The structure of interleukin-23 reveals the molecular basis of p40 subunit sharing with interleukin-12

Abstract

Interleukin (IL)-23 is a recently identified member of the IL-12 family of heterodimeric cytokines that modulate subpopulations of T helper cells, and both IL-12 and IL-23 are attractive targets for therapy of autoimmune diseases. IL-23 is a binary complex of a four-helix bundle cytokine (p19) and a soluble class I cytokine receptor p40. IL-12 and IL-23 share p40 as an alpha-receptor subunit, yet show only 15% sequence homology between their four-helix cytokines p19 and p35, respectively, and signal through different combinations of shared receptors. In order to elucidate the structural basis of p40 sharing, we have determined a 2.3-A crystal structure of IL-23 for comparison to the previously determined structure of IL-12. The docking mode of p19 to p40 is altered compared to p35, deviating by a 'tilt' and 'roll' that results in an altered footprint of p40 on the A and D helices of the respective cytokines. Binding of p19 to p40 is mediated primarily by an arginine residue on helix D of p19 that forms an extensive charge and hydrogen-bonding network with residues at the base of a pocket on p40. This 'arginine pocket' is lined with an inner shell of hydrophobic interactions that are ringed by an outer shell of polar interactions. Comparative analysis indicates that the IL-23 and IL-12 complexes 'mimic' the network of interactions constituting the central arginine pocket despite p19 and p35 having limited sequence homology. The majority of the structural epitopes in the two complexes are composed of unique p19 and p35 pairwise contacts with common residues on p40. Thus, while the critical hotspot is maintained in the two complexes, the majority of the interfaces are structurally distinct and, therefore, provide a basis for the therapeutic targeting of IL-12 versus IL-23 heterodimer formation despite their use of a common receptor subunit.

Fig. 1

Schematic representation of IL-23 and IL-12 signaling complexes. IgD stands for “Ig-like domain”, while CHR denotes “cytokine-binding homology region” and FnIII denotes “Fibronectin-type III domain”. Yellow lines denote two conserved disulfide linkages and the conserved WSXWS motif.

Fig. 2

Structure of IL-23. (a) Side view of the IL-23 crystal structure. p19 helices A to D are colored in magenta, and p40 domains 1 to 3 colored in blue. The single N-acetylglucosamine residue attached to Asn200 on p40 is shown in red. Disulfide linkages are colored in yellow. (b) Face-on view of IL-23. Receptor interaction sites 2 and 3 are highlighted in ovals, and the key site 3-interacting sidechain of Trp137 is displayed.

Fig. 3

Structural anatomy of the p19–p40 interface. (a) Close-up view of the secondary structure and amino acid contacts between p19 and p40. (b) Cut away view of the ‘Arginine pocket’ 2Fo-Fc electron density contoured at 1.5 σ. Several well-ordered waters are visible at the interface, stabilizing the interaction of p40 pocket residues with Arg159.

Fig. 4

Comparative analysis of IL-23 and IL-12. (a) p19 (pink) is ‘rolled’ toward the D2 of p40 (blue) by ~20° and (b) tilted by ~10° when compared to p35 (green). (c) Overlay of the p19–p40 and p35–p40 interaction interfaces when p40 is structurally superimposed. Note the intact side chain positioning of p19 Asp159 and p40 ‘Arginine pocket’ residues. All contact residues on p19 and p35 are drawn as sticks. (d) Comparison of the four-helix bundle interacting loops of p40 from the two structures (IL-23 p40 in cyan and IL-12 p40 in blue) reveals only slight distortions in the positioning of the loops when bound to their respective four-helix cytokines.

Fig. 5

Comparison of shared and distinct contact surfaces in the IL-23 and IL-12 complexes. (a) Histogram of buried surface area contributed by each p40 residue involved in p19 and/or p35 interaction. (b) Surface representation of p40 (middle panel) with residues interacting with p19 (pink), p35 (green), or p19 and p35 (yellow) highlighted. The p40-interacting surfaces of p19 (left panel in pink) and p35 (right panel in green) are shown with a transparent p40 overlay to demonstrate orientation.