The design of proteins that bind to a specific site on the surface of a target protein using no information other than the three-dimensional structure of the target remains a challenge1–5. Here we describe a general solution to this problem that starts with a broad exploration of the vast space of possible binding modes to a selected region of a protein surface, and then intensifies the search in the vicinity of the most promising binding modes. We demonstrate the broad applicability of this approach through the de novo design of binding proteins to 12 diverse protein targets with different shapes and surface properties. Biophysical characterization shows that the binders, which are all smaller than 65 amino acids, are hyperstable and, following experimental optimization, bind their targets with nanomolar to picomolar affinities. We succeeded in solving crystal structures of five of the binder–target complexes, and all five closely match the corresponding computational design models. Experimental data on nearly half a million computational designs and hundreds of thousands of point mutants provide detailed feedback on the strengths and limitations of the method and of our current understanding of protein–protein interactions, and should guide improvements of both. Our approach enables the targeted design of binders to sites of interest on a wide variety of proteins for therapeutic and diagnostic applications.

Although spontaneous protein crystallization is a rare event in vivo, Charcot-Leyden crystals (CLCs) consisting of galectin-10 (Gal10) protein are frequently observed in eosinophilic diseases, such as asthma. We found that CLCs derived from patients showed crystal packing and Gal10 structure identical to those of Gal10 crystals grown in vitro. When administered to the airways, crystalline Gal10 stimulated innate and adaptive immunity and acted as a type 2 adjuvant. By contrast, a soluble Gal10 mutein was inert. Antibodies directed against key epitopes of the CLC crystallization interface dissolved preexisting CLCs in patient-derived mucus within hours and reversed crystal-driven inflammation, goblet-cell metaplasia, immunoglobulin E (IgE) synthesis, and bronchial hyperreactivity (BHR) in a humanized mouse model of asthma. Thus, protein crystals may promote hallmark features of asthma and are targetable by crystal-dissolving antibodies.

The pro-inflammatory cytokine thymic stromal lymphopoietin (TSLP) is pivotal to the pathophysiology of widespread allergic diseases mediated by type 2 helper T cell (Th2) responses, including asthma and atopic dermatitis. The emergence of human TSLP as a clinical target against asthma calls for maximally harnessing its therapeutic potential via structural and mechanistic considerations. Here we employ an integrative experimental approach focusing on productive and antagonized TSLP complexes and free cytokine. We reveal how cognate receptor TSLPR allosterically activates TSLP to potentiate the recruitment of the shared interleukin 7 receptor α-chain (IL-7Rα) by leveraging the flexibility, conformational heterogeneity and electrostatics of the cytokine. We further show that the monoclonal antibody Tezepelumab partly exploits these principles to neutralize TSLP activity. Finally, we introduce a fusion protein comprising a tandem of the TSLPR and IL-7Rα extracellular domains, which harnesses the mechanistic intricacies of the TSLP-driven receptor complex to manifest high antagonistic potency.

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...

Intracellular signalling cascades initiated by class III receptor tyrosine kinases (RTK-IIIs) and their cytokine ligands contribute to haematopoiesis and mesenchymal tissue development. They are also implicated in a wide range of inflammatory disorders and cancers. Recent snapshots of RTK-III ectodomains in complex with cognate cytokines have revealed timely insights into the structural determinants of RTK-III activation, evolution and pathology. Importantly, candidate 'driver' and 'passenger' mutations that have been identified in RTK-IIIs can now be collectively mapped for the first time to structural scaffolds of the corresponding RTK-III ectodomains. Such insights will generate a renewed interest in dissecting the mechanistic effects of such mutations and their therapeutic relevance.

The class III receptor tyrosine kinase (RTKIII) Fms-like tyrosine kinase receptor 3 (Flt3) and its cytokine ligand (FL) play central roles in hematopoiesis and the immune system, by establishing signaling cascades crucial for the development and homeostasis of hematopoietic progenitors and antigen-presenting dendritic cells. However, Flt3 is also one of the most frequently mutated receptors in hematologic malignancies and is currently a major prognostic factor and clinical target for acute myeloid leukemia. IntroductionHematopoiesis is a finely regulated process during which diverse cell types originating from a limited and self-renewing population of hematopoietic stem cells (HSCs) are stimulated to proliferate and differentiate to create the cellular repertoire that sustains the mammalian hematopoietic and immune systems. 1 The Fms-like tyrosine kinase receptor 3 (Flt3) is the most recent addition to the diverse family of hematopoietic receptors. Flt3 is activated on HSCs and early myeloid and lymphoid progenitors by its cognate ligand (FL), to initiate downstream signaling via the phosphatidylinositol 3-kinase/AKT and the Ras/Raf/extracellular signal-regulated kinase pathways. 2,3 Consistent with the narrow expression profile of Flt3 in the bone marrow environment, signaling via the Flt3 ligand-receptor complex primarily impacts early hematopoiesis, particularly the proliferation and development of HSC and B-cell progenitors. 2,4 In recent years, Flt3 and FL emerged as potent regulators of dendritic cell (DC) development and homeostasis, [5][6][7] and DC-mediated natural killer cell activation, 8 thereby gaining an important role at the interface of innate and acquired immunity and in cancer immunotherapy. 9,10 Notably, Flt3/FL-driven DC generation yields both classic and plasmacytoid DCs from bone marrow progenitors regardless of myeloid or lymphoid commitment, a property that is currently unmatched by any other receptor-cytokine system relevant for DC physiology. 11,12 Flt3 is a class III receptor tyrosine kinase (RTKIII) together with the prototypic platelet-derived growth factor receptors-␣/␤, colony-stimulating factor 1 receptor (CSF-1R), and KIT. 13 Thus, Flt3 has been predicted to display a modular structure featuring an extracellular segment with 5 immunoglobulin (Ig)-like domains (residues 27-543), a single transmembrane (TM) helix (residues 544-563), a cytoplasmic juxtamembrane domain ([JM]; residues 572-603), and a split intracellular kinase module (residues 604-958). The RTKIII family is closely related to the RTKV family of vascular endothelial growth factor receptors (VEGFRs), which have 7 extracellular Ig-like domains. The hallmark of RTKIII/V signaling lies in the activation of the extracellular receptor segments on binding of the cognate cytokines, followed by intermolecular autophosphorylation and activation of the intracellular kinase domains. 14 Besides the clear role of Flt3 signaling in hematopoiesis and immune system development, overexpression of wild-type or oncogenic forms of Flt3 h...

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SUMMARY The hematopoietic Colony Stimulating Factor-1 receptor (CSF-1R or FMS) is essential for the development of diverse cell types central to the immune system. Here we report a structural and mechanistic consensus for the assembly of hematopoietic human and mouse CSF-1:CSF-1R complexes. The EM structure of the complete extracellular assembly of the human CSF-1:CSF-1R complex reveals how receptor dimerization by CSF-1 invokes a ternary complex featuring extensive homotypic receptor contacts that contribute 15-fold to the affinity of the complex, and striking structural plasticity at the extremities of the complex. Small-angle X-ray scattering analysis of unliganded hCSF-1R points to large domain rearrangements upon CSF-1 binding, and provides structural evidence for the relevance of receptor predimerization at the cell-surface. Comparative structural and binding studies of human and mouse CSF-1R complexes, including a quantification of the CSF-1/CSF-1R species cross-reactivity, show that bivalent cytokine binding to receptor is a common denominator in complex formation independent of receptor homotypic interactions.