Journal of Molecular Biology
Volume 382, Issue 4, 17 October 2008, Pages 931-941
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The Structure of Interleukin-23 Reveals the Molecular Basis of p40 Subunit Sharing with Interleukin-12

https://doi.org/10.1016/j.jmb.2008.07.051Get rights and content

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 α-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-Å 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.

Introduction

The interactions of four-helix bundle cytokines with their class I cytokine receptors control many aspects of cellular development, differentiation, and proliferation across the immune, nervous, and hematopoietic systems. Engagement of cytokines by their receptors results in receptor homo- or heterodimerization, leading to the activation of intracellular Janus kinase (Jak)/signal transducer and activator of transcription (Stat) signaling cascades.1 Four-helix bundle cytokines have a stereotypical up–up–down–down helical topology for both short- and long-chain members of the family.2 Cytokine receptors also have several conserved features, such as the presence of a cytokine-binding homology region that is characterized by tandem fibronectin-type III (FnIII) domains containing a hallmark pattern of disulfide bonds and a WSXWS motif in the second of the FnIII domains.3 The prototypical long-chain cytokine human growth hormone was shown to homodimerize its receptors using a ‘site I/site II’ paradigm through the sides of its helices, and the receptors engaged the cytokine through interstrand loops at the junction of the two FnIII domains.4, 5 While this basic building block has been found in structures of all cytokine receptor complexes, the gp130, or interleukin (IL)-6/IL-12 family of cytokines, has evolved an additional receptor binding epitope (termed site III) that requires the presence of a top-mounted Ig domain on the receptors to homo- or heterodimerize signaling complexes in the ‘tall’ receptor family (e.g., gp130, LIF-R, and IL-12).6, 7 Recently, several orphan members of the IL-12 class of cytokines, IL-23 and IL-27, have been paired with their cognate receptors, and their biological activities were clarified.8, 9 In particular, IL-23 has generated a great deal of excitement with regard to its potential role in immune modulation of different subpopulations of T helper (Th) cells in concert with IL-12.10, 11

IL-12 and IL-23 are heterodimeric cytokines that, unlike typical four-helix cytokines that are secreted alone, are secreted from dendritic cells and macrophages as disulfide-linked complexes between the helical cytokines p35 and p19, respectively (Fig. 1), and a shared binding protein termed p40.11, 12, 13, 14 Both p35 and p19 have sequence homology to IL-6 and granulocyte colony-stimulating factor (GCSF), marking them as members of the gp130 class of long-chain cytokines, and the p40 subunit is similar in structure to typical class I cytokine receptors such as the nonsignaling α-receptors for IL-6 and ciliary neurotrophic factor.15 In essence, IL-12 and IL-23 represent cytokines constitutively associated with a soluble α-receptor subunit. While many cytokines exist as naturally ‘shed’ soluble complexes with their α-receptors,16 IL-12 and IL-23 are unique in that they are secreted as binary complexes.

With regard to receptor activation, while IL-12 and IL-23 share p40, they signal through different heterodimeric cell surface complexes involving receptors homologous to gp130 (Fig. 1). IL-12 (p35/p40) signals through a heterodimer consisting of IL-12Rβ1 and IL-12Rβ2,17 whereas IL-23 (p19/p40) signals through a heterodimer consisting of IL-12Rβ1 and the IL-12Rβ2-like receptor called IL-23R.8 The architecture of the IL-12 and IL-23 receptor signaling complexes can be predicted to recapitulate features of the site I/II/III paradigm based on the organizing principles seen for other members of the tall receptor family such as gp130/IL-6, GCSF/GCSF-R, and LIF/LIF-R.7, 18 IL-23 also activates the same spectrum of Jak/Stat signaling molecules as IL-12: Jak2, Tyk2, Stat1, Stat3, Stat4, and Stat5.8

Biologically, it first seemed likely that these two cytokines would have redundant roles in immune homeostasis, namely, Th1-type responses that are important for cell-mediated antimicrobial and cytotoxic activities, based on their shared use of p40 as a subunit. However, it was quickly shown that their functions are nonredundant.11 Whereas IL-12 drives the typical Th1 responses such as interferon-γ production, IL-23 does not influence the development of Th1 cells, but instead drives the development of an alternate CD4+ T-cell population (now termed Th17 cells) that are notable for their production of proinflammatory IL-17 cytokines.19 Several studies have shown that IL-23 regulation of autoreactive Th17 cells plays a critical role in the development of chronic autoimmune disorders.20, 21, 22 Recently, IL-23 has also been shown to possess tumor-promoting proinflammatory activity and IL-23 blockade can render tumors susceptible to infiltration by IL-12-induced cytotoxic T cells.23 These studies have led to considerable interest in the possibility of therapeutic IL-23 blockade for treatment of autoimmune disorders and, potentially, cancer.

Because IL-12 and IL-23 share their p40 subunit, it is important to delineate common versus cytokine-specific protein–protein interactions to serve as guideposts for the potential development of IL-12- versus IL-23-specific antagonists. Here, we present a structural analysis of IL-23 that represents an important benchmark in elucidating the molecular basis of p40 subunit sharing with IL-12 and in understanding the assembly of their respective signaling complexes based on the organizing principles derived from other members of the gp130/IL-6/IL-12 family.

Section snippets

Results

We expressed IL-23 by coinfection of insect HiFive cells with recombinant baculoviruses carrying cDNAs for p19 and p40. The complex was purified by gel filtration as a single peak of disulfide-linked dimer (data not shown), confirming the integrity of the IL-23 heterodimer as shown for IL-12.15 IL-23 was crystallized and the structure was determined to a resolution of 2.3 Å by molecular replacement using the coordinates of p40,15 and p19 was then built from the partial phases in order to

Discussion

As both IL-12 and IL-23 are attractive therapeutic targets for various autoimmune diseases, one goal is to develop small-molecule inhibitors of heterodimer formation.15 Small-molecule antagonism of cytokine–receptor interactions remains a daunting, but important, challenge given how important many cytokine–receptor interactions are in human health and disease. While antibodies can be highly effective antagonists, production costs are high and special storage and handling are required. Despite

Protein expression and purification

Recombinant IL-23 used for crystallization was expressed using the baculovirus system. High-titer baculovirus stocks were prepared by transfection and amplification in Spodoptera frugiperda (SF9) cells cultured at 28 °C in SF900 II media (Invitrogen). Protein expression was carried out in Trichoplusia ni (Hi-Five, Invitrogen) cells growing in suspension in Insect Xpress media (Lonza).

Human p19 (residues 20–189) and p40 (residues 23–328) cDNAs were cloned into the insect cell expression vector

Acknowledgements

We thank Sean Juo for assistance with data collection, structure analysis, and helpful discussion of the manuscript. We thank Rob Kastelein and Fernando Bazan for helpful discussions. P.J.L. is a Damon Runyon Fellow, supported by the Damon Runyon Cancer Research Foundation (DRG-1928-06). This work was funded by National Institutes of Health grant (AI51321) and a Sandler Program in Asthma Research (SPAR) grant to K.C.G. K.C.G. is also supported by the Keck Foundation and the Howard Hughes

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