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J. Biol. Chem., Vol. 282, Issue 51, 37191-37204, December 21, 2007
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Complex
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From the
RIKEN Genomic Sciences Center and the RIKEN Research Center for Allergy and Immunology, RIKEN Yokohama Institute, 1-7-22 Suehiro-cho, Tsurumi, Yokohama, Kanagawa 230-0045, the ¶Tohoku University Biomedical Engineering Research Organization, Sendai 980-8575, and the ||Department of Biophysics and Biochemistry, University of Tokyo, Tokyo 113-0033, Japan
Received for publication, July 26, 2007 , and in revised form, September 28, 2007.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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(IL-15R) from one subset of cells presents IL-15 to neighboring IL-2Rβ/
c-expressing cells. Here we present the crystal structure of IL-15 in complex with the sushi domain of IL-15R. The structure reveals that the receptor-binding epitope of IL-15 adopts a unique conformation, which, together with amino acid substitutions, permits specific interactions with IL-15R that account for the exceptionally high affinity of the IL-15·IL-15R complex. Interestingly, analysis of the topology of IL-15 and IL-15R at the IL-15·IL-15R interface suggests that IL-15 should be capable of participating in a cis signaling mechanism similar to that of the related cytokine IL-2. Indeed, we present biochemical data demonstrating that IL-15 is capable of efficiently signaling in cis through IL-15R and IL-2Rβ/c expressed on the surface of a single cell. Based on our data we propose that cis presentation of IL-15 may be important in certain biological contexts and that flexibility of IL-15R permits IL-15 and its three receptor components to be assembled identically at the ligand-receptor interface whether IL-15 is presented in cis or trans. Finally, we have gained insights into IL-15·IL-15R·IL-2Rβ·c quaternary complex assembly through the use of molecular modeling. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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-helix bundle family of cytokines, which plays a pivotal role in both innate and adaptive immunity (1, 2). IL-15 was initially identified due to its ability to mimic the activity of IL-2 in vitro (3, 4), and despite a lack of significant sequence homology, IL-15 and IL-2 are able to similarly stimulate the proliferation and differentiation of natural killer (NK), T, and B cells (1, 2). Despite these many overlapping functions, IL-15 and IL-2 possess distinct biological activities in vivo, such as the ability of IL-15 to control the development of CD8+ memory T cells and the involvement of IL-2 in the maintenance of regulatory T cells (5).
The cell-surface receptor for IL-15 comprises three subunits: IL-15 receptor (IL-15R) , IL-2Rβ (also known as IL-15Rβ, CD122, and p75), and c (also known as CD132 and p65). The ectodomain of IL-15R consists of a single sushi domain (6) (also known as a short consensus repeat or complement control protein repeat), which is essential for IL-15 binding and IL-15R function (7, 8), a membrane-proximal proline-threonine-rich (PT) region, and a linker/hinge region that connects the sushi domain and the PT region (6). IL-2R is structurally related to IL-15R but contains an additional sushi domain that is connected to the first by a linker region. The ectodomain of IL-2Rβ and c each consist of two fibronectin-type III domains, which participate in IL-15 binding (9), and the cytoplasmic domains of IL-2Rβ and c sequester signaling molecules such as Jak1 and Jak3 (10, 11). In contrast, the short cytoplasmic domains of IL-15R and IL-2R are not thought to play a major role in signaling (12), although the cytoplasmic domain of IL-15R has recently been shown to play a role in receptor recycling (13). Because IL-2Rβ and c are shared subunits of the receptor for IL-2 (6, 12, 14), IL-15 and IL-2 are capable of activating similar signaling pathways, including Jak1/Jak3 and Stat3/Stat5, Ras/mitogen-activated protein kinase, and phosphatidylinositol 3-kinase pathways (1), which has been suggested to partially account for the overlapping functions of IL-15 and IL-2.
A possible mechanism by which IL-15 and IL-2 attain their distinct in vivo functions is through differences in the properties of their chain receptors. IL-15R binds specifically to IL-15 with high affinity (Kd = 30-100 pM) (6, 12, 15, 16), whereas IL-2R specifically binds to IL-2 with a comparatively lower affinity (Kd = 10-30 nM) (17). IL-15, IL-2, and their chain receptors also exhibit distinct expression profiles, which have been suggested to contribute to their distinct biological activities (18). Furthermore, in contrast to IL-2, which acts as a soluble cytokine, IL-15 is coordinately expressed with IL-15R by activated monocytes and dendritic cells and is extremely difficult to detect in free form in humans and mice due to stable complex formation with IL-15R on the cell surface (13, 19-23). Recent data have demonstrated that IL-15R from activated monocytes presents IL-15 in trans to IL-2Rβ/c and/or IL-15R/IL-2Rβ/c-expressing cells such as CD8+ memory T cells and NK cells (13, 21, 24). This finding suggests that the architecture of IL-15R subunits in the IL-15 quaternary complex differs from that of IL-2 receptor subunits in the IL-2 quaternary complex, which are assembled in cis (i.e. on the surface of the same cell) as revealed by the recently reported IL-2·IL-2R·IL-2Rβ·c crystal structure (25, 26). Although trans presentation is currently thought to be the major mechanism by which IL-15 exerts its biological effects in vivo, the possibility for the assembly of a cis IL-15 quaternary complex on cells capable of expressing IL-15R, IL-2Rβ, and c, has not been ruled out (18). However, considering the structural similarities between IL-15, IL-2, and their receptors (15, 27, 28), and data suggesting that IL-15 has only a single binding site for interaction with IL-15R (29), the molecular basis by which trans presentation occurs and can even co-exist with the formation of IL-2-like cis quaternary complex is not clear.
In this report, we present the crystal structure of murine IL-15 in complex with the sushi-domain fragment of IL-15R, refined to 2.2-Å resolution. The structure reveals the molecular determinants of the specific, high affinity IL-15·IL-15R interaction. Importantly, helix B and the hC-hD loop of IL-15 adopt unique conformations, which permit specific interactions with IL-15R that likely account for its significantly higher affinity relative to the IL-2·IL-2R interaction. We also present biochemical data demonstrating that, in addition to trans presentation, IL-15R is capable of presenting IL-15 to IL-2Rβ/c in cis, suggesting a potential role for cis IL-15 presentation in certain biological contexts. Finally, analysis of the structure suggests that flexibility of the linker and/or PT region of IL-15R permit IL-15 and its three receptor components to be assembled identically at the ligand-receptor interface whether IL-15 is presented in cis or trans.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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(residues 1-168; sIL-15R1-168), and the full-length mature murine IL-15 (residues 1-114) were cloned into the TA vector pCR2.1TOPO (Invitrogen) as fusion proteins containing an N-terminal histidine tag and a tobacco etch virus protease cleavage site. Selenomethionine-labeled proteins were synthesized using the large scale dialysis mode of the Escherichia coli cell-free reaction (30). Both IL-15 and sIL-15R1-168 precipitated during synthesis. The precipitated proteins were denatured in 25 mM Tris-HCl buffer (pH 8.3) containing 6 M guanidine hydrochloride and 1 mM dithiothreitol and were refolded separately or co-refolded by rapid dilution into 50 mM Tris buffer (pH 8.3) containing 1 M arginine hydrochloride, 5 mM reduced glutathione, and 0.5 mM oxidized glutathione. After stirring for 24 h at 4 °C, the protein solutions were filtered and dialyzed against 25 mM Tris-HCl buffer (pH 7.5) containing 150 mM NaCl for 12-24 h at 4 °C. The refolded proteins were purified to homogeneity by chromatography on affinity (HisTrap) and gel-filtration (Superdex 200 HR 10/30) columns. The histidine tags were removed by overnight digestion with tobacco etch virus protease at 4 °C. Due to the location of the tobacco etch virus cleavage site, seven residues from the histidine tag (GSSGSSG) remain at the N termini of the IL-15 and sIL-15R1-168 proteins after histidine tag cleavage. During the gelfiltration step with 25 mM Tris-HCl buffer (pH 7.5) containing 300 mM NaCl, IL-15 eluted as a mixture of trimeric and monomeric species, and only the monomeric protein was used for subsequent experiments. For complex formation, separately refolded monomeric IL-15 and sIL-15R1-168 were mixed at a 1.5:1 molar ratio and concentrated using an Amicon Ultra 15 filtration unit. The IL-15·sIL-15R1-168 mixture was loaded onto a Superdex 200 HR 10/30 column equilibrated in 25 mM Tris-HCl buffer (pH 7.5) containing 150 mM NaCl and eluted at a position consistent with 1:1 complex formation, which was confirmed via analytical ultracentrifugation. Co-refolded IL-15·sIL-15R1-168 complex behaved identically to the separately refolded and reconstituted complex during purification. The co-refolding method was utilized for large scale protein preparation due to its consistently higher yields. For large scale protein preparation, an anion exchange step (Mono Q HR 5/5) was added prior to the final gel-filtration step (which was performed using a Superdex 75 HR 16/60 column). All columns were purchased pre-packed from GE Healthcare.
Limited Proteolysis, Crystallization, and Data Collection—Initial crystallization trials of the IL-15·sIL-15R1-168 complex were performed using the standard sitting drop vapor diffusion method. Despite extensive screening efforts, we were unable to obtain crystals of the complex using the full-length ectodomain of IL-15R. During protein synthesis and purification, we noted that the C-terminal region of sIL-15R1-168 was extremely sensitive to proteolytic degradation, suggesting that this region of the receptor is highly flexible.
To obtain boundaries more suitable for crystallization, we performed limited proteolysis experiments on the IL-15·sIL-15R1-168 complex. Using elastase (Nacalai Tesque), a stable fragment of the complex was identified during small scale pilot experiments, and the reaction conditions were scaled-up. The digested protein was purified by gel-filtration chromatography and analyzed using MALDI-TOF and N-terminal sequencing. The results of this analysis showed that the PT region and most of the linker region of IL-15R are rapidly degraded and that the stable fragment of IL-15R corresponds to residues 1-71, which includes the entire sushi domain and the first five residues of the linker region. The results also showed that IL-15 was not proteolytically digested. On the basis of these results a modified IL-15R construct (sIL-15R1-71) was generated, synthesized, and purified in complex with IL-15 as described above for sIL-15R1-168.
Diffraction quality crystals of the selenomethionine-labeled IL-15·sIL-15R1-71 complex (hereafter referred to as IL-15·IL-15R) were grown by mixing 2 µl of IL-15·IL-15R (18 mg/ml in 25 mM Tris-HCl buffer (pH 7.5) containing 150 mM NaCl) with 2 µl of crystallization buffer (1.7 M sodium/potassium phosphate, pH 6.8, 3% benzamidine-HCl) and incubating at 20 °C using hanging drop vapor diffusion. Large, single crystals measuring 0.5 mm in the longest dimension appeared within 5-7 days. Crystals were flash frozen in a final cryoprotectant solution composed of the mother liquor and 25% glycerol. Diffraction data were measured on an ADSC Quantum210 detector at Photon Factory-Advanced Ring, Beamline NW12. The data were indexed, integrated, and scaled using HKL2000 (31). The IL-15·IL-15R crystals belong to the tetragonal space group P4212 with unit cell dimensions a = b = 118.5 Å, c = 76.11 Å. There are two IL-15·IL-15R complexes in the asymmetric unit, with a solvent content of 
58%.
Structure Determination and Refinement—The IL-15·IL-15R structure was solved using diffraction data collected at the selenium absorption peak wavelength (
= 0.9793 Å) of one selenomethionine-derivatized crystal. The program SOLVE (32) was used to locate the selenium sites and to calculate the phases using the single wavelength anomalous dispersion method. The program RESOLVE (33) was used for density modification, and ARP/wARP (34) was used to generate an initial model via automatic model building. Positional and B-factor refinements were performed using the program CNS (35), and model building into the 2Fo - Fc and Fo - Fc electron density maps was performed with the program O (36). The final refined atomic model contains residues 1-114 of both copies of IL-15 in the asymmetric unit, residues 3-71 in one copy of IL-15R, and residues 3-70 in the other copy of IL-15R in the asymmetric unit, 90 water molecules, and one benzamidine molecule. The remaining residues of the N-terminal histidine tag are completely disordered in both copies of IL-15R, whereas the last three residues of this region are ordered in both copies of IL-15 and are included in the atomic model. Additionally, residues 1-2 are disordered in both copies of IL-15R, and residue 71 is disordered in one copy of IL-15R in the asymmetric unit. The atomic coordinates and structure factors have been deposited into the Protein Data Bank under accession code 2PSM. Superimposition of the C trace of the two copies of IL-15 and the IL-15R shows that they are essentially identical (r.m.s.d. = 0.451 Å and 0.202 Å, respectively). Representations of the structure were created with the programs PyMOL3 and CCP4MG (38).
Generation of IL-15R- and IL-2Rβ-expressing FDC3 Cell Line—The cDNA of full-length murine IL-15R and IL-2Rβ were amplified by PCR and introduced into retroviral vectors, MSCV-IRES-GFPRV and pNX-IRES-hCD4RV, respectively. The murine mast cell line, FDC-P1 (39), was purchased from ATCC, re-cloned, and renamed as FDC3. This cell line was cultured in Dulbecco's modified Eagle's medium containing 10% calf serum and conditioned media of P815 cells expressing IL3. PLAT:E packaging cell lines were transfected with IL-15R/MSCV-IRES-GFPRV or IL-2Rβ/pMX-IRES-hCD4RV by FuGENE according to the manufacture's instructions. After 24 h, the medium was replaced, and the retroviral supernatant was generated by culturing the cells for a further 24 h. The FDC3 cell line was infected by overnight culture in retroviral culture supernatant containing 8 mg/ml Polybrene. Infected FDC3 cell lines were purified by cell sorting for expression of the green fluorescent protein and/or the hCD4 surface markers. Sorted cells were expanded, were >90% pure, and stably expressed the retroviral markers.
Single Cell Assay—FDC3β and FDC3β cells cultured in IL-3 containing Dulbecco's modified Eagle's medium for 4 days were washed with phosphate-buffered saline three times, and cultured in IL-15-free, IL3-free Dulbecco's modified Eagle's medium with 10% calf serum for 7 h. A single cell was picked up by micropipette and transferred into a well of a Terasaki plate (Nunc, Roskilde, Denmark) containing Dulbecco's modified Eagle's medium with 10% calf serum in the presence or absence of 30 ng/ml IL-15. Cell division was checked under the microscope at 0 and 22 h of incubation.
Thymidine Uptake Assay—The sIL-15R protein used for the thymidine uptake assay was produced using a modified method of a previously published protocol (40). Briefly, the cDNA coding full-length ectodomain of murine IL-15R was amplified by PCR and subcloned into the pQE30 bacterial expression vector (His-sIL-15R1-168). His-sIL-15R1-168 was expressed in E. coli strain M15 cells and was purified to homogeneity by nickel affinity and gel-filtration chromatography.
Proliferation assays were set up in 96-well microtiter plates. Murine IL-15 (R&D) was diluted across the plates in the presence or absence of 1 µg/ml His-sIL-15R1-168 protein. In the experiment using IL-15·IL-15R1-168 complex or IL-15·IL-15R1-71 complex, the concentrations of IL-15 were calculated based on the content of IL-15 in the complex. FDC3β or FDC3β cells were plated at 4000 cells in 96-well flat-bottom plate and incubated for 24 h. The plates were pulsed with [3H]thymidine (0.1 µCi/well, Amersham Biosciences) for the last 6 h of culture. The cells were harvested on glass fiber filters and counted on a mutidetector direct beta counter (Matrix 96, Packard Instrument Co., Meriden, CT). Dose-response curves were generated and analyzed using GraphPad Prism.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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, and to obtain insights into the architecture of the IL-15·IL-15R complex, we have determined the crystal structure of IL-15 in complex with IL-15R. Initial attempts to crystallize IL-15 in complex with the full-length ectodomain of IL-15R were unsuccessful. Limited proteolysis experiments performed on purified IL-15·IL-15R complex led to the identification of a stable IL-15R fragment (encompassing the sushi domain and the first five residues of the linker region) that was amenable to crystallization (see "Experimental Procedures"). The IL-15·IL-15R structure was solved using the single wavelength anomalous dispersion method and has been refined to 2.2-Å resolution with working and free R values of 22.4% and 23.6%, respectively (Table 1). As shown in Fig. 1A, IL-15R interacts with the upper edge of the IL-15 four-helix bundle.
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Despite sharing only 16% sequence identity overall, the helices that comprise the four-helix bundles of receptor bound IL-15 and IL-2 (PDB ID: 1Z92
[PDB]
and 2B5I) (25, 42) superimpose well, with an r.m.s.d. of 1.2 Å over 68 equivalent C positions (Figs. 1B and 2A). The most obvious structural differences between IL-15 and IL-2 result from differences in the length of the helices that form the core of the protein and length and conformational differences in the loop regions between the helices (hA-hB, hB-hC, and hC-hD loops) (Figs. 1B and 2A). Importantly, the hA-hB loop of IL-15 is shorter by four residues compared with that of IL-2, which prevents the formation of a helix corresponding to hA' of IL-2 (Figs. 1B and 2A). Another important difference is observed in hB of IL-15, which adopts a more rigid and extended conformation compared with the corresponding region of IL-2 (hB and hB'), which is bent due to the presence of a proline residue (Pro-65) at the start of hB' (Figs. 1B and 2A). IL-15 lacks a proline residue in its hB region, which permits the C-terminal extension of hB and more rigid conformation of the helix. Because hB and the hA-hB loop region account for a major portion of the ligand-receptor interface in both the IL-15·IL-15R and IL-2·IL-2R complexes, these conformational differences have an impact on the distinct modes of receptor binding observed for these ligands (see below).
A final structural difference worth noting is the fact that hA of IL-15 is significantly shorter at its N terminus compared with hA of free or receptor-bound IL-2 from previously determined crystal structures (PDB ID: 1Z92
[PDB]
, 2ERJ, and 2B5I) (Figs. 1B and 2A). hA of IL-2 plays a role in the assembly of the IL-2·IL-2R·IL-2Rβ·c complex (26) (IL-2 quaternary complex), so the significantly shorter N terminus of IL-15 relative to IL-2 has possible implications on the IL-15 mode of binding to IL-2Rβ/c, which will be discussed in detail below.
Structure of Ligand-bound IL-15R—The IL-15R sushi domain has an elliptical structure comprised of six β-strands (βB, βC, βC', βD, βE, and βE') that envelope a compact hydrophobic core (Fig. 1A). The central β-sheet of the IL-15R sushi domain is formed by strands βC, βD, and βE. In typical sushi domain structures two β-strands at the N-terminal region of the domain (βA and βB) pack against the central β-sheet to form a β-sandwich arrangement (43). As noted previously in the NMR structure of unliganded human IL-15R sushi domain (PDB ID: 2ERS), this arrangement is not observed in the IL-15R sushi domain fold due to the presence of three consecutive proline residues (Pro-5, Pro-6, and Pro-7) that prevent the formation of the βA strand (Fig. 1A) (27). The IL-15R sushi domain contains two disulfide bridges: the first between Cys-4 at the N terminus of the sushi domain and Cys-46 in βD, which serves to pin the highly flexible N-terminal region to the central β-sheet of the domain, and the second between Cys-30 at the C-terminal end of the βC strand and Cys-64 in the βE' strand, which seems to stabilize the sushi domain core (Fig. 1A). Comparison of our crystal structure of ligand-bound murine IL-15R to the NMR structure of unliganded human IL-15R (PDB ID: 2ERS) shows that the core of the IL-15R sushi domain does not undergo significant conformational changes upon ligand binding. However, the conformation of loop regions between the β-strands (outside of regions involved in ligand binding) are quite different, presumably due to inherent flexibility (data not shown).
Although IL-15R and IL-2R are structurally related in that they are composed of sushi domains, linker, and PT regions, IL-15R contains a single sushi domain that mediates ligand binding, whereas IL-2R contains two sushi domains separated by a linker region (Fig. 2B). The previously determined IL-2·IL-2R crystal structure (PDB ID: 1Z92
[PDB]
) (42) showed that the two sushi domains of IL-2R engage in strand exchange and that the N-terminal sushi domain (IL-2R D1) participates in most of the interactions with IL-2. Superimposition of IL-2R D1 onto the sushi domain of IL-15R from the IL-15·IL-15R structure shows that the conformation of most of the receptor regions involved in ligand binding adopt a similar conformation (βC-βE' region of the receptors superimpose with an r.m.s.d. of 1.73 Å) (Fig. 2C). In contrast, the strand-exchanged region of IL-2R D1 (residues 102-122), which is involved in some contacts with IL-2, adopts a significantly different conformation from the corresponding region of IL-15R (residues 3-20), which does not make any contacts with IL-15 (Fig. 2C). The effects of these structural differences on the distinct modes of IL-15·IL-15R and IL-2·IL-2R binding will be discussed below.
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Interface—The IL-15R binding surface of IL-15 comprises residues from hB, hC', and the hA-hB and hC-hD loops, whereas the IL-15 binding surface of IL-15R comprises residues from strands βC and βC' and the βC'-βD and βE-βE' loops (Fig. 1). Calculation of the surface electrostatic potential of IL-15 reveals a groove at the receptor-binding interface that is lined with acidic residues and thus possesses a striking degree of negative potential (Fig. 3A). Calculation of the surface electrostatic potential of IL-15R reveals three prominent "knobs" of positive potential located at its ligand-binding surface that are formed by Arg-25 and Arg-27 in βC, and Arg-36 in βC' (Fig. 3A). In forming the IL-15·IL-15R complex, the IL-15R knobs insert into the groove of IL-15 to form an interlocking interface with good charge and shape complementarity (Sc = 0.73) and high specificity (14 direct hydrogen bonds). 14 residues from IL-15 and 15 residues from IL-15R bury a total surface area of 1240 Å2 at the IL-15·IL-15R interface.
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interface reveal two adjacent concentrated clusters of primarily hydrogen bond-mediated interactions (termed sites 1 and 2, Fig. 4, A and B), which likely account for most of the binding energy of the complex. At the center of site 1, Arg-27 in βC of IL-15R forms salt bridges with Glu-53 at the C-terminal tip of hB and Asp-22 in the hA-hB loop of IL-15 (Fig. 4A). As shown in Fig. 4A, these central salt bridges are surrounded by additional contacts between residues in hB of IL-15 and the βC'-βD and βE-βE' loops of IL-15R. The set of interactions observed at site 1 is unique to the IL-15·IL-15R complex and constitutes a major determinant of the unique mode of IL-15·IL-15R binding (see below).
Arg-36 of IL-15R is at the center of the network of hydrogen bonds that take place at site 2 of the IL-15·IL-15R interface. Here, Arg-36 of IL-15R forms salt bridges with Glu-46 in the middle of hB and Glu-93 in the hC-hD loop of IL-15 (Fig. 4B). Adjacent to this set of interactions, Tyr-26 and Glu-46 of IL-15 engage in hydrogen bonds with the backbone oxygen of Arg-36 and the backbone nitrogen of Gly-39, respectively, in IL-15R (Fig. 4B). Although most of the contacts at the IL-15·IL-15R interface are hydrogen bond-mediated, there are a few important hydrophobic interactions that take place at site 2. The first of these hydrophobic contacts is mediated by the methyl group of Ala-38 at the tip of the βC'-βD loop of IL-15R, which inserts into a prominent hydrophobic pocket formed by Tyr-26, Leu-45, and Val-49 and a partially solvent-exposed disulfide bridge between Cys-42 and Cys-88 in IL-15 (Fig. 4B). This interaction strengthens the site 2 interface directly via binding and contributes to the exclusion of bulk solvent by capping one end of the IL-15·IL-15R interface. Adjacent to this interaction, Ile-65 and Pro-68 in the βE' and linker regions, respectively, of IL-15R, pack against a hydrophobic patch formed by the aliphatic portion of the side chains of Glu-87, Glu-89, and Glu-90 in hC' and the hC-hD loop of IL-15 (Fig. 4B). This set of hydrophobic interactions at site 2 is unique to the IL-15·IL-15R interface and, in addition to site 1 interactions, play a role in determining IL-15·IL-15R specificity (see below).
The residues involved in the key interactions identified in the murine IL-15·IL-15R crystal structure are in good agreement with published mutagenesis studies (15, 28). Scatchard analysis using human proteins shows that mutation of IL-15R Arg-36 to threonine results in an 80-fold decrease in affinity for IL-15 (28), and mutation of IL-15 Glu-46 to lysine results in a complete loss in affinity for IL-15R (15), underscoring the importance of the site 2 interactions to general binding affinity. Furthermore, sequence alignment shows that all of the key residues involved in murine IL-15 and IL-15R complex formation, including Arg-27, Arg-36, and Ala-38 of IL-15R and Tyr-26, Glu-46, and Glu-53 of IL-15, are fully conserved in all vertebrate IL-15 and IL-15R orthologs for which sequence information is available (Fig. 1, B and C). This suggests that the IL-15·IL-15R mode of binding has been conserved throughout evolution, and thus, our structure provides the molecular basis for the high affinity and specificity of the IL-15·IL-15R complex for all vertebrates.
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crystal structure, which was published by Chirifu et al. (44) during the preparation of this report (PDB ID: 2Z3Q and 2Z3R). The structures of the human and mouse orthologs of IL-15 and IL-15R are similar (supplemental Fig. S1, A and B), and as expected the interactions at sites 1 and 2 are conserved (supplemental Fig. S1, C and D). One unexpected structural difference between the human and mouse IL-15 proteins is that, although hB of both orthologs adopts the same extended conformation (which permits Glu-53 at the tip of hB to salt bridge with Arg-27 of IL-15R), the orientation of hB (IL-15R binding site) relative to helices A, C, and D (IL2Rβ/c binding site), is slightly different between the two structures (supplemental Fig. S1A). This appears to be due to sequence divergence in the hB-hC loop of the ligands, in particular Tyr-54 (which is Ser-54 in human IL-15) (Fig. 1B), which would severely clash with residues in hB, hC, and the hA-hB loop if murine IL-15 adopted the same structure as human IL-15 in this region (supplemental Fig. S2).
IL-15·IL-15R Mode of Binding Is Distinct from That of IL-2·IL-2R and Other Cytokine·Cytokine Receptor Complexes—In marked contrast to the IL-15·IL-15R interface, which is dominated by salt bridges/hydrogen bonds, the IL-2·IL-2R interface (PDB ID: 1Z92
[PDB]
) is dominated by hydrophobic interactions at its center, which are surrounded by polar interactions (42). As predicted by Lorenzen et al. (27) based on molecular modeling, calculation of surface electrostatic potential of IL-15 and IL-15R from our crystal structure shows that the IL-15·IL-15R interface contains more charged residues and exhibits a higher degree of charge and shape complementarity compared with the IL-2·IL-2R interface (compare Fig. 3, A and B). Rickert et al. identified two critical interaction sites at the IL-2·IL-2R interface: one centered around Phe-42 in the hA-hB loop of IL-2 (patch 1, Fig. 4C) and the second centered around Tyr-45 in the hA-hB loop of IL-2 (patch 2, Fig. 4D) (42). To compare their modes of binding, we superimposed IL-2 from the IL-2·IL-2R structure (PDB ID: 1Z92
[PDB]
) onto IL-15 from our IL-15·IL-15R structure. Interestingly, the superimposition shows that, despite the considerably different nature of their interactions, patch 1 and patch 2 of the IL-2·IL-2R interface overlap well with site 1 and site 2 of the IL-15·IL-15R interface (Fig. 4, compare A with C, and B with D).
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interface, hA' and hB' of IL-2 adopt a significantly different conformation than the corresponding regions at site 1 of IL-15 (Fig. 4, compare C and A). In the IL-2·IL-2R structure, hA' and hB' lie roughly parallel to one another and form an interface with the unique strand-swapped regions of sushi domain 1 (particularly βG) and sushi domain 2 (particularly the N terminus) of IL-2R (Fig. 4C). Although these interactions account for only a small portion of the IL-2·IL-2R interface and are slightly different in each of the three IL-2·IL-2R-containing crystal structures due to crystal packing (PDB IDs: 2B5I, 1Z92, and 2ERJ), they are significant in terms of binding mechanism, because they result in a slight rotation of IL-2R relative to IL-15R, which has only one sushi domain and thus cannot participate in equivalent interactions (Fig. 5). Importantly, Leu-42 and Tyr-43 of IL-2R βD are brought into the vicinity of Phe-42 due to the relative rotation of IL-2R, where they engage in a hydrophobic interaction that acts as the primary energetic determinant in the IL-2 binding epitope (Figs. 4C and 5) (42). The overall result of this relative rotation is that residues with different physicochemical properties and from different regions of the receptor and ligand form the functional epitopes at the IL-2·IL-2R and IL-15·IL-15R interfaces (Fig. 4, compare C and A).
Compared with site 1/patch 1, the superimposition shows that the nature of the interactions at site 2/patch 2 of the IL-15·IL-15R and IL-2·IL-2R interfaces are better conserved (Fig. 4, compare D and B). Patch 2 at the IL-2·IL-2R interface is centered around Tyr-45 in the hA-hB loop of IL-2, which packs against the aliphatic part of Arg-35 and Arg-36 from the βC' strand of IL-2R and engages in a hydrogen bond with the backbone nitrogen of Arg-35 (Fig. 4D). Surrounding this central hydrophobic cluster are several polar interactions, including a salt bridge between Arg-36 of IL-2R and Glu-62 of IL-2 (Fig. 4D). Interestingly, this salt bridge corresponds to the salt bridge between Arg-36 of IL-15R and Glu-46 of IL-15 (Fig. 4, compare D and B). Tyr-45 of IL-2 is also conserved in IL-15 (Tyr-26, Fig. 1B), however, Tyr-26 of IL-15 engages in a distinct set of interactions with the receptor in the IL-15·IL-15R structure (Fig. 4B). Superimposition shows that site 2/patch 2 serves as the pivot point of the relative rotation observed at the IL-2·IL-2R and IL-15·IL-15R interfaces (Fig. 5), which is not surprising considering the conservation of key interactions at this region mentioned above.
IL-15R and IL-2R have similar domain composition, share limited sequence homology, and together are unique among cytokine receptors in that sushi domains mediate interactions with their specific four-helix bundle cytokines. However, despite these similarities, comparison of the structures shows that the IL-15·IL-15R and IL-2·IL-2R modes of binding have diverged during evolution. The nature of the interactions at the IL-2·IL-2R interface resembles those of cytokine·cytokine receptor complexes such as growth hormone·growth hormone receptor (PDB ID: 3HHR
[PDB]
) (45) and GCSF·GCSFR (PDB ID: 1PGR) (46) complexes, which are dominated by hydrophobic contacts, whereas the nature of interactions at the IL-15·IL-15R interface resembles the hydrogen bond-dominated "avocado clusters" observed at the IL4·IL4R interface (PDB ID: 1IAR) (47), or the mosaic, interlocking interface of the p35·p40 complex of IL12 (PDB ID: 1F45
[PDB]
) (48). A functional consequence of this divergence between IL-15·IL-15R and IL-2·IL-2R is that it results in complexes with different affinities, which may account for the inherently different signaling properties and biological activities of IL-15 and IL-2 by modulating the strength and duration of signaling by the βc complex. Indeed, it has recently been demonstrated that IL-15 is unique among cytokines due to its persistent in vivo activity and ability to mediate prolonged signaling in CD8+ T cells (13). Consistent with this observation, IL-15 is predominantly detected in complex with IL-15R at the plasma membrane (effectively increasing its local concentration), and upon endocytosis these IL-15·IL-15R complexes are stable and can be effectively recycled back to the cell surface ("intercellular reservoir effect" (13, 22)). It has been suggested that both of these unique properties of IL-15 may be mediated by the high affinity and enhanced stability of the IL-15·IL-15R complex (13, 22, 29, 49).
The binding kinetics of the IL-15·IL-15R and IL-2·IL-2R interactions have previously been analyzed using surface plasmon resonance (16, 50). These studies have shown that, although the kon of the IL-15·IL-15R and IL-2·IL-2R interactions are comparable, the koff of the IL-15·IL-15R interaction is more that 4 orders of magnitude slower compared with the IL-2·IL-2R interaction. Because koff is determined by the strength of short range interactions, it is likely that the additional salt bridges observed in the IL-15·IL-15R complex at sites 1 and 2 and the excellent shape complementarity at the IL-15·IL-15R interface are the main determinants of its slower koff, and thus, higher affinity. Importantly, the IL-15·IL-15R crystal structure will provide an excellent molecular framework for the determination of the precise contributions of the individual interactions to the thermodynamics and kinetics of the IL-15·IL-15R interaction in future studies.
IL-15·IL-15R·IL-2Rβ·c Quaternary Complex Model and Biochemical Evidence for cis Presentation of IL-15—Previously published mutagenesis studies have suggested that IL-15 and IL-2 share similar interaction surfaces for IL-15Rβ and c (51). To gain insights into the architecture of IL-15 and IL-15R subunits during receptor activation, we created a model of the IL-15·IL-15R·IL-2Rβ·c complex by superimposing IL-15 and IL-15R from the IL-15·IL-15R crystal structure onto IL-2 and IL-2R from the IL-2 quaternary complex structure (PDB ID: 2B5I) (Fig. 6A). The IL-15·IL-15R·IL-2Rβ·c complex model shows that the interaction surface of IL-15 and IL-2 for their respective receptors is also shared, and comparison of the topology of IL-15·IL-15R and IL-2·IL-2R structures shows that, with respect to the polarity of their N and C termini, IL-15 and IL-15R are oriented similarly to IL-2 and IL-2R at the ligand-receptor interface. Together, these findings are somewhat surprising considering the ability of IL-15R to mediate trans IL-15 presentation and suggest that IL-15R should be capable of presenting IL-15 in cis to IL-2Rβ/c in a manner reminiscent of the IL-2·IL-2R·IL-2Rβ·c complex. With respect to previously published models of the IL-15·IL-15R complex, the topology of the IL-15·IL-15R complex revealed by our crystal structure most resemble the models proposed by Lorenzen et al. (27) and "docking solution 2" described by Quemener et al. (28).
To test the ability of IL-15R to present IL-15 in cis to IL-2Rβ/c on the surface of the same cell, we utilized the mouse mast cell line FDC3, which normally express the c subunit of IL-15R, but not IL-15R or IL-2Rβ. FDC3 cells were infected with retroviruses expressing full-length murine IL-2Rβ only (FDC3β) or murine IL-15R and IL-2Rβ (FDC3β), and we employed the use of a single cell proliferation assay in which the ability of a single cell to proliferate in response to IL-15 is tested, thereby eliminating the possibility of trans presentation. As shown in Fig. 6B, a single FDC3β cell was consistently able to proliferate in the presence of 30 ng/ml IL-15, whereas FDC3β did not, clearly demonstrating that cis presentation can indeed occur.
We next decided to analyze the efficiency of cis versus trans IL-15 presentation by comparing the effect that soluble IL-15R and soluble IL-15·IL-15R complex have on the ability of IL-15 to induce proliferation in FDC3β versus FDC3β cells. As shown in Fig. 6 (C and D), FDC3β cells could proliferate only in the presence of high concentration (EC50 
100 ng/ml) of murine IL-15 alone, whereas the proliferative capacity of IL-15 was markedly enhanced in the presence of His-sIL-15R1-168 or IL-15·sIL-15R1-168 complex (EC50 3 ng/ml). Importantly, the IL-15·sIL-15R1-71 complex behaved nearly identically to the IL-15·sIL-15R1-168 complex with respect to the enhancement of IL-15 activity on FDC3β cells (Fig. 6D), demonstrating that the IL-15R fragment crystallized in this study (residues 1-71) is responsible for the enhanced biological activity of IL-15. Interestingly, in contrast to the enhancing activity of soluble IL-15R on FDC3β cells, the proliferative capacity of IL-15 on FDC3β cells was decreased in the presence of His-sIL-15R1-168 (EC50 3 ng/ml) compared with IL-15 alone (EC50 0.05 ng/ml).
Cumulatively, the above results indicate that IL-15 is most active when it is presented in cis by membrane-bound IL-15R to IL-2Rβ/c on the surface of the same FDC3β cell. In this context, soluble IL-15R inhibits the activity of IL-15 presumably by competing with membrane-bound IL-15R for IL-15. When trans presentation is forced (on FDC3β cells), soluble IL-15R enhances the activity of IL-15 relative to IL-15 alone, but IL-15 signaling in this context is never as effective as cis presentation on FDC3β cells. The molecular basis by which soluble IL-15R enhances the activity of IL-15 on FDC3β cells is unclear, but has been suggested to be due to a conformational change induced in IL-15 upon receptor binding that enhances signaling through IL-2Rβ/c (29).
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able to engage in both cis and trans presentation of IL-15 to IL-2Rβ/c? The C terminus of the IL-15R ectodomain of consists of a 32-residue linker region and 74-residue PT region that exhibits a high degree of flexibility and thus were not included in the IL-15R protein used for crystallization (see "Experimental Procedures"). Analysis of the IL-15 quaternary complex model shows that the C-terminalmost ordered residue of IL-15R (Arg-71) lies in a plane that is nearly parallel with the cell surface (Fig. 6A). In the context of our structural and biochemical data, we propose a model in which the highly flexible nature of the linker and/or PT regions of IL-15R allow for the presentation of IL-15 in cis or trans, and that the orientation of IL-15 and its receptor components at the quaternary complex interface are the same whether IL-15 is presented in cis or trans (Fig. 6A).
According to this model, during cis presentation, the PT region of IL-15R serves to extend the IL-15-binding sushi domain far enough from the plasma membrane so that IL-15 can be productively presented to IL-2Rβ/c. During trans presentation, the IL-15R sushi domain must also be extended from the plasma membrane to present IL-15 to IL-2Rβ/c of the recipient cell. However, because the orientation of the IL-15 binding surface of the trans-acting IL-15R would be opposite to that of a cis-acting IL-15R molecule, a highly flexible region of the IL-15R (most likely the linker region or N-terminal portion of the PT region) must adopt a conformation that allows the proper presentation of IL-15 to the IL-2Rβ/c in trans. The precise role of the linker region and PT region in cis and trans presentation is currently unresolved and is the subject of ongoing biochemical and structural studies.
Interestingly, alternatively spliced isoforms of IL-15 lacking the linker region (exon 3), the linker region, and part of the PT region (exons 3 and 4) or the linker region and the entire PT region (exons 3-5) have been discovered (23, 52). It will be particularly interesting to test the biological activities of each of these isoforms in context of their ability to participate in trans versus cis presentation. More recently, it has been demonstrated that alternative splicing of IL-15R generates soluble IL-15R sushi domain that forms a complex with IL-15 and is capable of signaling through the IL-2Rβ/c (23). Bulanova et al. (23) suggest that, in addition to the membrane-bound IL-15·IL-15R complex, these alternatively spliced sushi domains may also play an important role in IL-15 trans presentation in vivo, and by extension of our model, we propose that these complexes exhibit the same architecture as presented in Fig. 6A.
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