R ESEARCH A RTICLES peptide, disordered in the structure, between the IL-2Ra globular head and the trans- Structure of the Quaternary membrane segment. This allows the IL-2Ra binding domain to extend away from the cell Complex of -2 with Its surface and reach the dorsally located binding site on IL-2 (Fig. 1B). The bases of the re- ceptors IL-2Rb and g , both class I–type a, b, and g Receptors c c receptors, converge to form a Y Xinquan Wang,* Mathias Rickert,* K. Christopher Garcia. shape and IL-2 binds in the fork (Fig. 1, A and B). Formation of the quaternary com- Interleukin-2 (IL-2) is an immunoregulatory cytokine that acts through a plex is mediated by four binding sites—IL-2/

quaternary receptor signaling complex containing alpha (IL-2Ra), beta (IL-2Rb), IL-2Ra, IL-2/IL-2Rb, IL-2/gc, and IL-2Rb/ )2 and common gamma chain (gc) receptors. In the structure of the quaternary gc—burying a total of 5700 of surface area ectodomain complex as visualized at a resolution of 2.3 angstroms, the binding (fig. S1). The IL-2/IL-2Ra and IL-2/IL-2Rb of IL-2Ra to IL-2 stabilizes a secondary binding site for presentation to IL-2Rb. contacts are independent, whereas IL-2 and

gc is then recruited to the composite surface formed by the IL-2/IL-2Rb IL-2Rb form a composite interface with gc, complex. Consistent with its role as a shared receptor for IL-4, IL-7, IL-9, IL-15, reflecting the cooperative nature of complex

and IL-21, gc forms degenerate contacts with IL-2. The structure of gc provides a assembly. rationale for loss-of-function mutations found in patients with X-linked severe IL-2Ra has been shown to deviate from combined immunodeficiency diseases (X-SCID). This complex structure provides typical structure and mode

a framework for other gc-dependent cytokine-receptor interactions and for the of interaction with IL-2 (17). It is composed engineering of improved IL-2 therapeutics. of two domain-swapped ‘‘sushi’’ modules, es- sentially miniature b-sheet sandwich domains.

The cytokine IL-2 is mainly produced by the heterodimerization of their cytoplasmic IL-2Rb and gc are prototypical members of antigen-activated T cells and promotes the domains and subsequent kinase activation of the class I cytokine receptor superfamily proliferation, differentiation, and survival of multiple signaling pathways (13, 14). A com- (19). Both are composed of N- and C-terminal mature T and B cells as well as the cytolytic plex with three subunits—IL-2Ra,IL-2b, and fibronectin-III domains (D1 and D2, respec- , activity of natural killer (NK) cells in the innate gc—binds with high affinity (Kd 10 pM) tively), which are characterized by a b-sandwich immune defense (1, 2). IL-2 is used thera- and is the receptor form on activated T cells sheet consisting of seven antiparallel strands peutically as an immune adjuvant in certain (10). The high-affinity receptor complex arranged in a three-on-four topology. In IL-2Rb, types of lymphoproliferative diseases and mediates most biological effects of IL-2 in the D1 and D2 domains are connected by a cancers, and IL-2 antagonists can prevent organ vivo (2). helical linker and are bent at È90-, whereas transplant rejection (3, 4). However, severe Whereas IL-2Ra isaspecificreceptorfor in gc the D1 and D2 domains are bent at È - dose-limiting toxicity has limited its effective- IL-2, IL-2Rb is also a component of the IL-15 120 (Fig. 1A). Both IL-2Rb and gc contain ness in the clinic. These deleterious side effects receptor and gc is shared by IL-4, the two disulfide bonds in the N-terminal are mediated through different combinations of IL-7, IL-9, IL-15, and IL-21 (15). Mutations domain (D1) and a ‘‘WSXWS’’ motif (20)in

IL-2 receptors, which suggests that structure- in gc can abolish the activity of all gc- the C-terminal domain (D2) that are char- based engineering of receptor-selective var- dependent cytokines and result in X-linked acteristic of class I cytokine receptors (19). iants could have clinical benefit (5). severe combined immunodeficiency diseases However, a third disulfide bond in the gc IL-2 exerts its pleiotropic activities through (X-SCID), in which the T and NK cells are D2 domain is unusual because of its cen- binding to different receptor complexes, absent or profoundly reduced in number (16). tral position in the interface with IL-2 and depending on which of the components are Because the six gc-dependent cytokines have its role in enabling degenerate cytokine expressed on the cell surface: the alpha chain low , structural information recognition (Cys160 to Cys209) (discussed (IL-2Ra), beta chain (IL-2Rb), and common will be helpful to delineate shared versus below). cytokine receptor gamma chain (gc)(6–10). ligand-specific binding determinants that could IL-2/IL-2Ra. In the ‘‘low-affinity’’ com- Isolated IL-2Ra has been termed the Blow- be exploited therapeutically. Previously, we plex, the atomic interactions between IL-2 [ , ) affinity IL-2 receptor (binding affinity Kd reported the structure of the binary complex of and IL-2Ra, now visualized at 2.3 , are 10 nM) and is not involved in signal transduc- IL-2 with IL-2Ra (17). We now present the unchanged from the binary complex at 2.8 ) ) tion (11). A complex of IL-2Rb and gc binds crystal structure, at 2.3 resolution, of the (17). The binding interface between IL-2 and , with intermediate affinity (Kd 1nM)andisthe quaternary complex of IL-2 with the extra- IL-2Ra in the quaternary complex is com- receptor form on NK cells, macrophages, and cellular domains of receptors IL-2Ra,IL-2Rb, posed of helices A¶ and B¶ and part of the AB resting T cells (2), although IL-2Rb alone and gc. loop in IL-2 and strands G, C, and D in the , has very low affinity (Kd 100 nM) and gc Overall structure. Because of the heter- D1 domain and strand A in the D2 domain in alone has no detectable binding affinity for ogeneity of the fully glycosylated IL-2Ra (table S2A). The two prominent 42 IL-2 (12). The association of IL-2Rb and gc in expressed from insect cells, we crystallized a hydrophobic ridges around residues Phe and the presence of IL-2 is necessary and suffi- glycan-minimized quaternary complex, which Tyr 45 of IL-2 insert into grooves between the cient for effective signal transduction through had five potential Asn-linked glycosylation IL-2Ra beta strands. Superposition of the two sites mutated (18). This material behaved IL-2Ra structures in the binary and quater- Howard Hughes Medical Institute, Department of identically to the fully glycosylated proteins nary complexes shows a significant shift in Microbiology and Immunology, and Department of and yielded crystals that diffracted to 2.3 ) the D2 domain of IL-2Ra (È2 )), which is Structural Biology, Stanford University School of Medi- resolution (18). most likely a result of crystal packing and cine, 299 Campus Drive, Fairchild D319, Stanford, CA 94305, USA. The quaternary complex is composed of reflects some flexibility in the D1-D2 one copy each of IL-2, IL-2Ra, IL-2Rb,and junction. *These authors contributed equally to this work. .To whom correspondence should be addressed. gc (Fig. 1A). The orientation of IL-2Ra ex- IL-15 is the only other cytokine that uses an E-mail: [email protected] plains the necessity for the long connecting atypical sushi-domain alpha receptor (IL-

www.sciencemag.org SCIENCE VOL 310 18 NOVEMBER 2005 1159 R ESEARCH A RTICLES

123 49 62 137 Fig. 1. Structure of the human IL-2/Rabg quaternary signaling Asn of IL-2Rb and at Asn ,Asn ,andAsn of gc are shown in complex. (A and B) Ribbon diagram of the complex structure shown gray (20). Disulfide bonds are shown in red. The disordered peptides in two views related by a È90- rotation about the vertical axis. IL-2 is connecting the C terminus of the receptors to the cell membrane are showninviolet,andthereceptorsareshownincyan(IL-2Ra), blue shown as dotted lines in their respective colors. The program PyMol

(IL-2Rb), and gold (gc). The observed N-linked carbohydrates at (43) was used to make all figures.

15Ra), which is expressed primarily on NK binary and quaternary complexes, several Two residues of IL-2 that have been shown by cells (21). However, IL-15Ra is only a single turns of helix C are slightly unwound and mutagenesis to be critical for IL-2Rb binding, sushi domain analogous to the IL-2Ra D1 translated forward by È1.0 ) toward IL-2Rb Asp20 and Asn88, are involved in hydrogen domain (22, 23). By analogy with IL-2, the (Fig. 2A). This local conformational adjust- bonding networks to both water molecules and IL-15/IL-15Ra complex likely forms first, fol- ment moves IL-2 residue Asn88 into hydrogen- side chains on IL-2Rb (Fig. 3A). The side chains 42 133 134 lowed by binding to IL-2Rb and gc to form the bonding distance to IL-2Rb residue Arg of IL-2Rb residues His and Tyr insert into quaternary signaling complex. (Fig. 2B). This movement possibly ‘‘primes’’ a complementary cavity in IL-2 to form IL-2Ra does not appear to make any the next step in complex assembly by forming hydrogen and ionic bonds with Asp20 of IL-2

contact with either IL-2Rb or gc.Thisis a more complementary IL-2Rb binding site. (Fig. 3B). rather surprising, given that the IL-2/IL-2Ra Consistent with this, mutation of Asn88 in IL-2 IL-2Rb is also used by IL-15 to form a complex binds with much higher affinity to ablates binding to IL-2Rb (5). IL-2Ra may quaternary complex along with IL-15Ra and , IL-2Rb (Kd 30 pM) than does IL-2 binding stabilize a favorable IL-2Rb–binding confor- gc (15). IL-15 has limited sequence identity , to IL-2Rb alone (Kd 100 nM) (12, 24) mation of IL-2 helix C, reducing a conforma- (19%) with IL-2, so its contact with IL-2Rb is and the on-rate of IL-2 for IL-2Rb is faster in tional entropy penalty that would be incurred probably through a unique set of interactions. the presence of IL-2Ra by a factor of 3 to 20 during binding to IL-2Rb. This priming of a The bridging water molecules may contribute (11, 25). We see no evidence of a composite ‘‘quiescent’’ IL-2Rb binding site in IL-2 by to the ability of IL-2Rb to cross-react by ac- receptor binding surface for IL-2, so what is IL-2Ra could effectively increase the on-rate commodating the different IL-15 residues the basis of the cooperativity? One possibility for the IL-2 interaction with IL-2Rb,ashas through remodeling of the intervening solvent would be simple entropy reduction, wherein been observed. layer.

IL-2Ra captures and concentrates free IL-2 at IL-2/IL-2Rb. The interface between IL-2 IL-2/gc. Neither IL-2 nor IL-2Rb alone È )2 the cell surface for presentation to IL-2Rb and IL-2Rb buries 1350 formed by have measurable affinities for gc (12). There- and gc. Another possibility would be an IL- residues from helices A and C in IL-2 and fore, two very weak interactions, IL-2/gc and ¶ 2Ra–induced conformational change in IL-2 residues from loops CC 1, EF1, BC2, and IL-2Rb/gc, combine to produce an intermediate- that stabilizes the formation of the ternary FG2 in IL-2Rb (table S2B). The interface is affinity IL-2/IL-2Rb/gc complex. In the qua- complex. highly polar, with eight hydrogen bonds ternary complex structure, the interaction

To address the latter mechanism, we directly between IL-2 and IL-2Rb residues. surface of the IL-2/Rab complex with gc is compared IL-2 structures in the quaternary Strikingly, there are seven water molecules composed of two interfaces: a small one be-

complex, binary complex, and unbound buried in the interface that bridge interactions tween IL-2 and gc, and a larger one between states. The root mean square deviations for between IL-2 and IL-2Rb by forming bonds IL-2Rb and gc. È )2 Ca atoms in the helical core between the with atoms (Fig. 3A) (table S2B). The IL-2/gc interface buries 970 of different IL-2 molecules indicate nearly Solvent exchange with the layer of water surface area and is the smallest of the four identical structures, ranging from 0.29 ) to molecules between IL-2Rb andIL-2could protein-protein interfaces in the complex. The ) 0.57 . One notable exception is at the explain the fast on- and off-rates and the weak gc binding surface is striking in its absence of beginning of helix C of IL-2, where, in the affinity of the IL-2/IL-2Rb binary complex. extended side chain–specific interactions with

1160 18 NOVEMBER 2005 VOL 310 SCIENCE www.sciencemag.org R ESEARCH A RTICLES Fig. 2. IL-2Ra binding results in local conformational changes within IL-2 helix C. (A) Backbone superposition of IL-2 structures in quaternary complex (violet), bi- nary complex (orange) (PDB 1Z92) (17), and three unbounded states: PDB 1M4C (green), 1M47 (dark green), and 3INK (gray) (44, 45). (B) IL-2 residue Asn88 in helix C forms a hydrogen bond with Arg42 from IL-2Rb in quater- nary and binary complexes as a result of closer proximity induced by IL-2Ra binding.

Fig. 3. A polar interface and hydra- tion layer between IL-2 and IL-2Rb. (A) All interactions between IL-2 and IL-2Rb. The buried water molecules in the interface are shown as green spheres. The hydrogen bonds be- tween IL-2 and IL-2Rb are in black; those between water molecules and protein atoms are in green. (B) Close-up view of the shape comple- mentarity in the interface, as viewed from above.

IL-2 and in the preponderance of main-chain and Ser130 from IL-2. The Tyr103 aromatic their flat surfaces, and that the peripheral contacts. Although there are several apparent ring packs flat against the side chains of polar interactions modulate specificity for 127 130 ‘‘hotspots,’’ the gc binding surface is remark- Ser and Ser in IL-2 (Fig. 4, A and B). individual cytokines. 126 able in its flatness and almost tangential The second is around residue Gln in IL-2, IL-2Rb/gc. The second part of the com- contact with IL-2 (Fig. 4A). The gc structure which has been shown by mutagenesis to be posite interface between IL-2/Rb and gc is contains an unusual disulfide bond in the a critical energetic hotspot. Similar to Tyr103, formed by extensive interactions between the 126 heart of the interface with IL-2 that connects the side chain of Gln is almost parallel to D2 domains of IL-2Rb and gc, burying more loops FG2 with BC2 and supports the the surface formed by main-chain atoms of than 1750 )2 of surface area (Fig. 5A). The 207 211 207 211 conformations of Ser to Pro that form residues Pro to Ser in gc, and this D2 domains from IL-2Rb and gc are related by direct contacts with IL-2 (Fig. 4A). The di- orientation is further fixed by two hydrogen almost exact two-fold symmetry, and the sulfide also contributes to the apparent bonds with the receptor, to Pro207 O and interfaceisformedby21residuesfrom 211 rigidity of the cytokine-binding surface, Ser OG (Fig. 4A). A single bridged water IL-2Rb and 19 residues from gc from strands ¶ ¶ which is surprising given that one prevailing molecule in the IL-2/gc interface forms C2, C 2, and E2 and loop C E2 (table S2D). assumption for receptor cross-reactivity is hydrogen bonds with Gln126 of IL-2 and The interface is highly polar, with a peripheral 127 128 structural plasticity (26). The gc binding sur- with Gln and Asn of gc, respectively ring of 17 hydrogen bonds surrounding a face does not appear to contain mobile (table S2C). hydrophobic stripe in the center dominated by 166 167 structural elements, although we do not have Previous mutagenesis studies have found Trp from IL-2Rb and Tyr from gc (Fig. a structure of the unliganded receptor for that two of the flat gc patches we see in the 5B) (table S2D). comparison. structure that are involved in binding IL-2— The D2-D2 interaction between IL-2Rb 103 208 The overall interface involves residues residue Tyr and residues from Leu to and gc is the largest buried surface seen so 211 from helices A and D in IL-2 and residues Ser in gc—are also important for binding far in cytokine-receptor complexes, and it from loops CC¶1, EF1, BC2, FG2, and the IL-4, IL-7, IL-15, and IL-21 (27–29). There underscores the role of receptor-receptor linker between strands G1 and A2 in gc is also evidence that gc binding sites for dif- contact in the cooperative assembly of the (table S2C). In contrast to the broad array of ferent cytokines overlap but are not identical quaternary complex. Although it is surprising specific polar interactions between IL-2 and (30). We propose that these two patches that IL-2Rb and gc have no measurable IL-2Rb, small contact patches dominate the form the central degenerate recognition affinity toward one another given this ex-

IL-2/gc interface. The first one is composed surfaces that participate in binding all cyto- tensive contact surface, a lack of interaction 103 127 of residue Tyr from gc and residues Ser kines in the gc-dependent family by using would prevent the receptors from heterodi-

www.sciencemag.org SCIENCE VOL 310 18 NOVEMBER 2005 1161 R ESEARCH A RTICLES

Fig. 4. Interactions between gc and IL-2. (A) Contacting residues

in the IL-2/gc interface. (B)Surface representation of the relative con- 103 tact patch around Tyr from gc, as viewed from above.

merizing and signaling in the absence of cytokine. Given the structural observations

of a small IL-2/gc interface and a large and tightly packed IL-2Rb/gc interface, we suggest that the receptor-receptor (i.e., D2- D2) contact may serve as an important energetic determinant. If so, the role of the cytokine would be to stabilize complex for- mation by guiding a perfect geometrical alignment of the numerous interatomic con- tacts (hydrogen bonds, van der Waals con- tacts, etc.) in the D2-D2 interface. In this respect, considering the relatively flat and

chemically inert IL-2/gc interface, it may be that specificity is largely provided by receptor- receptor contact with IL-2Rb rather than cytokine. Fig. 5. Extensive receptor-receptor contact between IL-2Rb and g .(A) Surface representation of the This model can in part be rationalized by c quaternary complex shows the shape complementarity in the IL-2Rb/g interface. (B) Hydrogen- the ‘‘shared’’ function of g . g is expressed c c c bonding interactions between IL-2Rb and g . on most immune cell types, but the tissue c and cytokine specificity are regulated by the coordinate expression of different a recep- thus, T, B, and NK cells can be affected by a on average, 19% sequence identity to one

tors (or, in the case of IL-2, the b receptor). single mutation. We mapped extracellular gc another, with most of the homology con- Given the lack of sequence homology mutations that have been found in X-SCID centrated inside the helical cores. Although

between gc-dependent cytokines, the capac- patients in which gc is expressed but is not we currently know the structures of only ity of gc to discriminate among (and to cross- competent for activation by any of the gc IL-2 and IL-4 in the gc-dependent cytokine react with) these ligands would be more cytokines. Many mutations appear to concen- family, we sought to identify conserved res-

easily achieved by spreading the energetics trate near the gc cytokine-binding site, and idues that might serve as a recognition code È of the interaction over the combined 2600 several of these—Y103N, Y103C, L208P, for gc binding throughout the family. Se- )2 of surface area presented by the IL-2/IL- C209R, C209Y, G210R, G210V, and quence alignment between IL-2 and other

2Rb composite surface, rather than focusing C160R (20, 33)—map to the gc binding gc-dependent cytokines (fig. S3) indicates it all on the small portion of this surface interface with IL-2 (Fig. 6A). Mutation of that residue Gln126, which plays a key È )2 209 160 contributed by cytokine alone ( 970 ). By Cys or Cys , which participate in the structural role in IL-2 interactions with gc,is comparison, in the structures of more ligand- disulfide bond in the gc cytokine-binding conserved in IL-2, IL-9, IL-15, and IL-21, specific cytokine receptors such as human surface, would be particularly destabilizing. whereas IL-4 and IL-7 have Arg121 and growth (hGHbp) and eryth- The interface mutations would effectively Lys139 in this position, respectively. Super-

ropoietin receptor (epoR) complexed with ablate cytokine recognition by gc, but it position of the IL-4/IL-4Ra complex (34) their ligands, there is much less receptor- seems likely that the receptor would still ap- with the IL-2 quaternary complex indicates receptor contact (È900 )2 for hGHbp, no pear to be competent to signal if heterodi- that Arg121 may play a structural role similar 126 contact for epoR) (31, 32). merized. Although the database X-SCID to that of Gln in IL-2 in contacting gc. X-SCID mutations. X-linked severe com- mutations map to all other parts of the gc Although position 126 in helix D may serve bined immunodeficiency (SCID) is a syn- structure, none of the X-SCID mutations map as a common contact point with gc, there are drome of profoundly impaired cellular and to the IL-2Rb/gc interface, possibly implying not obvious constellations of conserved humoral immunity caused by mutations in the a structural necessity for this area to be residues that allow us to dock the different

encoding the common gamma chain preserved in the expressed receptor (Fig. 6B). cytokines with gc. It appears that each (33). The mutated gene results in faulty Degenerate cytokine recognition cytokine uses distinct structural solutions

signaling through several cytokine receptors; by gc. The gc-dependent cytokines have, for gc recognition.

1162 18 NOVEMBER 2005 VOL 310 SCIENCE www.sciencemag.org R ESEARCH A RTICLES

3. J. Theze, P. M. Alzari, J. Bertoglio, Immunol. Today 17, 481 (1996). 4. A. C. Church, QJM 96, 91 (2003). 5. A. B. Shanafelt et al., Nat. Biotechnol. 18, 1197 (2000). 6. W. J. Leonard et al., Nature 311, 626 (1984). 7. T. Nikaido et al., Nature 311, 631 (1984). 8. D. Cosman et al., Nature 312, 768 (1984). 9. M. Hatakeyama et al., Science 244, 551 (1989). 10. T. Takeshita et al., Science 257, 379 (1992). 11. H. M. Wang, K. A. Smith, J. Exp. Med. 166, 1055 (1987). 12. M. Rickert, M. J. Boulanger, N. Goriatcheva, K. C. Garcia, J. Mol. Biol. 339, 1115 (2004). 13. Y. Nakamura et al., Nature 369, 330 (1994). 14. B. H. Nelson, J. D. Lord, P. D. Greenberg, Nature 369, 333 (1994). 15. K. Ozaki, W. J. Leonard, J. Biol. Chem. 277, 29355 (2002). 16. W. J. Leonard, Nat. Rev. Immunol. 1, 200 (2001). 17. M. Rickert, X. Wang, M. J. Boulanger, N. Goriatcheva, K. C. Garcia, Science 308, 1477 (2005). 18. See supporting data on Science Online. 19. J. F. Bazan, Proc.Natl.Acad.Sci.U.S.A.87, 6934 (1990). 20. Single-letter abbreviations for amino acid residues: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; Y, Tyr. Fig. 6. Mapping known X-SCID mutations in the structure of gc.(A) Five missense mutations that 21. B. Becknell, M. A. Caligiuri, Adv. Immunol. 86, 209 (2005). are located in the gc cytokine-binding epitope, and make contact with IL-2 in the structure, are shown as red sticks. (B) Distribution of all missense mutations in the X-SCID mutation database 22. D. M. Anderson et al., J. Biol. Chem. 270, 29862 (1995). (http://genome.nhgri.nih.gov/scid) in gc. The gc area participating in the D2-D2 interaction with IL-2Rb is free of mutations and is indicated within the dashed line. 23. J. G. Giri et al., EMBO J. 14, 3654 (1995). 24. N. Arima et al., J. Exp. Med. 176, 1265 (1992). 25. S. F. Liparoto, T. L. Ciardelli, J. Mol. Recognit. 12, 316 Cytokine recognition by shared recep- the high-affinity quaternary complex con- (1999). 26. S. Atwell, M. Ultsch, A. M. De Vos, J. A. Wells, Science tors. The flat and apparently rigid surface taining IL-2Rabg expressed on T cells, where- 278, 1125 (1997). in the common binding epitope of gc as the toxic side effects are mediated through 27. F. Olosz, T. R. Malek, J. Biol. Chem. 277, 12047 suggests that it uses somewhat chemically the IL-2Rbg form of the receptor on NK cells (2002). 28. J. L. Zhang, M. Buehner, W. Sebald, Eur. J. Biochem. inert complementary surfaces to interact (42). This hypothesis suggests that it might be 269, 1490 (2002). with divergent cytokine residues. Although possible to dissociate efficacy and toxicity by 29. J. L. Zhang, D. Foster, W. Sebald, Biochem. Biophys. this contrasts with notions of receptor generating an IL-2 variant with selectivity for Res. Commun. 300, 291 (2003). promiscuity through binding site flexibility the IL-2Rabg receptor complex, versus the 30. N. Raskin et al., J. Immunol. 161, 3474 (1998). 31. O. Livnah et al., Science 273, 464 (1996). (26), it parallels structural results for gp130, IL-2Rbg complex of NK cells (5). Proof-of- 32. R. S. Syed et al., Nature 395, 511 (1998). the shared cytokine receptor for long-chain concept was demonstrated with an IL-2 33. J. M. Puck, Immunol. Today 17, 507 (1996). cytokines (35), in complex with three dif- variant bearing an Asn88 Y Arg mutation 34. T. Hage, W. Sebald, P. Reinemer, Cell 97, 271 (1999). 35. M. J. Boulanger, K. C. Garcia, Adv. Protein Chem. 68, ferent cytokines: LIF, viral IL-6, and human that conferred a factor of 3000 selectivity 107 (2004). IL-6 (36–38). In the gp130 system, thermo- increase for the IL-2Rabg complex by 36. M. J. Boulanger, A. J. Bankovich, T. Kortemme, D. dynamic compensation between rigid sur- crippling the interaction between IL-2 and Baker, K. C. Garcia, Mol. Cell 12, 577 (2003). 37. D. Chow, X. He, A. L. Snow, S. Rose-John, K. C. Garcia, faces, rather than conformational change, IL-2Rb (5). Science 291, 2150 (2001). enables cross-reactivity with a broad range In the structure we see that Asn88 is the 38. M. J. Boulanger, D. C. Chow, E. E. Brevnova, K. C. of chemically diverse cytokine surfaces (35). side chain brought into hydrogen-bonding Garcia, Science 300, 2101 (2003). 39. B. J. McFarland, R. K. Strong, Immunity 19, 803 We predict, on the basis of direct thermo- distance to IL-2Rb by the structural pertur- (2003). dynamic measurements of the quaternary bation of helix C in response to IL-2Ra 40. J. Dutcher, Oncology (Huntingt.) 16, 4 (2002). complex assembly (12), that g also uses binding, and is involved in an extensive 41. K. A. Smith, B. A. Boyle, AIDS Read. 13, 365 (2003). c 42. K. A. Smith, Blood 81, 1414 (1993). such a mechanism for cross-reactivity. Such hydrogen-bonding network (Fig. 3A). Such 43. W. L. DeLano, The PyMOL Molecular Graphics System a large range of energetic compensation an energetically critical residue may not be (DeLano Scientific, San Carlos, CA, 2002). appears to be a property of binding sites the most desirable choice for generating a 44. M. R. Arkin et al., Proc. Natl. Acad. Sci. U.S.A. 100, found in shared receptors, which are tuned receptor-selective IL-2, because it may not 1603 (2003). 45. D. B. McKay, Science 257, 412 (1992). for degenerate recognition through a mech- be necessary to completely ablate binding to 46. We thank N. Goriatcheva for expert technical assist- anism that bypasses the entropic penalty for the IL-2Rbg receptors. Rather, weakening ance; K. Smith, T. Springer, and W. Leonard for helpful conformational change (39). the IL-2Rb interaction, or even contact with discussions; and the Advanced Light Source (UC- Berkeley) and Stanford Synchrotron Radiation Labora- Therapeutic implications. A recombi- gc, while maintaining near wild-type affinity tory for synchrotron beamtime. Supported by the Keck nant human IL-2 (rIL-2) analog (Aldesleukin, for the IL-2Rabg complex appears tenable Foundation, HHMI, and NIH grant AI51321. Co- Proleukin, Chiron Inc., Emeryville, CA) is through structure-guided engineering. It is ordinates and structure factors have been deposited in the with accession number 2B5I. currently licensed in the United States for the our hope that this quaternary complex treatment of metastatic melanoma and renal structure can be used to design IL-2 variants Supporting Online Material cell carcinoma and is undergoing clinical that will allow its powerful clinical potential www.sciencemag.org/cgi/content/full/310/5751/1159/ trials for patients with HIV/AIDS (40, 41). to be more fully realized. DC1 Materials and Methods Treatment of cancer patients with rIL-2 Figs. S1 to S3 results in robust responses but is associated Tables S1 and S2 with life-threatening toxicity, which limits References and Notes References 1. K. A. Smith, Science 240, 1169 (1988). its use (40). The antitumor efficacy of rIL-2 2. B. H. Nelson, D. M. Willerford, Adv. Immunol. 70,1 25 July 2005; accepted 14 October 2005 therapy has been shown to be mediated by (1998). 10.1126/science.1117893

www.sciencemag.org SCIENCE VOL 310 18 NOVEMBER 2005 1163