RESUMEN
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.
Asunto(s)
Aprendizaje Profundo , Ingeniería de Proteínas , Proteínas/metabolismo , Unión ProteicaRESUMEN
Anaplastic lymphoma kinase (ALK)1 and the related leukocyte tyrosine kinase (LTK)2 are recently deorphanized receptor tyrosine kinases3. Together with their activating cytokines, ALKAL1 and ALKAL24-6 (also called FAM150A and FAM150B or AUGß and AUGα, respectively), they are involved in neural development7, cancer7-9 and autoimmune diseases10. Furthermore, mammalian ALK recently emerged as a key regulator of energy expenditure and weight gain11, consistent with a metabolic role for Drosophila ALK12. Despite such functional pleiotropy and growing therapeutic relevance13,14, structural insights into ALK and LTK and their complexes with cognate cytokines have remained scarce. Here we show that the cytokine-binding segments of human ALK and LTK comprise a novel architectural chimera of a permuted TNF-like module that braces a glycine-rich subdomain featuring a hexagonal lattice of long polyglycine type II helices. The cognate cytokines ALKAL1 and ALKAL2 are monomeric three-helix bundles, yet their binding to ALK and LTK elicits similar dimeric assemblies with two-fold 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 these diverse structure-function findings, we propose a structural and mechanistic blueprint for complexes of ALK family receptors, and thereby extend the repertoire of ligand-mediated dimerization mechanisms adopted by receptor tyrosine kinases.
Asunto(s)
Quinasa de Linfoma Anaplásico/química , Quinasa de Linfoma Anaplásico/metabolismo , Citocinas/química , Citocinas/metabolismo , Proteínas Tirosina Quinasas Receptoras/química , Proteínas Tirosina Quinasas Receptoras/metabolismo , Quinasa de Linfoma Anaplásico/clasificación , Quinasa de Linfoma Anaplásico/genética , Sitios de Unión , Activación Enzimática , Factor de Crecimiento Epidérmico/química , Glicina , Células HEK293 , Humanos , Modelos Moleculares , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Mutación , Unión Proteica , Dominios Proteicos , Multimerización de Proteína , Especificidad por SustratoRESUMEN
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.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/química , Proteínas de Unión al Calcio/química , Endocitosis , Proteínas de Plantas/química , Unión Proteica , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas de Arabidopsis , Proteínas de Unión al Calcio/genética , Membrana Celular/metabolismo , Cristalografía por Rayos X , Proteínas de la Membrana/química , Simulación de Dinámica Molecular , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente , Dominios Proteicos , Transporte de Proteínas , Alineación de Secuencia , Nicotiana/genéticaRESUMEN
Structural studies of glycoproteins and their complexes provide critical insights into their roles in normal physiology and disease. Most glycoproteins contain N-linked glycosylation, a key post-translation modification that critically affects protein folding and stability and the binding kinetics underlying protein interactions. However, N-linked glycosylation is often an impediment to yielding homogeneous protein preparations for structure determination by X-ray crystallography or other methods. In particular, obtaining diffraction-quality crystals of such proteins and their complexes often requires modification of both the type of glycosylation patterns and their extent. Here, we demonstrate the benefits of producing target glycoproteins in the GlycoDelete human embryonic kidney 293 cell line that has been engineered to produce N-glycans as short glycan stumps comprising N-acetylglucosamine, galactose and sialic acid. Protein fragments of human Down syndrome cell-adhesion molecule and colony-stimulating factor 1 receptor were obtained from the GlycoDelete cell line for crystallization. The ensuing reduction in the extent and complexity of N-glycosylation in both protein molecules compared with alternative glycoengineering approaches enabled their productive deployment in structural studies by X-ray crystallography. Furthermore, a third successful implementation of the GlycoDelete technology focusing on murine IL-12B is shown to lead to N-glycosylation featuring an immature glycan in diffraction-quality crystals. It is proposed that the GlycoDelete cell line could serve as a valuable go-to option for the production of homogeneous glycoproteins and their complexes for structural studies by X-ray crystallography and cryo-electron microscopy.
Asunto(s)
Glicoproteínas/química , Polisacáridos/química , Microscopía por Crioelectrón/métodos , Cristalografía por Rayos X/métodos , Glicosilación , Células HEK293 , Humanos , Procesamiento Proteico-PostraduccionalRESUMEN
As the last enzyme in nucleotide synthesis as precursors for DNA replication, thymidylate kinase of M. tuberculosis (MtbTMPK) attracts significant interest as a target in the discovery of new anti-tuberculosis agents. Earlier, we discovered potent MtbTMPK inhibitors, but these generally suffered from poor antimycobacterial activity, which we hypothesize is due to poor bacterial uptake. To address this, we herein describe our efforts to equip previously reported MtbTMPK inhibitors with targeting moieties to increase the whole cell activity of the hybrid analogues. Introduction of a simplified Fe-chelating siderophore motif gave rise to analogue 17 that combined favorable enzyme inhibitory activity with significant activity against M. tuberculosis (MIC of 12.5 µM). Conjugation of MtbTMPK inhibitors with an imidazo[1,2-a]pyridine or 3,5-dinitrobenzamide scaffold afforded analogues 26, 27 and 28, with moderate MtbTMPK enzyme inhibitory potency, but sub-micromolar activity against mycobacteria without significant cytotoxicity. These results indicate that conjugation with structural motifs known to favor mycobacterial uptake may be a valid approach for discovering new antimycobacterial agents.
Asunto(s)
Diseño de Fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/enzimología , Nucleósido-Fosfato Quinasa/antagonistas & inhibidores , Antituberculosos/química , Antituberculosos/farmacología , Línea Celular , Humanos , Pruebas de Sensibilidad Microbiana , Relación Estructura-ActividadRESUMEN
Histiocytoses are clonal hematopoietic disorders frequently driven by mutations mapping to the BRAF and MEK1 and MEK2 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. In this study, we uncovered activating mutations in CSF1R and rearrangements in RET and ALK that conferred dramatic responses to selective inhibition of RET (selpercatinib) and crizotinib, respectively, in patients with histiocytosis.
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Quinasa de Linfoma Anaplásico/genética , Histiocitosis/genética , Proteínas Proto-Oncogénicas c-ret/genética , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Adolescente , Adulto , Aminopiridinas/farmacología , Benzotiazoles/farmacología , Niño , Preescolar , Femenino , Genoma Humano , Neoplasias Hematológicas/tratamiento farmacológico , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/patología , Histiocitosis/tratamiento farmacológico , Histiocitosis/patología , Humanos , Lactante , Masculino , Mutación , Ácidos Picolínicos/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Pirazoles/farmacología , Piridinas/farmacología , Pirroles/farmacología , Proteínas Tirosina Quinasas Receptoras/genética , Gemelos Monocigóticos , Secuenciación del Exoma , Adulto JovenRESUMEN
Clathrin-mediated endocytosis (CME) is a highly conserved and essential cellular process in eukaryotic cells, but its dynamic and vital nature makes it challenging to study using classical genetics tools. In contrast, although small molecules can acutely and reversibly perturb CME, the few chemical CME inhibitors that have been applied to plants are either ineffective or show undesirable side effects. Here, we identify the previously described endosidin9 (ES9) as an inhibitor of clathrin heavy chain (CHC) function in both Arabidopsis and human cells through affinity-based target isolation, in vitro binding studies and X-ray crystallography. Moreover, we present a chemically improved ES9 analog, ES9-17, which lacks the undesirable side effects of ES9 while retaining the ability to target CHC. ES9 and ES9-17 have expanded the chemical toolbox used to probe CHC function, and present chemical scaffolds for further design of more specific and potent CHC inhibitors across different systems.
Asunto(s)
Derivados del Benceno/farmacología , Cadenas Pesadas de Clatrina/antagonistas & inhibidores , Endocitosis/efectos de los fármacos , Arabidopsis , Derivados del Benceno/química , Cadenas Pesadas de Clatrina/metabolismo , Humanos , Modelos Moleculares , Estructura Molecular , Tiofenos/farmacologíaRESUMEN
Small GTP-binding proteins from the ADP-ribosylation factor (ARF) family are important regulators of vesicle formation and cellular trafficking in all eukaryotes. ARF activation is accomplished by a protein family of guanine nucleotide exchange factors (GEFs) that contain a conserved catalytic Sec7 domain. Here, we identified and characterized Secdin, a small-molecule inhibitor of Arabidopsis thaliana ARF-GEFs. Secdin application caused aberrant retention of plasma membrane (PM) proteins in late endosomal compartments, enhanced vacuolar degradation, impaired protein recycling, and delayed secretion and endocytosis. Combined treatments with Secdin and the known ARF-GEF inhibitor Brefeldin A (BFA) prevented the BFA-induced PM stabilization of the ARF-GEF GNOM, impaired its translocation from the Golgi to the trans-Golgi network/early endosomes, and led to the formation of hybrid endomembrane compartments reminiscent of those in ARF-GEF-deficient mutants. Drug affinity-responsive target stability assays revealed that Secdin, unlike BFA, targeted all examined Arabidopsis ARF-GEFs, but that the interaction was probably not mediated by the Sec7 domain because Secdin did not interfere with the Sec7 domain-mediated ARF activation. These results show that Secdin and BFA affect their protein targets through distinct mechanisms, in turn showing the usefulness of Secdin in studies in which ARF-GEF-dependent endomembrane transport cannot be manipulated with BFA.
Asunto(s)
Arabidopsis/efectos de los fármacos , Factores de Intercambio de Guanina Nucleótido/antagonistas & inhibidores , Ftalazinas/farmacología , Piperazinas/farmacología , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Brefeldino A/farmacología , Endocitosis/efectos de los fármacos , Endosomas/efectos de los fármacos , Endosomas/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Plantas Modificadas Genéticamente , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Transporte de Proteínas , Vacuolas/efectos de los fármacos , Vacuolas/metabolismoRESUMEN
Subversion of the host immune system by viruses is often mediated by molecular decoys that sequester host proteins pivotal to mounting effective immune responses. The widespread mammalian pathogen parapox Orf virus deploys GIF, a member of the poxvirus immune evasion superfamily, to antagonize GM-CSF (granulocyte macrophage colony-stimulating factor) and IL-2 (interleukin-2), two pleiotropic cytokines of the mammalian immune system. However, structural and mechanistic insights into the unprecedented functional duality of GIF have remained elusive. Here we reveal that GIF employs a dimeric binding platform that sequesters two copies of its target cytokines with high affinity and slow dissociation kinetics to yield distinct complexes featuring mutually exclusive interaction footprints. We illustrate how GIF serves as a competitive decoy receptor by leveraging binding hotspots underlying the cognate receptor interactions of GM-CSF and IL-2, without sharing any structural similarity with the cytokine receptors. Our findings contribute to the tracing of novel molecular mimicry mechanisms employed by pathogenic viruses.
Asunto(s)
Factor Estimulante de Colonias de Granulocitos y Macrófagos/inmunología , Interleucina-2/inmunología , Parapoxvirus/inmunología , Proteínas Virales/inmunología , Cristalografía por Rayos X , Factor Estimulante de Colonias de Granulocitos y Macrófagos/química , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Células HEK293 , Interacciones Huésped-Patógeno/inmunología , Humanos , Interleucina-2/química , Interleucina-2/metabolismo , Modelos Moleculares , Complejos Multiproteicos/química , Complejos Multiproteicos/inmunología , Complejos Multiproteicos/metabolismo , Parapoxvirus/metabolismo , Infecciones por Poxviridae/inmunología , Infecciones por Poxviridae/metabolismo , Infecciones por Poxviridae/virología , Unión Proteica , Proteínas Virales/química , Proteínas Virales/metabolismoRESUMEN
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.