Structural Activation of Pro-inflammatory Human Cytokine IL-23 by Cognate IL-23 Receptor Enables Recruitment of the Shared Receptor IL-12Rβ1

Abstract

Interleukin-23 (IL-23), an IL-12 family cytokine, plays pivotal roles in pro-inflammatory T helper 17 cell responses linked to autoimmune and inflammatory diseases. Despite intense therapeutic targeting, structural and mechanistic insights into receptor complexes mediated by IL-23, and by IL-12 family members in general, have remained elusive. We determined a crystal structure of human IL-23 in complex with its cognate receptor, IL-23R, and revealed that IL-23R bound to IL-23 exclusively via its N-terminal immunoglobulin domain. The structural and functional hotspot of this interaction partially restructured the helical IL-23p19 subunit of IL-23 and restrained its IL-12p40 subunit to cooperatively bind the shared receptor IL-12Rβ1 with high affinity. Together with structural insights from the interaction of IL-23 with the inhibitory antibody briakinumab and by leveraging additional IL-23:antibody complexes, we propose a mechanistic paradigm for IL-23 and IL-12 whereby cognate receptor binding to the helical cytokine subunits primes recruitment of the shared receptors via the IL-12p40 subunit.

Keywords: Crohn’s disease; IL-12Rβ1; IL-23; IL-23 receptor; cytokine-receptor complex; inflammation; inflammatory bowel disease; psoriasis; rheumatoid arthritis; structure.

Figure 1. Structure of the IL-23:IL-23R complex

A) Schematic representation of protein components participating in IL-23 and IL-12 signaling complexes. EC: Extracellular, PM: Plasma Membrane, CP: Cytoplasm, p19: IL-23p19, p40: IL-12p40. B) Cartoon representation of the IL-23:IL-23R crystal structure. Nanobody 22E11 used as a crystallization adjuvant is shown in gray surface representation. C) Close-up view of the IL-23R:IL-23p19 interface in the vicinity of W156 in IL-23p19. D) Restructuring of IL-23 upon binding to IL-23R. E) Close-up view of the interactions around the AB-loop of IL-23p19. F) Top-down view of the IL-23:IL-23R complex with IL-23R (surface). See also Figure S1, Figure S2, Figure S3, Table S1, and Table S2.

Figure 2. Conformational selection in IL-23 upon IL-23R binding

A) Structural superposition of IL12-p40 subunits as visualized in all available crystal structures (herein and PDB codes 1f42, 1f45, 3d85, 3d87, 3duh, 3hmx, 3qwr, 4grw, 5mj3 and 5mj4) with respect to the D2 domain. B) Schematic recapitulation of the IL-23:IL-23R binding event. C) Structural transition of the N-terminus of helix D in IL-23p19 from a canonical α-helix (grey, PDB 5mj3) to a 3₁₀ helix (orange) in the IL-23:IL-23R complex. See also

Figure 3. W156 in mouse IL-23p19 is a functional hotspot

A) Sequence alignment of human and mouse IL-23p19. Residues at the human IL-23:IL-23R interface and positions chosen for mutagenesis in mouse IL-23 are shown in blue and red, respectively. Predicted secretion signal sequences are underlined. B) Structural context of interrogated positions (violet) at the human IL-23:IL-23R interface. C) Experimental BLI setup and representative response curves fitted with a 1:1 binding model (red) to quantify the kinetics (k_a, k_d) and binding affinity (K_D) of wildtype and mutant IL-23 to IL-23R. D, E) IL-17A and IL-22 secretion by Th17 cells upon differentiation from purified naive CD4+ T by the addition of mouse IL-23. Error bars represent the standard deviation calculated from a technical replicate. F) Representative H&E staining of cutaneous biopsies obtained from C57BL/6J mice treated with PBS, IL-23 or IL-23 mutants. Diffuse epidermal hyperplasia (acanthosis) with associated compact hyperkeratotic and parakeratosis of the stratum corneum caused by wildtype IL-23 is indicated with an asterisk. The vertical black bar in the mIL-23^WT panel indicates epidermal thickness measurement. G) Quantification of epidermal thickness by microscopy. H–J) Expression of Krt16, S100a8, and S100a9 in the dorsal skin of C57BL/6J mice injected with PBS, IL-23, or IL-23 mutants. p-values were determined using one-way ANOVA followed by Holm-Sidak's multiple comparisons test (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). Data are presented as ± S.E.M. n=[3–6]. See also Figure S4 and Figure S5A.

Figure 4. The IL-23:IL-23R binary complex enables high affinity binding of IL-12Rβ1

A) Titration of IL-23 (72 µM) into IL-12Rβ1 (6.8 µM) B) Titration of IL-23 (55 µM) into IL-23R (6.5 µM). The stoichiometry of this experiment was set to 1 and the concentration in the cell was fitted to account for inactive IL-23R. The stoichiometry before correction is provided in parentheses. C) Titration of IL-12Rβ1 (38.8 µM) into preformed IL-23:IL-23R (3.6 µM). Fitted values are provided with their fitting errors. See also Figure S5.

Figure 5. The IL-12Rβ1 binding epitope maps to IL-12p40

A) Cartoon representation of IL-23 structures (vermillion) in complex with antagonists: IL-23:briakinumab-Fab (reported herein); IL-23 in complex with three nanobodies (PDB code: 4grw), IL-23:Ustekinumab-Fab (pdb code: 3hmx) B) Cropped Coomassie-stained reducing SDS-PAGE gel of purified IL-23:IL-12Rβ1 complex crosslinked in the absence (control) or presence of IL-23 antagonists. Protein bands corresponding to crosslinked species (arrow with star) and IL-12Rβ1 in the absence of IL-23 antagonists (arrow) are indicated. C) Previously proposed model for the IL-23:receptor complex. D) Herein proposed model of the IL-23:IL-23R:IL-12Rβ1 ternary complex. See also Figure S6.

Figure 6. Assembly mechanism of the receptor complex mediated by IL-23

The signaling complex mediated by IL-23 proceeds via the sequential recruitment of the two cognate receptors, and involves conformational selection, and restructuring of IL-23 by IL-23R to recruit IL-12Rβ1 with high affinity. Such cytokine-receptor ternary complex is poised to support phosphorylation of intracellular Jak2 and Tyk2 tyrosine kinases associated with the intracellular parts of the receptors, to initiate signaling cascades. Extracellular antagonism of such assemblies can be achieved at two different stages along the ternary complex itinerary depending on the specificity of IL-23 targeting. See also Figure S6A.