Structural insights into the interaction of IL-33 with its receptors

Abstract

Interleukin (IL)-33 is an important member of the IL-1 family that has pleiotropic activities in innate and adaptive immune responses in host defense and disease. It signals through its ligand-binding primary receptor ST2 and IL-1 receptor accessory protein (IL-1RAcP), both of which are members of the IL-1 receptor family. To clarify the interaction of IL-33 with its receptors, we determined the crystal structure of IL-33 in complex with the ectodomain of ST2 at a resolution of 3.27 Å. Coupled with structure-based mutagenesis and binding assay, the structural results define the molecular mechanism by which ST2 specifically recognizes IL-33. Structural comparison with other ligand-receptor complexes in the IL-1 family indicates that surface-charge complementarity is critical in determining ligand-binding specificity of IL-1 primary receptors. Combined crystallography and small-angle X-ray-scattering studies reveal that ST2 possesses hinge flexibility between the D3 domain and D1D2 module, whereas IL-1RAcP exhibits a rigid conformation in the unbound state in solution. The molecular flexibility of ST2 provides structural insights into domain-level conformational change of IL-1 primary receptors upon ligand binding, and the rigidity of IL-1RAcP explains its inability to bind ligands directly. The solution architecture of IL-33-ST2-IL-1RAcP complex from small-angle X-ray-scattering analysis resembles IL-1β-IL-1RII-IL-1RAcP and IL-1β-IL-1RI-IL-1RAcP crystal structures. The collective results confer IL-33 structure-function relationships, supporting and extending a general model for ligand-receptor assembly and activation in the IL-1 family.

Keywords: SAXS; X-ray crystallography; cytokine signaling; protein–protein interaction.

Fig. 1.

Crystal structure of IL-33 in complex with ST2 receptor. IL-33 and ST2 are colored with purple and blue, respectively, and N-linked glycans in ST2 are presented as “sticks” with yellow color.

Fig. 2.

IL-33/ST2 interface. There are two distinct binding sites between IL-33 and ST2. At site 1, IL-33 acidic residues Glu144, Glu148, Asp149, and Asp244 form salt-bridge interactions with ST2 basic residues Arg38, Lys22, Arg198, and Arg35, respectively. Glu144 and Asp149 of IL-33 also have hydrogen-bonding interactions with ST2 main chain atoms. At site 2, IL-33 acidic residue Glu165 has salt-bridge interaction with Arg313 of ST2. A significant hydrophobic cluster at site 2 involves residues Tyr163 and Leu182 of IL-33 and Leu246, Leu306, and Leu311 of ST2.

Fig. 3.

Surface electrostatic-potential analyses of binding site 1. (A) Crystal structures of IL-33–ST2, IL-1β–IL-1RI, IL-1β–IL-1RII, and IL-1Ra–IL-1RI pairs after alignment based on the bound ligand. (B) IL-33 and ST2 have charge complementarity at binding site 1 (yellow dotted circle), with the IL-33 surface negatively charged (red) and the ST2 surface positively charged (blue). (C) There is no obvious charge complementarity at binding site 1 of IL-1β/IL-1RI, IL-1β/IL-1RII, and IL-1Ra/IL-1RI interfaces. (D) IL-18 has a negatively charged patch on the surface of potential binding site 1.

Fig. 4.

Flexibility of ST2 (A) and rigidity of IL-1RAcP (B) in solution. Comparison of experimental data and calculated scattering profiles for ST2 (A) and IL-1RAcP (B). Experimental data are represented in blue dots. Cyan, red, and light orange lines represent the theoretical scattering curves of the initial model derived from crystal structure (cyan), the single best-fit conformation (red), and the assembly from MES (light orange). Residuals calculated as I(q)_experimental/I(q)_model are shown below the scattering curves.

Fig. 5.

Solution architecture of IL-33–ST2 (A) and IL-33–ST2–IL-1RAcP (B) complexes. Good fit of the experimental data (blue dot) and the full-atomic models derived from crystal structures (red line). Residuals calculated as I(q)_experimental/I(q)_model are shown below the scattering curves.