Crystal structure of the IL-2 signaling complex: Paradigm for a heterotrimeric cytokine receptor

Assembly of the Quaternary IL-2 Signaling Complex.

The high-affinity complex of IL-2 with its extracellular IL-2Rα, IL-2Rβ, and γ_c receptor domains was assembled from the individual receptor ectodomains expressed in insect cells. Throughout purification of γ_c, this receptor subunit existed as a stable homotrimer, as determined by gel filtration. When incubated at 37°C in the presence of IL-2Rα, IL-2Rβ, and IL-2, the γ_c trimer could dissociate and incorporate into the stable quaternary IL-2 signaling complex. However, upon chemical deglycosylation, γ_c aggregated and could not be dissociated for engagement in the high-affinity complex. Thus, self interaction through trimerization and surface carbohydrate sites on γ_c may serve an important solubility role, masking epitopes for IL-2 and IL-2Rβ recognition until formation of a productive signaling complex.

Structure Determination of the Quaternary IL-2 Signaling Complex.

The 3.0-Å crystal structure was determined by molecular replacement by using the coordinates from an independently assembled IL-2/IL-2R complex (17), in which mutations at Asn-17^βGln and Asn-45^βGln eliminated two glycosylation sites and which crystallized in a different space group (C2). A brief comparison of the IL-2/IL-2R complexes in the triclinic (here) and the monoclinic crystal form (17) is presented in Supporting Materials and Methods, which is published as supporting information on the PNAS web site. The x-ray structure was refined to R_free and R_cryst of 26% and 22%, respectively (Table 2, which is published as supporting information on the PNAS web site). Two independent complexes in the asymmetric unit offered a valuable cross validation of key architectural features of the signaling complex (Table 1). In the following analyses, however, only values pertaining to the first complex (chain IDs A, B, C, and D under PDB ID code 2ERJ) are cited unless structural differences are explicitly highlighted or the values differ significantly.

Architecture of the Quaternary IL-2 Receptor Signaling Complex.

The assembly of the IL-2Rβ and γ_c ectodomains bound to IL-2 recapitulate architectural features of the prototypic, human growth hormone (GH) receptor dimer in that a four-helix bundle cytokine is clamped between the elbow regions of two receptor subunits that are roughly related by pseudo-2-fold symmetry, but with a slight translation of γ_c relative to IL-2Rβ (Fig. 1) (18). The interdomain region of γ_c contacts IL-2 helices A and D (classical binding site I), and the elbow region of IL-2Rβ contacts IL-2 helices A and C (classical binding site II) (18). IL-2Rα interacts mainly with the long connection between helices A and B that include helices A′ and B′ of IL-2, in a region recently termed “site IV” (19). IL-2Rα surprisingly does not contact IL-2Rβ or γ_c, and the three interaction sites on IL-2 generally do not overlap with each other, except for a small, but significant, region (see below). The IL-2 molecule in this quaternary complex undergoes only minimal conformational changes, the most significant of which is the ordering of the BC loop (residues 74–81) due to crystal-packing interactions. The IL-2Rβ and γ_c constructs terminate just 6 and 7 residues from the transmembrane domains, respectively (20). Thus, we can estimate the distance from the observed C terminus (Gly-165^α) of IL-2Rα to the membrane to be ≈40 Å, consistent with the predicted unstructured 54-residue linker between Gly-165^α and the putative transmembrane domain (20).

Structure of IL-2Rα and Its Interface with IL-2.

IL-2Rα folds into two “sushi-like” domains D1 and D2, which form five-stranded β-sheet sandwiches. These domains are disposed at ≈75° to each other, as in the crystal structure of IL-2Rα complexed with IL-2 (19). D1 accounts for the majority (82%) of the total buried surface area (1,590 Ų) between IL-2Rα and IL-2, whereas D2 contributes only to a minor extent. The nature of the IL-2/IL-2Rα interface reveals a striking dichotomy of a hydrophobic center dominated by IL-2Rα residues Leu-2^α, Met-25^α, Leu-42^α, and Tyr-43^α and IL-2 residues Phe-42^IL-2, Phe-44^IL-2, Tyr-45^IL-2, Pro-65^IL-2, and Leu-72^IL-2, and a polar periphery featuring five ion pairs (Lys-38^α/Glu-61^IL-2, Arg-36^α/Glu-62^IL-2, Glu-1^α/Lys-35^IL-2, Asp-6^α/Arg-38^IL-2, and Glu-29^α/Lys-43^IL-2; Fig. 2). Seven hydrogen bonds are formed between IL-2 and IL-2Rα (Table 3, which is published as supporting information on the PNAS web site). No significant differences in side- and main-chain conformations are observed at the core of the IL-2/IL-2Rα interface, but some variation in total buried surface area and shape correlation S_c in the two complexes (0.71 and 0.64, by using a 1.7-Å probe) is noted (Table 1). A slight rigid-body rotation (10°) of IL-2Rα with respect to IL-2 seems to result from different crystal packing environments that would give rise to the slight variations in shape complementarity and buried surface.

Architecture of IL-2Rβ and γ_c.

The ectodomains of IL-2Rβ and γ_c possess a cytokine-binding homology region, which is divided in two fibronectin type-III (FN-III) domains termed D1 and D2. Each domain contains seven β-strands that form a sandwich of two antiparallel β-sheets. The N-terminal D1 domains of IL-2Rβ and γ_c both conform to the hybrid type of the Ig fold (21) where the C′ strand hydrogen bonds with both faces of the β-sandwich (Fig. 1), similar to erythropoietin receptor (EPOR) (22). In contrast, the D2 domains belong to the switched type of Ig folds, where strand C′ hydrogen bonds only with strand C. Domain D1 (residues 6–99) and D2 (103–209) of IL-2Rβ are disposed at an elbow angle of 73°, characteristic of class I cytokine receptors (GHR, 64°; IL-4R, 82°; EPOR, 62° and 77°), whereas in γ_c the elbow angles between D1 (32–125) and D2 (129–226) increases to 92° and 98°, respectively, more typical of class II cytokine receptors (IL-10R1, 93°; IFN-αR2, 100°; tissue factor, 118°) (23). The distinct elbow angle in γ_c is also accompanied by a more extended interdomain region. Finally, the D1 and D2 linker regions of IL-2Rβ (residues 100–102) and γ_c (126–128) both adopt a 3₁₀ helical conformation.

A common feature of class I cytokine receptor domains includes two highly conserved disulfide bridges in D1 that link strands A to B, and strands C′ to E, as in IL-2Rβ and γ_c. IL-2Rβ D1 contains an additional disulfide between strands C and F (Cys-33^β-Cys-84^β). D2 of γ_c exhibits another unique disulfide that connects loops BC2 and FG2 (Cys-160^γ-Cys-209^γ) and significantly contributes to the interface with IL-2 (Fig. 4). In D2 of both IL-2Rβ and γ_c, a tryptophan-arginine ladder on sheet C′-C-F-G, that contains the highly conserved WSXWS motif, is present, as in other cytokine receptors (18, 24).

In IL-2Rβ, a particularly well defined glycan structure, consisting of four sugars N-linked to Asn-17, is wedged between D1 and D2 (Fig. 1A and Fig. 6, which is published as supporting information on the PNAS web site). The α1,6-linked core fucose forms four hydrogen bonds with Arg-105^β and Leu-106^β of D2. Moreover, the four sugars make numerous van der Waals contacts with D1 and D2, which may help stabilize a precise orientation of the respective fibronectin type-III domains to each other.

Interface Between IL-2Rβ and IL-2.

IL-2 forms the second largest interface with IL-2Rβ (1,150 Ų, Table 1) with good shape complementarity (0.68) and high specificity via 10 hydrogen bonds (Table 3). Recognition of IL-2 by IL-2Rβ is dominated by the central, protruding Tyr-134^β, which contributes 17% of the buried surface area on IL-2Rβ (Fig. 3). For IL-2, Asp-20^IL2 and His-16^IL2 seem to be the most critical residues; Asp-20^IL2 hydrogen bonds to His-133^β and Tyr-134^β, whereas His-16^IL2 is tucked into a slot created by Tyr-134^β, Gln-188^β, and the methyl groups of Thr-74^β and Thr-73^β. Major van der Waals contacts in the interface are also made with Arg-41^β, Val-75^β, His-133^β, Leu-19^IL2, Asp-84^IL2, Asn-88^IL2, and Val-91^IL2.

Interface Between γ_c and IL-2.

The γ_c/IL-2 interaction (970 Ų) is the smallest of the three IL-2/IL-2R interfaces, although with good surface complementarity (0.72). Furthermore, this contact surface features only two hydrogen bonds (Table 3) and weak electrostatic interactions (Fig. 4), indicating low specificity. The contribution of main-chain atoms to the buried surface area is intermediate (17% on average) between those of IL-2Rα (21%) and IL-2Rβ (12%) (Table 1). Tyr-103^γ, His-159^γ, and Leu-208^γ together with the disulfide (Cys-160^γ-Cys-209^γ) contribute 53% of the buried surface on γ_c and represent the major binding determinant. The importance of the disulfide bridge was previously appreciated from mutagenesis data (25, 26), but its direct involvement in ligand binding was unexpected. With a buried surface area of 72 Ų and one hydrogen bond, Gln-126^IL2 is the most critical IL-2 residue that contacts γ_c in accordance with biochemical data (27). Glu-15^IL2, Thr-123^IL2, and Ile-129^IL2 contribute other significant interactions to the IL-2/γ_c interface.

Interface Between IL-2Rβ and γ_c.

In stark contrast to the relatively small IL-2/γ_c interface, the IL-2R structure reveals a surprisingly large and specific interaction between the D2 receptor domains of IL-2Rβ and γ_c (1,640 Ų). The predominantly polar contacts are evenly distributed among the 22 residues of IL-2Rβ and 31 residues of γ_c without any obvious hot-spot residues. The low percentage of aromatic residues among the side-chain contributions (12%), in combination with 14 hydrogen bonds, seems to enforce much higher specificity of γ_c with IL-2Rβ than of γ_c to IL-2. Glu-136^β and His-138^β form the hinge of the D1/D2 IL-2Rβ elbow and significantly interact with both IL-2 and γ_c. Similarly, Tyr-182^γ, Pro-207^γ, and Leu-208^γ form the hinge of the D1/D2 γ_c elbow and interact with both IL-2 and IL-2Rβ.

Interface Between IL-2Rα and γ_c: Crystal Artifact or Physiologically Relevant.

A crystal contact of considerable size and specificity is formed between γ_c and a symmetry-related IL-2Rα molecule in the neighboring unit cell. Remarkably, this interaction is observed in both copies of the asymmetric unit, as well as in another crystal form (17). IL-2Rα D2 strands H, I, and J interact with the surface of the γ_c elbow region that is not involved in IL-2 binding. Structural features include high shape complementarity (0.68 and 0.78, respectively), a large buried surface area (1,150 Ų) and nine interdomain hydrogen bonds. In this arrangement, the D2 domains of IL-2Rβ/γ_c are parallel to each other, with their C termini toward the membrane (Fig. 7, which is published as supporting information on the PNAS web site). Such lateral interactions would lead to one-dimensional lattices of IL-2R complexes on the cell surface that could promote higher-order signaling assemblies, although no measurable affinity between soluble IL-2Rα and γ_c has been detected (28). However, this same set of experiments also did not detect any binding between IL-2Rβ and γ_c, which in fact form an extensive interface in the fully assembled IL-2R.