reveals that the homodimer interface forms at the elbow loop residues that interact with its ligands. Third, the structure can be easily positioned on a cell surface of a bilayer (Fig. 1B). Although our IL-7Rα ECD consists of residues 1–219 of the fulllength receptor, the two chains were built to residues 211 and 212. Modeling the unseen residues (4 Å per residue) onto the structure, the C termini of the chains can easily reach the bilayer. There is also ample space to accommodate the glycans of IL7Rα. Fourth, although homodimerization of the IL-7Rα ECDs is PNAS | February 14, 2012 | vol. 109 | no. 7 | 2507

weak in the millimolar range, translating this solution affinity to a binding constant in the cell membrane would give a value in the micromolar range because of the reduced dimensionality of localization in a lipid bilayer. A similar argument was presented for EPOR homodimer association, which displayed no solution selfassociation of the EPOR ECDs (21, 38). Fifth, IL-7 binds a homodimeric IL-7Rα–Fc fusion (NS0 cells; R&D Systems) and monomeric IL-7Rα (insect cells) with similar binding affinities (Kds ∼60 nM) (19). Sixth, the ECD and full-length IL-7Rα exhibit the same trend of binding γc stronger than itself (13). Finally, experiments performed on other homodimeric α/β-receptors in the γc family have shown that the ECDs, and not the transmembrane or intracellular domains, are responsible for dimerization. For instance, removal of the IL-2Rβ and γc ECDs from the fulllength receptors abrogates IL-2Rβ homodimerization and IL2Rβ/γc heterodimerization (14). Although not tested (13), a similar result would likely occur with IL-7Rα and γc. Thus, although we cannot completely rule out crystallization forces, the IL-7Rα homodimer structure agrees well with observed biology and provides a structural mechanism describing signal quiescence and activation for normal and aberrant IL-7 signaling and may be applicable to the γc family. 1. Mazzucchelli R, Durum SK (2007) Interleukin-7 receptor expression: Intelligent design. Nat Rev Immunol 7(2):144–154. 2. Ozaki K, Leonard WJ (2002) Cytokine and cytokine receptor pleiotropy and redundancy. J Biol Chem 277:29355–29358. 3. Kovanen PE, Leonard WJ (2004) Cytokines and immunodeficiency diseases: Critical roles of the γ(c)-dependent cytokines interleukins 2, 4, 7, 9, 15, and 21, and their signaling pathways. Immunol Rev 202(1):67–83. 4. Watanabe M, et al. (1998) Interleukin 7 transgenic mice develop chronic colitis with decreased interleukin 7 protein accumulation in the colonic mucosa. J Exp Med 187: 389–402. 5. Gregory SG, et al.; Multiple Sclerosis Genetics Group (2007) Interleukin 7 receptor α chain (IL7R) shows allelic and functional association with multiple sclerosis. Nat Genet 39:1083–1091. 6. Hafler DA, et al.; International Multiple Sclerosis Genetics Consortium (2007) Risk alleles for multiple sclerosis identified by a genomewide study. N Engl J Med 357: 851–862. 7. Barata JT, Cardoso AA, Nadler LM, Boussiotis VA (2001) Interleukin-7 promotes survival and cell cycle progression of T-cell acute lymphoblastic leukemia cells by downregulating the cyclin-dependent kinase inhibitor p27(kip1). Blood 98:1524–1531. 8. Zenatti PP, et al. (2011) Oncogenic IL7R gain-of-function mutations in childhood T-cell acute lymphoblastic leukemia. Nat Genet 43:932–939. 9. Rochman Y, Spolski R, Leonard WJ (2009) New insights into the regulation of T cells by γ(c) family cytokines. Nat Rev Immunol 9:480–490. 10. Goodwin RG, et al. (1990) Cloning of the human and murine interleukin-7 receptors: Demonstration of a soluble form and homology to a new receptor superfamily. Cell 60:941–951. 11. Wells JA (1996) Binding in the growth hormone receptor complex. Proc Natl Acad Sci USA 93(1):1–6. 12. Rose T, Lambotte O, Pallier C, Delfraissy JF, Colle JH (2009) Identification and biochemical characterization of human plasma soluble IL-7R: Lower concentrations in HIV-1-infected patients. J Immunol 182:7389–7397. 13. Rose T, et al. (2010) Interleukin-7 compartmentalizes its receptor signaling complex to initiate CD4 T lymphocyte response. J Biol Chem 285:14898–14908. 14. Pillet AH, et al. (2010) IL-2 induces conformational changes in its preassembled receptor core, which then migrates in lipid raft and binds to the cytoskeleton meshwork. J Mol Biol 403:671–692. 15. Kammer W, et al. (1996) Homodimerization of interleukin-4 receptor α chain can induce intracellular signaling. J Biol Chem 271:23634–23637. 16. Malka Y, et al. (2008) Ligand-independent homomeric and heteromeric complexes between interleukin-2 or -9 receptor subunits and the γ chain. J Biol Chem 283: 33569–33577. 17. Yoshimura A, Longmore G, Lodish HF (1990) Point mutation in the exoplasmic domain of the erythropoietin receptor resulting in hormone-independent activation and tumorigenicity. Nature 348:647–649. 18. Schreiber G, Walter MR (2010) Cytokine-receptor interactions as drug targets. Curr Opin Chem Biol 14:511–519. 19. McElroy CA, Dohm JA, Walsh ST (2009) Structural and biophysical studies of the human IL-7/IL-7Rα complex. Structure 17:54–65. 20. Wang X, Rickert M, Garcia KC (2005) Structure of the quaternary complex of interleukin-2 with its α, β, and γc receptors. Science 310:1159–1163.

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Materials and Methods Detailed methods describing protein production, crystal structure determination, biophysical measurements, and mass spectrometry are given in SI Appendix, Materials and Methods. Briefly, the IL-7Rα and γc ECDs were expressed from insect cells and purified as described previously (24). A crystal of the IL-7Rα ECD grown from 1.26 M (NH4)2SO4, 0.1 M Tris·HCl (pH 8.5), and 0.2 M Li2SO4 diffracted X-rays at the General Medicine and Cancer Institutes Collaborative Access Team (GM/CA-CAT) beamline 23ID-D at the Advanced Photon Source. The structure was solved using the liganded IL-7Rα structure [Protein Data Bank (PDB) ID code 3di3] as a search model for molecular replacement. SPR binding kinetics and affinities of the IL-7Rα and γc ECDs were determined using modified and similar methods described previously (19). Models of the complexes of IL-7Rα–γc and IL-7–IL-7Rα–γc along with the IL-7Rα cancer mutations were generated using MacPyMOL (39) and energy-minimized with CNS (40). Structural figures were rendered with MacPyMOL (39). ACKNOWLEDGMENTS. We thank R. Mariuzza and M. Walter for comments on the manuscript. C.A.M. was supported by a postdoctoral fellowship from the American Heart Association (725595B). X-ray data collection was done at the National Institutes of Health (NIH) GM/CA 23ID beamline at the Advanced Photon Source at Argonne National Laboratory, which is operated by UChicago Argonne, LLC under Contract DE-AC02-06CH11357. This work was supported in part by NIH Grant P41RR018502 (to L.W.). This research was supported by NIH Grant AI72142 (to S.T.R.W.).

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