Structural reorganization of the interleukin-7signaling complexCraig A. McElroya, Paul J. Hollandb, Peng Zhaoc, Jae-Min Limc, Lance Wellsc, Edward Eisensteinb,d,and Scott T. R. Walshb,e,1

aBattelle Biomedical Research Center, Columbus, OH 43201; bInstitute for Bioscience and Biotechnology Research, W. M. Keck Laboratory for StructuralBiology, 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 ofGeorgia, Athens, GA 30602

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

We report here an unliganded receptor structure in the commongamma-chain (γc) family of receptors and cytokines. The crystalstructure of the unliganded form of the interleukin-7 alpha receptor(IL-7Rα) extracellular domain (ECD) at 2.15 Å resolution revealsa homodimer forming an “X” geometry looking down onto the cellsurface with the C termini of the two chains separated by 110 Å andthe dimer interface comprising residues critical for IL-7 binding. Fur-ther biophysical studies indicate a weak association of the IL-7RαECDs but a stronger association between the γc/IL-7Rα ECDs, similarto previous studies of the full-length receptors onCD4+ T cells. Basedon these and previous results, we propose a molecular mechanismdetailing the progression from the inactive IL-7Rα homodimer andIL-7Rα–γc heterodimer to the active IL-7–IL-7Rα–γc ternary complexwhereby the two receptors undergo at least a 90° rotation awayfrom the cell surface, moving the C termini of IL-7Rα and γc froma distance of 110 Å to less than 30 Å at the cell surface. This molec-ular mechanism can be used to explain recently discovered IL-7– andγc-independent gain-of-function mutations in IL-7Rα from B- and T-cell acute lymphoblastic leukemia patients. Themechanismmay alsobe applicable to other γc receptors that form inactive homodimersand heterodimers independent of their cytokines.

X-ray crystallography | biophysics | homodimerization | cancer mutations

Interleukin (IL)-7Rα (CD127) is an essential pleiotropic re-ceptor 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 thegamma chain (γc) (CD132), forming the signaling complex. Thispathway effects extracellular matrix remodeling and the de-velopment 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 signalingpathway induces dendritic cell activation and B-cell development(reviewed in ref. 2). These pathways using IL-7Rα have beenimplicated in several diseases including severe combined im-munodeficiency (reviewed in ref. 3), autoimmune conditions(colitis and multiple sclerosis) (4–6), and cancers (7, 8). Thus, itis imperative to dissect the molecular mechanisms of IL-7Rαassembly with its cognate ligands.IL-7Rα is a transmembrane receptor belonging to the cytokine

receptor homology class 1 (CRH1) family. IL-7Rα is a hetero-typic cytokine receptor belonging to the γc family. γc functions asthe activating receptor for IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21 (9). Full-length human IL-7Rα consists of a 219-residue ex-tracellular domain (ECD), a 25-residue transmembrane domain(TMD), and a 195-residue intracellular domain (ICD) (SI Ap-pendix, Fig. 1) (10). IL-7 signaling is thought to function via theclassical stepwise cytokine-induced receptor heterodimerizationparadigm (reviewed in ref. 11). In this mechanism, IL-7 interactswith the IL-7Rα ECD, forming a 1:1 assembly, which sub-sequently recruits γc, producing the signaling complex. The as-sociation of the two receptors by IL-7 activates intracellular

phosphorylation events through the JAK/Stat, PI3/Akt, and SRCsignaling cascades, ultimately leading to enhanced gene tran-scription and cellular responses (reviewed in ref. 1).The stepwise cytokine-induced heterodimerization mechanism

of IL-7Rα has been called into question. Coimmunoprecipitationand fluorescence correlation spectroscopy studies demonstratean IL-7–independent preassembly of IL-7Rα homodimers andIL-7Rα–γc heterodimers on T cells (12, 13). Neither the pre-assembly of IL-7Rα homodimers nor IL-7Rα–γc heterodimersinduces signaling (12, 13). Similarly, preformed receptor homo-and heterodimers have been reported for IL-2Rβ (14), IL-4Rα(15), and IL-9Rα (16). The preassembly model of cytokinereceptors, first reported for the homotypic erythropoietin re-ceptor (EPOR) (17), represents a new cytokine receptor sig-naling paradigm (reviewed in ref. 18). It remains an openquestion as to whether the two binding mechanisms and/or otherundiscovered mechanisms function independently or synergisti-cally. Ultimately, the structural details describing preassembly ofpreformed receptors of short-chain heterotypic receptors like theγc 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 theasymmetric unit, with the dimer interface comprising many ofthe same residues involved in IL-7 binding. We compare theunliganded IL-7Rα structure with the structures of IL-7Rα withIL-7 (19), other γc receptors (20–23), and the EPOR homodimerstructure (21). Biophysical studies indicate weak self-associationof the IL-7Rα ECDs but a stronger association between the IL-7Rα and γc ECDs. These results provide the basis for structuralmodels of normal and aberrant IL-7 signaling. Finally, the pro-posed structural mechanism may be applicable to the γc family.

ResultsArchitecture of the Unliganded IL-7Rα Structure. The IL-7Rα ECD(residues 1–219) was expressed and purified as described pre-viously (24). The crystal structure of IL-7Rα was solved usingmolecular replacement to a resolution of 2.15 Å. The structurewas refined with Rcryst and Rfree values of 0.214 and 0.258, re-spectively (SI Appendix, Table 1). Surprisingly, the asymmetricunit 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 theEditorial Board.

Data deposition: The atomic coordinates and structure factors reported in this paper havebeen deposited in the Protein Data Bank, www.pdb.org (PDB ID code 3up1).1To whom correspondence should be addressed. E-mail: swalsh12@umd.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1116582109/-/DCSupplemental.

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with pseudotwofold symmetry (Fig. 1). The receptor chains aresimilar, with a Cα backbone root-mean-square deviation (rmsd) of0.93 Å for residues 18–209 (SI Appendix, Figs. 2–5). An O-linkedglycan attached to S133 of chain B (S133B) causes backbonechanges on the order of 1.4–7.7 Å relative to chain A. This con-formational change also slightly shifts the main chain of the twopreceding β-strands, A2 and B2. Other than the small differencesof the interfacial region, the remaining differences correspond toloop regions and are likely a result of crystal-packing differences.IL-7Rα consists of two fibronectin type-3 (FN3) domains (D1

andD2) connected by a 310-helical linker with an overall L-shapedarchitecture similar to structures of IL-7Rα in complex with IL-7(19) and other CRH1 members (25). Chains A and B have elbowangles of 77° and 79°, respectively. IL-7Rα contains three disulfidebonds in the D1 domain connecting β-strands A1-B1 (C22-C3),F1-G1 (C88-C98), and C1-C′1 (C54-C62). Electron density wasabsent for theN- (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 withPNGase F. PNGase F cleaves the whole N-glycan, but cannotremove the N-glycan if an α(1→3) fucose is attached to the firstGlcNAc. No other N-glycans were visualized for the other Asnsites of IL-7Rα (N29, N45, N162, N212, and N213). Interestingly,the region surrounding the side chain of S133B contained signif-icant difference density, suggesting an O-glycan of this Ser (SIAppendix, Fig. 3). The O-glycan of S133B forms numerous con-tacts with the receptor residues. Chain A of IL-7Rα displayed noevidence of this modification, indicating that it was either notO-glycosylated or is dynamic. Mass spectrometry confirmed S133to be glycosylated with an N-acetyl galactosamine and furtheridentified an O-linked glycan of T132 (SI Appendix, Figs. 6–8 andTable 2). There was no crystallographic evidence of an O-glycanattached to T132 of either chain. Future studies will explore theimportance of O-glycosylation of IL-7Rα.

IL-7Rα Dimer Interface. A surprising feature of the unliganded IL-7Rα 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 disulfidebonds 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 interfaceformed 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 thetwo views. The two insets are related by a 180° rotation around the y axis.

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looking down onto the cell surface: Their N and C termini arelocated on opposite ends, and the binding interface consists ofsimilar elbow loop residues that were previously shown to becritical for IL-7 interaction (19) (Fig. 1). The IL-7Rα bindinginterface uses 19 and 20 residues and buries 568 and 600 Å2 ofsurface area for chain A and B, respectively (average of 584 Å2).There is one main-chain hydrogen bond (h-bond) betweenK138B O and E59A N (2.7 Å). The interface comprises 28% ofcharged residues, 17% of polar residues, and 56% of apolarresidues, 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 sixloop regions, contacts are formed only from loops L2, L3, L5, andL6 of chain A and loops L2–L6 of chain B (SI Appendix, Fig. 4).The asymmetry of the dimer interface primarily results from side-chain orientation differences, but there are two notable main-chain conformational changes between the two chains. The firstmain-chain difference is the conformation of loop L5. Not con-tacting the binding interface directly, theO-glycan of S133B altersthe L5 loop conformation of chain B, adopting a single turn ofα-helix, which does not occur in chain A (SI Appendix, Figs. 2 and5). The second main-chain difference is the conformation of loopL6. 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, resultingin side-chain rotamer changes of H191 and Y192.

Biophysical Measurements of IL-7Rα Associations. The self-associa-tion of the IL-7Rα ECD was assayed using size-exclusion chroma-tography (SEC), analytical ultracentrifugation (AUC), and surfaceplasmon resonance (SPR). Both unglycosylated (5 μM) and glyco-sylated (10 μM) IL-7Rα eluted as monomers by SEC (24). Morequantitative assessment of potential self-association of the IL-7RαECD involved sedimentation equilibrium experiments using AUC.Sedimentation equilibrium curves of IL-7Rα at multiple concen-trations and rotor speeds yielded a single, homogeneous specieswith a buoyant molecular weight (Mb) of 9,277 ± 120 (SI Appendix,Fig. 9). IL-7Rα showed no tendency to aggregate into higher-orderoligomers at loading concentrations of 23 μM, and the confidencevalues for the molecular weight were reasonable for a glycoprotein.The molecular weight (Mw) of IL-7Rα was estimated by comparingthe Mw based on composition with that obtained from the com-puted partial specific volume (ν) and from Mb determined by sed-imentation equilibrium. This approach converged on a value of∼20% (wt/wt) glycosylation, and indicates reasonable values for a νof 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-stateSPR analysis (Fig. 2A). A stronger self-association of 3 μM wasreported for the homodimerization of the IL-2Rβ ECDs, the re-ceptor homologous to IL-7Rα (26). Fig. 2B displays the bindingkinetics of the γc–IL-7Rα interaction. Analysis of these curves fitbest to a two-step conformational exchange binding model (SIAppendix, Fig. 10) with k1, k−1, k2, and k−2 rates of 2.6× 103M−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 alsoobserved for the interaction of full-length IL-7Rα with γc than IL-7Rα self-association on the cell surface of T cells (13). Likewise, ahigher binding affinity was observed for the interaction of full-lengthIL-2Rβ with γc than IL-2Rβ self-association on cells (14, 27). Fur-ther structural studies of IL-7Rα and γc will shed light on the in-dividual contributions of the ECD and TMD to IL-7–independentassociation.

Structural Comparison of Unliganded and Liganded States of IL-7Rα.The unliganded IL-7Rα structure is similar to the liganded IL-7Rα structure bound to IL-7. There are liganded structures of IL-7 bound to unglycosylated (two complexes) and glycosylated (one

complex) forms of IL-7Rα (19). Chain A of the unliganded IL-7Rα is more closely related to the structures of IL-7Rα in complexwith IL-7 than it is to chain B of the unliganded IL-7Rα. Back-bone superimpositions of residues 18–209 gives an average rmsdof 0.55 Å for chain A to the liganded IL-7Rα structures (SI Ap-pendix, Figs. 2, 4, and 5). When chain B is superimposed onto theliganded IL-7Rα structures, the average backbone rmsd increasesto 1.06 Å. Not surprisingly, the largest differences between chainB and the complex structures are in the same regions seen incomparison with chain A. These results suggest that either thereare no large conformational changes upon IL-7 binding to IL-7Rαor that the dimerization of IL-7Rα somehowmimics 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 h-bonds, and is less specific than the interface of IL-7Rα bound toIL-7. Depending on the chain, the unliganded IL-7Rα homo-dimer structure does not use all of the six loop regions L1–L6,whereas in the liganded IL-7Rα structures all loop regions engageIL-7 (SI Appendix, Fig. 4). Thus, the amount of buried surfacearea for the unliganded IL-7Rα homodimer is reduced by 134 Å2

compared with the liganded state of IL-7Rα (584 vs. 718 Å2; welist the average buried surface area of the two proteins). Finally,the unliganded IL-7Rα interface has only one h-bond versus fiveh-bonds for the liganded IL-7Rα structures and a poorer shapecomplementarity of 0.60 versus 0.68 for the liganded IL-7Rα

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

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structures [Sc values varied by 0.04 for the liganded IL-7Rαstructures, 0.65–0.69 (19)].

DiscussionStructural Comparisons to CRH1 Members. Comparison of the IL-7Rα homodimer structure to other CRH1 members reveals sig-nificant angular changes of the two FN3 domains. The FN3domain geometries were analyzed by three angles: elbow (ε;angle between the D1 and D2 domains), twist (τ; angle betweenthe x axis of the D1 and D2 domains), and swivel (σ; spin of theD2 domain in the x-z plane) (SI Appendix, Table 3) (28). Theunliganded IL-7Rα structure displays more obtuse ε and τ angles(Δε = 3°, Δτ = 4°) but a more acute σ angle (Δσ = 5°) to theliganded IL-7Rα structures (19). In comparison with IL-2Rβfrom IL-2–activating structures (20, 29), the unliganded IL-7Rαstructure 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 σangles (Δε= 6°, Δσ= 9°) but a more obtuse τ angle (Δτ= 3°) toIL-4Rα from an IL-4–activating structure (23). There are binaryand ternary complex structures of IL-4Rα, and this receptordisplays similar ε and τ angles, but the σ angle increases by 39°when IL-4Rα/IL-4 binds γc (22, 23). Future structural studies ofγc family members (e.g., IL-7 ternary complex) will be essentialin understanding nonactivating and activating receptor geome-tries and also receptor pleiotropy from a structural perspective.The only definitive unliganded homodimer structure of the

CRH1 family is EPOR (21). [Although the unliganded growthhormone receptor structure revealed a homodimer assembly,homodimerization was triggered through the transmembraneregion, not the ECD (30).] The binding interfaces between IL-7Rα and EPOR display significant variations (SI Appendix, Fig. 12and Table 3). The EPOR dimer interface has a larger amount ofburied surface area, 722 Å2, and more h-bonds, 9 (21), than theIL-7Rα dimer interface (584 Å2 and one h-bond). Althoughlargely apolar (EPOR = 43%, IL-7Rα = 56%), the bindinginterfaces do vary in their polarity, with polar and charged resi-dues comprising 33% and 24% of the EPOR dimer interface and17% and 28% of the IL-7Rα dimer interface, respectively. Ad-ditionally, the EPOR dimer interface is almost perfectly sym-metric, 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 issuperimposed onto chain A of EPOR using secondary structuralelements (SI Appendix, Fig. 13), there is a 47° rotation and a 12-Å

translation (using the conserved disulfide bond) of chain B of IL-7Rα to chain B of EPOR. Large differences are also observed forthe FN3 geometries of IL-7Rα compared with EPOR. IL-7Rαdisplays more obtuse ε and σ angles (Δε = 10°, Δσ = 4°) butsimilar τ angles. These large conformational changes of thehomodimeric structures reveal a level of structural complexitythat the receptors undergo before and after binding their ligandsthat allows us to speculate on IL-7Rα signaling mechanisms.

IL-7Rα Signaling Model. The IL-7Rα dimer structure explains whyhomodimerization of IL-7Rα does not activate the IL-7 or TSLPpathways. Although there is currently no structure of the IL-7ternary complex, there are activating structures of other γcmembers, namely IL-2 and IL-4 (20, 23, 29). The average distancebetween the C-terminal domains of these structures between theα/β receptors and γc is 27 Å. We predict a similar distance for theC-terminal domains of IL-7Rα and γc in an activating complex;however, the distance between the C-terminal domains of thehomodimeric IL-7Rα structure is 110 Å. Thus, the JAK1 mole-cules bound to the IL-7Rα ICDs are kept physically apart,inhibiting self-activation. A similar mechanism was presented forthe EPOR homodimer (21, 31). In this case, the C-terminal do-mains of the EPOR homodimer are separated by a smaller dis-tance of 73 Å relative to the IL-7Rα homodimer. Upon EPObinding, the distance between the two C-terminal domainsreduces to 39 Å, bringing the two ICDs in close proximity, leadingto activation. Based on the IL-7Rα homodimer structure, wepresent a structural mechanism for signal inactivation in the li-gand-independent association of IL-7Rα and γc.We demonstrate a stronger binding affinity for hetero-

dimerization of the IL-7Rα/γc ECDs than homodimerization ofthe IL-7Rα ECDs (Kds of 3.1 μM vs. >610 μM at 25 °C). Previousstudies also reported stronger binding affinity for full-length IL-7Rα and γc than the IL-7Rα homodimer on T cells at 37 °C (13).The IL-2– and IL-4–activating structures revealed extensivebinding surfaces between the C-terminal D2 domains of the α/βreceptors with γc (20, 23, 29); the results presented here disfavorthe ligand-independent association of IL-7Rα and γc throughtheir C-terminal D2 domains. Rather, the loop residues of IL-7Rα that are used to bind IL-7 and itself would likely be in-volved, leading us to propose the structural mechanism of theIL-7 signaling pathway highlighted in Fig. 3. The IL-7Rαhomodimer keeps the JAK1 molecules bound to the ICDs sep-arated by 110 Å. Likewise, the IL-7Rα–γc heterodimer [wemodeled 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 andIL-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 distancebetween theD2 domains of IL-7Rα and γc was an average of the γc structureswith IL-2 [PDB ID code 2bfi (20); PDB ID code 2erj (29)] and IL-4 [PDB ID code 3bpl (23)].

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elements of the γc ECD onto chain B (rmsd = 1.9 Å)] wouldkeep JAK1 and JAK3 separated by a large distance preventingactivation. When IL-7 binds both IL-7Rα and γc, the receptorsundergo an ∼90° rotation away from the cell surface, bringing thetwo C termini of IL-7Rα and γc to within less than 30 Å andactivating signal transduction.

IL-7Rα Signaling in Gain-of-Function Muteins. Gain-of-functionmutations in IL-7Rα have been identified in patients with B- andT-cell acute lymphoblastic leukemias (B-ALL and T-ALL) (8,32). In one study, B-ALL patients contained insertions anddeletions of residues in the N-terminal region of the IL-7RαTMD, with all of the sequences containing an extra Cys residue(32). In other B-ALL patients, the IL-7Rα ECD contains anS165-to-C165 mutation (32). The same study also reported T-ALL patients containing 30 different sequence insertions anddeletions in the N-terminal region of the IL-7Rα TMD, 27 ofwhich have an extra Cys (32). The mutations in the TMD of IL-7Rα were different among the B-ALL and T-ALL patients. Ina different study, T-ALL patients contained 17 different inser-tions and deletions in the N-terminal region of the IL-7Rα TMD,with 13 of them containing an additional Cys (8). These T-ALLmutations were different between the two studies (8, 32). Bothstudies tested several of the B- and T-ALL muteins in IL-7Rαand demonstrated IL-7– and γc-independent activation of thesignaling pathway (8, 32). The structural mechanism presentedabove can be further extended to speculate on the molecularbasis of the ALL muteins.We posit the B- and T-ALL muteins of IL-7Rα function in-

dependent of ligands by positioning at least two copies of IL-7Rαin geometries such that the C-terminal ends of their ECDs orTMDs are less than 30 Å apart. To provide structural support forthis hypothesis, we constructed energy-minimized structuralmodels of the S165C disulfide-linked homodimer of the B-ALLmutein of the IL-7Rα ECD and one of the T-ALL insertionsequences (JPSP7) in the IL-7Rα TMD (Fig. 4). The S165C B-ALL mutein of IL-7Rα forms an additional disulfide bond be-tween two IL-7Rα molecules on the cell surface (32). S165 ispositioned in a solvent-exposed loop region of the D2 domain,allowing for easy disulfide-bond formation between two IL-7Rαmolecules (Fig. 4A). The structural model of the S165C muteinreveals that the C-terminal ends of the D2 domains of the IL-7Rα ECDs would be 29 Å apart, well within the distance pre-dicted to activate the pathway.The JPSP7 T-ALL mutein contains a three-residue inser-

tion highlighted in bold into the IL-7Rα TMD sequence:PILLTCPTISILSFFSVALLVILACVLW (8). The IL-7Rα TMDis predicted to adopt a single α-helix spanning the lipid bilayer.The peptide sequences were fed into an algorithm developed byDeGrado and coworkers that optimizes the depth and angulargeometry of transmembrane α-helical segments in a lipid bilayerbased on known membrane protein structures (33). The algo-rithm predicts the first transmembrane residue of the T-ALLmutein to be I228, with residues PILLTCPT solvent-exposed.Results led to the conclusion that this T-ALL mutein forms anIL-7Rα homodimer/oligomer at the cell surface (8). A structuralmodel of the C225 disulfide-linked IL-7Rα homodimer wasconstructed (Fig. 4B). The C-terminal residues from 209 to 219of the wild-type juxtamembrane region are highly mobile. Thedistance between the Cαs of W247 of the two TMDs is 11 Å, wellwithin the distance required to bring the JAK1s bound to theICDs together, leading to activation.The linking of two IL-7Rαs through a disulfide bond in the

juxtamembrane region is not a unique mechanism. Similarmechanisms have been reported for TSLPR (34), EPOR (35,36), and epidermal growth factor receptors (37). However, notall of the T-ALL muteins contained an unpaired cysteine in thisregion of IL-7Rα. Therefore, we posit that the insertions and/or

deletions of the T-ALL muteins alter the orientation of the IL-7Rα TMD, enabling higher-order oligomeric states capable ofself-activating the IL-7 pathway. Future studies of the T-ALLmuteins will delineate the molecular mechanisms of IL-7Rα andaid in developing new diagnostic and treatment strategies forthese cancers.There are many reasons why the IL-7Rα homodimer structure

likely represents a physiologically relevant conformation ona cell surface. First, self-association of the full-length IL-7Rα hasbeen reported on the T-cell surface (13). Second, the structurereveals that the homodimer interface forms at the elbow loopresidues that interact with its ligands. Third, the structure can beeasily positioned on a cell surface of a bilayer (Fig. 1B). Al-though our IL-7Rα ECD consists of residues 1–219 of the full-length receptor, the two chains were built to residues 211 and212. Modeling the unseen residues (4 Å per residue) onto thestructure, the C termini of the chains can easily reach the bilayer.There is also ample space to accommodate the glycans of IL-7Rα. Fourth, although homodimerization of the IL-7Rα ECDs is

Fig. 4. Structural models of two IL-7Rα mutations found from patients witheither B-ALL or T-ALL. (A) Ribbon diagram of the disulfide-linked homodimerof 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 trans-membrane regions. For clarity, only one of the IL-7Rα ECDs is shown. Insertionof three amino acids by the T-ALL mutation from patient JPSP7 likely causesextension of the transmembrane region into the extracellular face, resultingin a disulfide-linked homodimer of two receptor molecules by C225.

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weak in the millimolar range, translating this solution affinity toa binding constant in the cell membrane would give a value in themicromolar range because of the reduced dimensionality of lo-calization in a lipid bilayer. A similar argument was presented forEPOR homodimer association, which displayed no solution self-association of the EPOR ECDs (21, 38). Fifth, IL-7 binds ahomodimeric IL-7Rα–Fc fusion (NS0 cells; R&D Systems) andmonomeric IL-7Rα (insect cells) with similar binding affinities(Kds ∼60 nM) (19). Sixth, the ECD and full-length IL-7Rα exhibitthe same trend of binding γc stronger than itself (13). Finally,experiments performed on other homodimeric α/β-receptors inthe γc family have shown that the ECDs, and not the transmem-brane or intracellular domains, are responsible for dimerization.For instance, removal of the IL-2Rβ and γc ECDs from the full-length receptors abrogates IL-2Rβ homodimerization and IL-2Rβ/γc heterodimerization (14). Although not tested (13), a sim-ilar result would likely occur with IL-7Rα and γc. Thus, althoughwe cannot completely rule out crystallization forces, the IL-7Rαhomodimer structure agrees well with observed biology andprovides a structural mechanism describing signal quiescence andactivation for normal and aberrant IL-7 signaling and may beapplicable to the γc family.

Materials and MethodsDetailed methods describing protein production, crystal structure de-termination, biophysicalmeasurements, andmass spectrometry are given in SIAppendix, Materials and Methods. Briefly, the IL-7Rα and γc ECDs wereexpressed from insect cells and purified as described previously (24). A crystalof the IL-7Rα ECD grown from 1.26 M (NH4)2SO4, 0.1 M Tris·HCl (pH 8.5), and0.2 M Li2SO4 diffracted X-rays at the General Medicine and Cancer InstitutesCollaborative Access Team (GM/CA-CAT) beamline 23ID-D at the AdvancedPhoton Source. The structure was solved using the liganded IL-7Rα structure[Protein Data Bank (PDB) ID code 3di3] as a search model for molecular re-placement. SPR binding kinetics and affinities of the IL-7Rα and γc ECDs weredetermined 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-7Rαcancer mutations were generated using MacPyMOL (39) and energy-mini-mized with CNS (40). Structural figures were rendered with MacPyMOL (39).

ACKNOWLEDGMENTS. We thank R. Mariuzza and M. Walter for commentson the manuscript. C.A.M. was supported by a postdoctoral fellowship fromthe American Heart Association (725595B). X-ray data collection was done atthe National Institutes of Health (NIH) GM/CA 23ID beamline at theAdvanced Photon Source at Argonne National Laboratory, which is operatedby UChicago Argonne, LLC under Contract DE-AC02-06CH11357. This workwas supported in part by NIH Grant P41RR018502 (to L.W.). This research wassupported by NIH Grant AI72142 (to S.T.R.W.).

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 re-dundancy. J Biol Chem 277:29355–29358.

3. Kovanen PE, Leonard WJ (2004) Cytokines and immunodeficiency diseases: Criticalroles of the γ(c)-dependent cytokines interleukins 2, 4, 7, 9, 15, and 21, and theirsignaling pathways. Immunol Rev 202(1):67–83.

4. Watanabe M, et al. (1998) Interleukin 7 transgenic mice develop chronic colitis withdecreased 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 αchain (IL7R) shows allelic and functional association with multiple sclerosis. Nat Genet39:1083–1091.

6. Hafler DA, et al.; International Multiple Sclerosis Genetics Consortium (2007) Riskalleles 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 sur-vival and cell cycle progression of T-cell acute lymphoblastic leukemia cells by down-regulating 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-cellacute lymphoblastic leukemia. Nat Genet 43:932–939.

9. Rochman Y, Spolski R, LeonardWJ (2009) New insights into the regulation of T cells byγ(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. Cell60:941–951.

11. Wells JA (1996) Binding in the growth hormone receptor complex. Proc Natl Acad SciUSA 93(1):1–6.

12. Rose T, Lambotte O, Pallier C, Delfraissy JF, Colle JH (2009) Identification and bio-chemical characterization of human plasma soluble IL-7R: Lower concentrations inHIV-1-infected patients. J Immunol 182:7389–7397.

13. Rose T, et al. (2010) Interleukin-7 compartmentalizes its receptor signaling complex toinitiate CD4 T lymphocyte response. J Biol Chem 285:14898–14908.

14. Pillet AH, et al. (2010) IL-2 induces conformational changes in its preassembled re-ceptor core, which then migrates in lipid raft and binds to the cytoskeleton mesh-work. J Mol Biol 403:671–692.

15. Kammer W, et al. (1996) Homodimerization of interleukin-4 receptor α chain caninduce intracellular signaling. J Biol Chem 271:23634–23637.

16. Malka Y, et al. (2008) Ligand-independent homomeric and heteromeric complexesbetween 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 domainof the erythropoietin receptor resulting in hormone-independent activation and tu-morigenicity. Nature 348:647–649.

18. Schreiber G, Walter MR (2010) Cytokine-receptor interactions as drug targets. CurrOpin Chem Biol 14:511–519.

19. McElroy CA, Dohm JA, Walsh ST (2009) Structural and biophysical studies of the hu-man IL-7/IL-7Rα complex. Structure 17:54–65.

20. Wang X, Rickert M, Garcia KC (2005) Structure of the quaternary complex of in-terleukin-2 with its α, β, and γc receptors. Science 310:1159–1163.

21. Livnah O, et al. (1999) Crystallographic evidence for preformed dimers of erythro-

poietin receptor before ligand activation. Science 283:987–990.22. Hage T, Sebald W, Reinemer P (1999) Crystal structure of the interleukin-4/receptor α

chain complex reveals a mosaic binding interface. Cell 97:271–281.23. LaPorte SL, et al. (2008) Molecular and structural basis of cytokine receptor pleiotropy

in the interleukin-4/13 system. Cell 132:259–272.24. Wickham J, Jr., Walsh ST (2007) Crystallization and preliminary X-ray diffraction of

human interleukin-7 bound to unglycosylated and glycosylated forms of its α-receptor.Acta Crystallogr Sect F Struct Biol Cryst Commun 63:865–869.

25. Wang X, Lupardus P, Laporte SL, Garcia KC (2009) Structural biology of shared cy-

tokine receptors. Annu Rev Immunol 27:29–60.26. Rose T, Moreau JL, Eckenberg R, Thèze J (2003) Structural analysis and modeling of

a synthetic interleukin-2 mimetic and its interleukin-2Rβ2 receptor. J Biol Chem 278:

22868–22876.27. Pillet AH, et al. (2008) Human IL-Rβ chains form IL-2 binding homodimers. Eur Cyto-

kine Netw 19(1):49–59.28. Deivanayagam CC, et al. (2000) Novel fold and assembly of the repetitive B region of

the Staphylococcus aureus collagen-binding surface protein. Structure 8(1):67–78.29. Stauber DJ, et al. (2006) Crystal structure of the IL-2 signaling complex: Paradigm for

a heterotrimeric cytokine receptor. Proc Natl Acad Sci USA 103:2788–2793.30. Brown RJ, et al. (2005) Model for growth hormone receptor activation based on

subunit rotation within a receptor dimer. Nat Struct Mol Biol 12:814–821.31. Remy I, Wilson IA, Michnick SW (1999) Erythropoietin receptor activation by a ligand-

induced conformation change. Science 283:990–993.32. Shochat C, et al. (2011) Gain-of-function mutations in interleukin-7 receptor-α (IL7R)

in childhood acute lymphoblastic leukemias. J Exp Med 208:901–908.33. Senes A, et al. (2007) E(z), a depth-dependent potential for assessing the energies of

insertion of amino acid side-chains into membranes: Derivation and applications to

determining the orientation of transmembrane and interfacial helices. J Mol Biol 366:

436–448.34. Yoda A, et al. (2010) Functional screening identifies CRLF2 in precursor B-cell acute

lymphoblastic leukemia. Proc Natl Acad Sci USA 107:252–257.35. Constantinescu SN, et al. (2001) Ligand-independent oligomerization of cell-surface

erythropoietin receptor is mediated by the transmembrane domain. Proc Natl Acad

Sci USA 98:4379–4384.36. Lu X, Gross AW, Lodish HF (2006) Active conformation of the erythropoietin receptor:

Random and cysteine-scanning mutagenesis of the extracellular juxtamembrane and

transmembrane domains. J Biol Chem 281:7002–7011.37. Lemmon MA, Schlessinger J (2010) Cell signaling by receptor tyrosine kinases. Cell

141:1117–1134.38. Philo JS, Aoki KH, Arakawa T, Narhi LO, Wen J (1996) Dimerization of the extracellular

domain of the erythropoietin (EPO) receptor by EPO: One high-affinity and one low-

affinity interaction. Biochemistry 35:1681–1691.39. DeLano W (2002) The PyMOL Molecular Graphics System (DeLano Scientific, San

Carlos, CA).40. Brünger AT, et al. (1998) Crystallography & NMR system: A new software suite for

macromolecular structure determination. Acta Crystallogr D Biol Crystallogr 54:

905–921.

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