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.

Across different kingdoms of life, ATP citrate lyase (ACLY, also known as ACL) catalyses the ATP-dependent and coenzyme A (CoA)-dependent conversion of citrate, a metabolic product of the Krebs cycle, to oxaloacetate and the high-energy biosynthetic precursor acetyl-CoA 1. The latter fuels pivotal biochemical reactions such as the synthesis of fatty acids, cholesterol and acetylcholine 2 , and the acetylation of histones and proteins 3,4. In autotrophic prokaryotes, ACLY is a hallmark enzyme of the reverse Krebs cycle (also known as the reductive tricarboxylic acid cycle), which fixates two molecules of carbon dioxide in acetyl-CoA 5,6. In humans, ACLY links carbohydrate and lipid metabolism and is strongly expressed in liver and adipose tissue 1 and in cholinergic neurons 2,7. The structural basis of the function of ACLY remains unknown. Here we report high-resolution crystal structures of bacterial, archaeal and human ACLY, and use distinct substrate-bound states to link the conformational plasticity of ACLY to its multistep catalytic itinerary. Such detailed insights will provide the framework for targeting human ACLY in cancer 8-11 and hyperlipidaemia 12,13. Our structural studies also unmask a fundamental evolutionary relationship that links citrate synthase, the first enzyme of the oxidative Krebs cycle, to an ancestral tetrameric citryl-CoA lyase module that operates in the reverse Krebs cycle. This molecular transition marked a key step in the evolution of metabolism on Earth. Human ACLY (hACLY) is a 1,101-residue polypeptide forming a functional 0.5-MDa tetramer and featuring an N-terminal citryl-CoA synthetase (CCS) module, consisting of CCSβ and CCSα regions, and a C-terminal citryl-CoA lyase (CCL) domain 14,15 (Fig. 1a). To determine the structural basis for the multistep ACLY reaction mechanism 16 (Extended Data Fig. 1a), we initially performed negative-stain electron microscopy analysis on crystallization-recalcitrant hACLY. These studies revealed that hACLY displays flexible arms around a compact core but also suggested substantial conformational heterogeneity under such experimental conditions (Extended Data Figs. 1b, 9). To facilitate structural studies by X-ray crystallography, we produced a variant of hACLY, termed hACLY-A/B, that lacked the linker region that connects the ancestral ACLY-A and ACLY-B parts (residues 426-486) and which contains the regulatory phosphorylation sites 17,18 (Fig. 1a, Extended Data Fig. 1c, d). Notably, hACLY-A/B displayed a twofold higher activity in vitro than full-length hACLY (Extended Data Fig. 1e). This indicates that the long linker region between CSSβ and CSSα might have an auto-inhibitory role, at least in the unphosphorylated state. Subsequent crystal structures to 3.3 Å resolution in space groups P1 and C2 for hACLY-A/B in complex with ADP, citrate and CoA show that the CCL domains of four hACLY chains form an intertwined, D2-symmetric 120-kDa CCL module that serves as the oligomerization platform of the ACLY enzyme (Fig. 1b, Extended Data Fig. 1f, g, Exte...

Thymic stromal lymphopoietin (TSLP), a cytokine produced by epithelial cells at barrier surfaces, is pivotal for the development of widespread chronic inflammatory disorders such as asthma and atopic dermatitis. The structure of the mouse TSLP-mediated signaling complex reveals how TSLP establishes extensive interfaces with its cognate receptor (TSLPR) and the shared interleukin 7 receptor α-chain (IL-7Rα) to evoke membrane-proximal receptor-receptor contacts poised for intracellular signaling. Binding of TSLP to TSLPR is a mechanistic prerequisite for recruitment of IL-7Rα to the high-affinity ternary complex, which we propose is coupled to a structural switch in TSLP at the crossroads of the cytokine-receptor interfaces. Functional interrogation of TSLP-receptor interfaces points to putative interaction hotspots that could be exploited for antagonist design. Finally, we derive the structural rationale for the functional duality of IL-7Rα and establish a consensus for the geometry of ternary complexes mediated by interleukin 2 (IL-2)-family cytokines.

Protein scaffolds can provide a promising alternative to antibodies for various biomedical and biotechnological applications, including therapeutics. Here we describe the design and development of the Alphabody, a protein scaffold featuring a single-chain antiparallel triple-helix coiled-coil fold. We report affinity-matured Alphabodies with favourable physicochemical properties that can specifically neutralize human interleukin (IL)-23, a pivotal therapeutic target in autoimmune inflammatory diseases such as psoriasis and multiple sclerosis. The crystal structure of human IL-23 in complex with an affinity-matured Alphabody reveals how the variable interhelical groove of the scaffold uniquely targets a large epitope on the p19 subunit of IL-23 to harness fully the hydrophobic and hydrogen-bonding potential of tryptophan and tyrosine residues contributed by p19 and the Alphabody, respectively. Thus, Alphabodies are suitable for targeting protein–protein interfaces of therapeutic importance and can be tailored to interrogate desired design and binding-mode principles via efficient selection and affinity-maturation strategies.