Histiocytoses are clonal hematopoietic disorders frequently driven by mutations in BRAF and MEK1/2 kinases. Currently, however, the developmental origins of histiocytoses in patients are not well understood, and clinically meaningful therapeutic targets outside of BRAF and MEK are undefined. Here we uncover activating mutations in CSF-1R, as well as rearrangements in RET and ALK which confer dramatic responses to selective inhibition of RET (selpercatinib) and crizotinib, respectively, in histiocytosis patients.

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Recently it has become possible to de novo design high affinity protein binding proteins from target structural information alone. There is, however, considerable room for improvement as the overall design success rate is low. Here, we explore the augmentation of energy-based protein binder design using deep learning. We find that using AlphaFold2 or RoseTTAFold to assess the probability that a designed sequence adopts the designed monomer structure, and the probability that this structure binds the target as designed, increases design success rates nearly 10-fold. We find further that sequence design using ProteinMPNN rather than Rosetta considerably increases computational efficiency.

We explore the improvement of energy-based protein binder design using deep learning. We find that using AlphaFold2 or RoseTTAFold to assess the probability that a designed sequence adopts the designed monomer structure, and the probability that this structure binds the target as designed, increases design success rates nearly 10-fold. We find further that sequence design using ProteinMPNN rather than Rosetta considerably increases computational efficiency.

Clathrin-mediated endocytosis (CME) is the gatekeeper of the plasma membrane. In contrast to animals and yeasts, CME in plants depends on the TPLATE complex (TPC), an evolutionary ancient adaptor complex. The mechanistic contribution of the individual TPC subunits to plant CME remains however elusive. In this study, we used a multidisciplinary approach to elucidate the structural and functional roles of the evolutionary conserved N-terminal Eps15 homology (EH) domains of the TPC subunit AtEH1/Pan1. By integrating high-resolution structural information obtained by X-ray crystallography and NMR spectroscopy with all-atom molecular dynamics simulations, we provide structural insight into the function of both EH domains. Whereas one EH domain binds negatively charged PI(4,5)P2 lipids, unbiased peptidome profiling by mass-spectrometry revealed that the other EH domain interacts with the double N-terminal NPF motif of a novel TPC interactor, the integral membrane protein Secretory Carrier Membrane Protein 5 (SCAMP5). Furthermore, we show that AtEH/Pan1 proteins control the internalization of SCAMP5 via this double NPF peptide interaction motif. Collectively, our structural and functional studies reveal distinct but complementary roles of the EH domains of AtEH/Pan1 have in plant CME and connect the internalization of SCAMP5 to the TPLATE complex.

Human colony-stimulating factor 1 receptor (hCSF-1R) is unique among the hematopoietic receptors because it is activated by two distinct cytokines, CSF-1 and interleukin-34 (IL-34). Despite ever-growing insights into the central role of hCSF-1R signaling in innate and adaptive immunity, inflammatory diseases, and cancer, the structural basis of the functional dichotomy of hCSF-1R has remained elusive. Here, we report crystal structures of ternary complexes between hCSF-1 and hCSF-1R, including their complete extracellular assembly, and propose a mechanism for the cooperative human CSF-1:CSF-1R complex that relies on the adoption by dimeric hCSF-1 of an active conformational state and homotypic receptor interactions. Furthermore, we trace the cytokine-binding duality of hCSF-1R to a limited set of conserved interactions mediated by functionally equivalent residues on CSF-1 and IL-34 that play into the geometric requirements of hCSF-1R activation, and map the possible mechanistic consequences of somatic mutations in hCSF-1R associated with cancer.

Clathrin-mediated endocytosis (CME) is the gatekeeper of the plasma membrane. In contrast to animals and yeasts, CME in plants depends on the TPLATE complex (TPC), an evolutionary ancient adaptor complex. However, the mechanistic contribution of the individual TPC subunits to plant CME remains elusive. In this study, we used a multidisciplinary approach to elucidate the structural and functional roles of the evolutionary conserved N-terminal Eps15 homology (EH) domains of the TPC subunit AtEH1/Pan1. By integrating high-resolution structural information obtained by X-ray crystallography and NMR spectroscopy with all-atom molecular dynamics simulations, we provide structural insight into the function of both EH domains. Both domains bind phosphatidic acid with a different strength, and only the second domain binds phosphatidylinositol 4,5-bisphosphate. Unbiased peptidome profiling by mass-spectrometry revealed that the first EH domain preferentially interacts with the double N-terminal NPF motif of a previously unidentified TPC interactor, the integral membrane protein Secretory Carrier Membrane Protein 5 (SCAMP5). Furthermore, we show that AtEH/Pan1 proteins control the internalization of SCAMP5 via this double NPF peptide interaction motif. Collectively, our structural and functional studies reveal distinct but complementary roles of the EH domains of AtEH/Pan1 in plant CME and connect the internalization of SCAMP5 to the TPLATE complex.

Anaplastic lymphoma kinase (ALK) 1 and the related leukocyte tyrosine kinase (LTK) 2 are recently deorphanized receptor tyrosine kinases (RTK) 3 . Together with their activating cytokines, ALKAL1 and ALKAL2 (also called FAM150A/FAM150B and AUGβ/AUGα) 4-6 , they are involved in neural development 7 , cancer [7][8][9] , and autoimmune diseases 10 . Furthermore, mammalian ALK recently emerged as a key regulator of energy expenditure and weight gain 11 , consistent with a metabolic role in Drosphila 12 . Despite such functional pleiotropy and growing therapeutic relevance 13,14 , structural insights into ALK and LTK and their complexes with cognate cytokines had remained elusive. Here, we show that the cytokine-binding segments of human ALK and LTK comprise an unprecedented architectural chimera of a permuted TNF-like module that braces a Glycine-rich subdomain featuring a hexagonal lattice of long polyglycine-II helices. The cognate cytokines ALKAL1 and ALKAL2 are monomeric three-helix bundles, yet their binding to ALK and LTK elicit similar dimeric assemblies with twofold symmetry, that tent a single cytokine molecule proximal to the cell membrane. We show that the membrane-proximal EGF-like domain dictates the apparent cytokine preference of ALK. Assisted by diverse structure-function findings, we propose a structural and mechanistic blueprint for complexes of ALK family receptors, thereby extending the repertoire of ligand-mediated dimerization mechanisms adopted by RTK. MainALK is an evolutionarily ancient RTK with the vertebrate orthologues uniquely endowed with an Nterminal heparin binding domain (HBD) (Fig. 1a and Extended Data Fig. 1a,b). Gene duplication in vertebrates spawned LTK as a second ALK-like receptor 16 , which evolved divergently with loss of the HBD and additionally the MAM-LDLa-MAM module in mammals (Extended Data Fig. 1c). The common architectural hallmark in the ectodomains of ALK and LTK comprises their cytokine-binding segment, which is unique among cytokine receptors and features an intriguing array of a TNF-like (TNFL) module, a glycine-rich (GR) region, and a membrane-proximal EGF-like module (EGFL) (Fig. 1a). Whereas ALKAL1 and ALKAL2 are both strong activators of LTK 4 , only ALKAL2 potently activates ALK 5,6 coupled to additional regulation via glycosaminoglycan binding to its HBD 15 .