RESUMEN
Neural migration is a critical step during brain development that requires the interactions of cell-surface guidance receptors. Cancer cells often hijack these mechanisms to disseminate. Here, we reveal crystal structures of Uncoordinated-5 receptor D (Unc5D) in complex with morphogen receptor glypican-3 (GPC3), forming an octameric glycoprotein complex. In the complex, four Unc5D molecules pack into an antiparallel bundle, flanked by four GPC3 molecules. Central glycan-glycan interactions are formed by N-linked glycans emanating from GPC3 (N241 in human) and C-mannosylated tryptophans of the Unc5D thrombospondin-like domains. MD simulations, mass spectrometry and structure-based mutants validate the crystallographic data. Anti-GPC3 nanobodies enhance or weaken Unc5-GPC3 binding and, together with mutant proteins, show that Unc5/GPC3 guide migrating pyramidal neurons in the mouse cortex, and cancer cells in an embryonic xenograft neuroblastoma model. The results demonstrate a conserved structural mechanism of cell guidance, where finely balanced Unc5-GPC3 interactions regulate cell migration.
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Movimiento Celular , Glipicanos/química , Receptores de Netrina/química , Animales , Glipicanos/metabolismo , Humanos , Ratones , Proteínas Mutantes , Receptores de Netrina/metabolismo , Receptores de Superficie Celular/metabolismo , Anticuerpos de Dominio Único , TrombospondinasRESUMEN
Folates (also known as vitamin B9) have a critical role in cellular metabolism as the starting point in the synthesis of nucleic acids, amino acids and the universal methylating agent S-adenylsmethionine1,2. Folate deficiency is associated with a number of developmental, immune and neurological disorders3-5. Mammals cannot synthesize folates de novo; several systems have therefore evolved to take up folates from the diet and distribute them within the body3,6. The proton-coupled folate transporter (PCFT) (also known as SLC46A1) mediates folate uptake across the intestinal brush border membrane and the choroid plexus4,7, and is an important route for the delivery of antifolate drugs in cancer chemotherapy8-10. How PCFT recognizes folates or antifolate agents is currently unclear. Here we present cryo-electron microscopy structures of PCFT in a substrate-free state and in complex with a new-generation antifolate drug (pemetrexed). Our results provide a structural basis for understanding antifolate recognition and provide insights into the pH-regulated mechanism of folate transport mediated by PCFT.
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Microscopía por Crioelectrón , Antagonistas del Ácido Fólico/química , Antagonistas del Ácido Fólico/metabolismo , Pemetrexed/química , Pemetrexed/metabolismo , Transportador de Folato Acoplado a Protón/química , Transportador de Folato Acoplado a Protón/metabolismo , Apoproteínas/química , Apoproteínas/metabolismo , Apoproteínas/ultraestructura , Transporte Biológico , Humanos , Modelos Moleculares , Transportador de Folato Acoplado a Protón/ultraestructura , ProtonesRESUMEN
Camelid single-domain antibodies, also known as nanobodies, can be readily isolated from naïve libraries for specific targets but often bind too weakly to their targets to be immediately useful. Laboratory-based genetic engineering methods to enhance their affinity, termed maturation, can deliver useful reagents for different areas of biology and potentially medicine. Using the receptor binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein and a naïve library, we generated closely related nanobodies with micromolar to nanomolar binding affinities. By analyzing the structure-activity relationship using X-ray crystallography, cryoelectron microscopy, and biophysical methods, we observed that higher conformational entropy losses in the formation of the spike protein-nanobody complex are associated with tighter binding. To investigate this, we generated structural ensembles of the different complexes from electron microscopy maps and correlated the conformational fluctuations with binding affinity. This insight guided the engineering of a nanobody with improved affinity for the spike protein.
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Anticuerpos Neutralizantes , Anticuerpos Antivirales , Afinidad de Anticuerpos , SARS-CoV-2 , Anticuerpos de Dominio Único , Glicoproteína de la Espiga del Coronavirus , Anticuerpos Neutralizantes/química , Anticuerpos Neutralizantes/genética , Anticuerpos Antivirales/química , Anticuerpos Antivirales/genética , Afinidad de Anticuerpos/genética , Microscopía por Crioelectrón , Entropía , Ingeniería Genética , Humanos , Unión Proteica , Dominios Proteicos , SARS-CoV-2/inmunología , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/genética , Glicoproteína de la Espiga del Coronavirus/inmunologíaRESUMEN
Heavy chain-only antibodies can offer advantages of higher binding affinities, reduced sizes, and higher stabilities than conventional antibodies. To address the challenge of SARS-CoV-2 coronavirus, a llama-derived single-domain nanobody C5 was developed previously that has high COVID-19 virus neutralization potency. The fusion protein C5-Fc comprises two C5 domains attached to a glycosylated Fc region of a human IgG1 antibody and shows therapeutic efficacy in vivo. Here, we have characterized the solution arrangement of the molecule. Two 1443 Da N-linked glycans seen in the mass spectra of C5-Fc were removed and the glycosylated and deglycosylated structures were evaluated. Reduction of C5-Fc with 2-mercaptoethylamine indicated three interchain Cys-Cys disulfide bridges within the hinge. The X-ray and neutron Guinier RG values, which provide information about structural elongation, were similar at 4.1 to 4.2 nm for glycosylated and deglycosylated C5-Fc. To explain these RG values, atomistic scattering modeling based on Monte Carlo simulations resulted in 72,737 and 56,749 physically realistic trial X-ray and neutron structures, respectively. From these, the top 100 best-fit X-ray and neutron models were identified as representative asymmetric solution structures, similar to that of human IgG1, with good R-factors below 2.00%. Both C5 domains were solvent exposed, consistent with the functional effectiveness of C5-Fc. Greater disorder occurred in the Fc region after deglycosylation. Our results clarify the importance of variable and exposed C5 conformations in the therapeutic function of C5-Fc, while the glycans in the Fc region are key for conformational stability in C5-Fc.
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Anticuerpos Antivirales , Cadenas Pesadas de Inmunoglobulina , SARS-CoV-2 , Humanos , Inmunoglobulina G/química , Cadenas Pesadas de Inmunoglobulina/química , Modelos Moleculares , Polisacáridos , Anticuerpos Antivirales/química , Anticuerpos de Dominio Único/químicaRESUMEN
The SARS-CoV-2 receptor, ACE2, is found on pericytes, contractile cells enwrapping capillaries that regulate brain, heart and kidney blood flow. ACE2 converts vasoconstricting angiotensin II into vasodilating angiotensin-(1-7). In brain slices from hamster, which has an ACE2 sequence similar to human ACE2, angiotensin II evoked a small pericyte-mediated capillary constriction via AT1 receptors, but evoked a large constriction when the SARS-CoV-2 receptor binding domain (RBD, original Wuhan variant) was present. A mutated non-binding RBD did not potentiate constriction. A similar RBD-potentiated capillary constriction occurred in human cortical slices, and was evoked in hamster brain slices by pseudotyped virions expressing SARS-CoV-2 spike protein. This constriction reflects an RBD-induced decrease in the conversion of angiotensin II to angiotensin-(1-7) mediated by removal of ACE2 from the cell surface membrane and was mimicked by blocking ACE2. The clinically used drug losartan inhibited the RBD-potentiated constriction. Thus, AT1 receptor blockers could be protective in COVID-19 by preventing pericyte-mediated blood flow reductions in the brain, and perhaps the heart and kidney.
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COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/metabolismo , COVID-19/metabolismo , Pericitos/metabolismo , Angiotensina II/farmacología , Angiotensina II/metabolismo , Enzima Convertidora de Angiotensina 2/química , Enzima Convertidora de Angiotensina 2/metabolismo , Capilares , Constricción , Receptores Virales/química , Receptores Virales/metabolismo , Peptidil-Dipeptidasa A/genética , Peptidil-Dipeptidasa A/metabolismo , Unión ProteicaRESUMEN
Many bacteria, including the major human pathogen Pseudomonas aeruginosa, are naturally found in multicellular, antibiotic-tolerant biofilm communities, in which cells are embedded in an extracellular matrix of polymeric molecules. Cell-cell interactions within P. aeruginosa biofilms are mediated by CdrA, a large, membrane-associated adhesin present in the extracellular matrix of biofilms, regulated by the cytoplasmic concentration of cyclic diguanylate. Here, using electron cryotomography of focused ion beam-milled specimens, we report the architecture of CdrA molecules in the extracellular matrix of P. aeruginosa biofilms at intact cell-cell junctions. Combining our in situ observations at cell-cell junctions with biochemistry, native mass spectrometry, and cellular imaging, we demonstrate that CdrA forms an extended structure that projects from the outer membrane to tether cells together via polysaccharide binding partners. We go on to show the functional importance of CdrA using custom single-domain antibody (nanobody) binders. Nanobodies targeting the tip of functional cell-surface CdrA molecules could be used to inhibit bacterial biofilm formation or disrupt preexisting biofilms in conjunction with bactericidal antibiotics. These results reveal a functional mechanism for cell-cell interactions within bacterial biofilms and highlight the promise of using inhibitors targeting biofilm cell-cell junctions to prevent or treat problematic, chronic bacterial infections.
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Adhesinas Bacterianas/metabolismo , Biopelículas/crecimiento & desarrollo , Pseudomonas aeruginosa/fisiología , Adhesinas Bacterianas/genética , Adhesión Bacteriana , Membrana Celular , Matriz Extracelular , Regulación Bacteriana de la Expresión Génica , Anticuerpos de Dominio ÚnicoRESUMEN
Adipose triglyceride lipase (ATGL) plays a key role in intracellular lipolysis, the mobilization of stored triacylglycerol. This work provides an important basis for generating reproducible and detailed data on the hydrolytic and transacylation activities of ATGL. We generated full-length and C-terminally truncated ATGL variants fused with various affinity tags and analyzed their expression in different hosts, namely E.coli, the insect cell line Sf9, and the mammalian cell line human embryonic kidney 293T. Based on this screen, we expressed a fusion protein of ATGL covering residues M1-D288 flanked with N-terminal and C-terminal purification tags. Using these fusions, we identified key steps in expression and purification protocols, including production in the E. coli strain ArcticExpress (DE3) and removal of copurified chaperones. The resulting purified ATGL variant demonstrated improved lipolytic activity compared with previously published data, and it could be stimulated by the coactivator protein comparative gene identification-58 and inhibited by the protein G0/G1 switch protein 2. Shock freezing and storage did not affect the basal activity but reduced coactivation of ATGL by comparative gene identification 58. In vitro, the truncated ATGL variant demonstrated acyl-CoA-independent transacylation activity when diacylglycerol was offered as substrate, resulting in the formation of fatty acid as well as triacylglycerol and monoacylglycerol. However, the ATGL variant showed neither hydrolytic activity nor transacylation activity upon offering of monoacylglycerol as substrate. To understand the role of ATGL in different physiological contexts, it is critical for future studies to identify all its different functions and to determine under what conditions these activities occur.
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Expresión Génica , Lipasa , Acilación , Animales , Células HEK293 , Humanos , Hidrólisis , Lipasa/biosíntesis , Lipasa/química , Lipasa/genética , Lipasa/aislamiento & purificación , Ratones , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Células Sf9 , SpodopteraRESUMEN
The knowledge of interactions between different molecules is undoubtedly the driving force of all contemporary biomedical and biological sciences. Chemical biology/biological chemistry has become an important multidisciplinary bridge connecting the perspectives of chemistry and biology to the study of small molecules/peptidomimetics and their interactions in biological systems. Advances in structural biology research, in particular linking atomic structure to molecular properties and cellular context, are essential for the sophisticated design of new medicines that exhibit a high degree of druggability and very importantly, druglikeness. The authors of this contribution are outstanding scientists in the field who provided a brief overview of their work, which is arranged from in silico investigation through the characterization of interactions of compounds with biomolecules to bioactive materials.
Asunto(s)
Biología MolecularRESUMEN
Collagen VI is a ubiquitous heterotrimeric protein of the extracellular matrix (ECM) that plays an essential role in the proper maintenance of skeletal muscle. Mutations in collagen VI lead to a spectrum of congenital myopathies, from the mild Bethlem myopathy to the severe Ullrich congenital muscular dystrophy. Collagen VI contains only a short triple helix and consists primarily of von Willebrand factor type A (VWA) domains, protein-protein interaction modules found in a range of ECM proteins. Disease-causing mutations occur commonly in the VWA domains, and the second VWA domain of the α3 chain, the N2 domain, harbors several such mutations. Here, we investigate structure-function relationships of the N2 mutations to shed light on their possible myopathy mechanisms. We determined the X-ray crystal structure of N2, combined with monitoring secretion efficiency in cell culture of selected N2 single-domain mutants, finding that mutations located within the central core of the domain severely affect secretion efficiency. In longer α3 chain constructs, spanning N6-N3, small-angle X-ray scattering demonstrates that the tandem VWA array has a modular architecture and samples multiple conformations in solution. Single-particle EM confirmed the presence of multiple conformations. Structural adaptability appears intrinsic to the VWA domain region of collagen VI α3 and has implications for binding interactions and modulating stiffness within the ECM.
Asunto(s)
Colágeno Tipo VI/química , Enfermedades Musculares , Mutación , Colágeno Tipo VI/genética , Cristalografía por Rayos X , Humanos , Dominios ProteicosRESUMEN
Protein-protein interactions are essential for the control of cellular functions and are critical for regulation of the immune system. One example is the binding of Fc regions of IgG to the Fc gamma receptors (FcγRs). High sequence identity (98%) between the genes encoding FcγRIIIa (expressed on macrophages and natural killer cells) and FcγRIIIb (expressed on neutrophils) has prevented the development of monospecific agents against these therapeutic targets. We now report the identification of FcγRIIIa-specific artificial binding proteins called "Affimer" that block IgG binding and abrogate FcγRIIIa-mediated downstream effector functions in macrophages, namely TNF release and phagocytosis. Cocrystal structures and molecular dynamics simulations have revealed the structural basis of this specificity for two Affimer proteins: One binds directly to the Fc binding site, whereas the other acts allosterically.
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Complejo Antígeno-Anticuerpo/química , Inmunoglobulina G/química , Simulación de Dinámica Molecular , Receptores de IgG/química , Regulación Alostérica , Complejo Antígeno-Anticuerpo/inmunología , Humanos , Inmunoglobulina G/inmunología , Receptores de IgG/inmunologíaRESUMEN
Rift Valley fever virus (RVFV) is a mosquito-transmitted virus from the Bunyaviridae family that causes high rates of mortality and morbidity in humans and ruminant animals. Previous studies indicated that DEAD-box helicase 17 (DDX17) restricts RVFV replication by recognizing two primary non-coding RNAs in the S-segment of the genome: the intergenic region (IGR) and 5' non-coding region (NCR). However, we lack molecular insights into the direct binding of DDX17 with RVFV non-coding RNAs and information on the unwinding of both non-coding RNAs by DDX17. Therefore, we performed an extensive biophysical analysis of the DDX17 helicase domain (DDX17135-555) and RVFV non-coding RNAs, IGR and 5' NCR. The homogeneity studies using analytical ultracentrifugation indicated that DDX17135-555, IGR, and 5' NCR are pure. Next, we performed small-angle X-ray scattering (SAXS) experiments, which suggested that DDX17 and both RNAs are homogenous as well. SAXS analysis also demonstrated that DDX17 is globular to an extent, whereas the RNAs adopt an extended conformation in solution. Subsequently, microscale thermophoresis (MST) experiments were performed to investigate the direct binding of DDX17 to the non-coding RNAs. The MST experiments demonstrated that DDX17 binds with the IGR and 5' NCR with a dissociation constant of 5.77 ± 0.15 µM and 9.85 ± 0.11 µM, respectively. As DDX17135-555 is an RNA helicase, we next determined if it could unwind IGR and NCR. We developed a helicase assay using MST and fluorescently-labeled oligos, which suggested DDX17135-555 can unwind both RNAs. Overall, our study provides direct evidence of DDX17135-555 interacting with and unwinding RVFV non-coding regions.
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ARN Helicasas DEAD-box/metabolismo , Interacciones Huésped-Patógeno , ARN no Traducido , ARN Viral , Fiebre del Valle del Rift/metabolismo , Fiebre del Valle del Rift/virología , Virus de la Fiebre del Valle del Rift/genética , Adenosina Trifosfato , Animales , ARN Helicasas DEAD-box/química , Humanos , Modelos Moleculares , Unión Proteica , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Relación Estructura-ActividadRESUMEN
BACKGROUND: Zika virus (ZIKV) has become a global threat with immediate need for accurate diagnostics, efficacious vaccines and therapeutics. Several ZIKV envelope (Env)-based vaccines have been developed recently. However, many commercially available ZIKV Env are based on the African lineage and produced in insect cells. Here, we sought to produce Asian-lineage ZIKV Env in mammalian cells for research and clinical applications. METHODS: We designed various gene expression constructs to optimize the production of ZIKV using prM-Env and full or C-terminal truncations of Env; with or without a rat CD4 fusion partner to allow large-scale production of soluble protein in mammalian HEK293 cells. Protein expression was verified by mass spectrometry and western-blot with a pan-flavivirus antibody, a ZIKV Env monoclonal antibody and with immune sera from adenoviral (ChAdOx1) ZIKV Env-vaccinated mice. The resulting Env-CD4 was used as a coating reagent for immunoassay (ELISA) using both mouse and human seropositive sera. RESULTS: Replacement of the C-terminus transmembrane Env domain by a rat CD4 and addition of prM supported optimal expression and secretion of Env. Binding between the antigens and the antibodies was similar to binding when using commercially available ZIKV Env reagents. Furthermore, antibodies from ZIKV patients bound ZIKV Env-CD4 in ELISA assays, whereas sera from healthy blood donors yielded minimal OD background. The serological outcomes of this assay correlated also with ZIKV neutralisation capacity in vitro. CONCLUSIONS: Results obtained from this study indicate the potential of the Asian-lineage Zika Env-CD4 and Env proteins in ELISA assays to monitor humoral immune responses in upcoming clinical trials as well as a sero-diagnostic tool in ZIKV infection.
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Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Proteínas Recombinantes de Fusión/inmunología , Proteínas del Envoltorio Viral/inmunología , Proteínas del Envoltorio Viral/aislamiento & purificación , Virus Zika/inmunología , Animales , Antígenos CD4/genética , Ensayo de Inmunoadsorción Enzimática/métodos , Células HEK293 , Humanos , México , Ratones , Pruebas de Neutralización/métodos , Ratas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismo , Virus Zika/genéticaRESUMEN
The production of recombinant integral membrane proteins for structural and functional studies remains technically challenging due to their relatively low levels of expression. To address this problem, screening strategies have been developed to identify the optimal membrane sequence and expression host for protein production. A common approach is to genetically fuse the membrane protein to a fluorescent reporter, typically Green Fluorescent Protein (GFP) enabling expression levels, localization and detergent solubilisation to be assessed. Initially developed for screening the heterologous expression of bacterial membrane proteins in Escherichia coli, the method has been extended to eukaryotic hosts, including insect and mammalian cells. Overall, GFP-based expression screening has made a major impact on the number of membrane protein structures that have been determined in the last few years.
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Genes Reporteros , Proteínas Luminiscentes/análisis , Proteínas de la Membrana/análisis , Animales , Células Cultivadas , Escherichia coli/metabolismo , Células Eucariotas/metabolismo , Expresión Génica , Proteínas Fluorescentes Verdes/análisis , Células HEK293/metabolismo , Humanos , Insectos/citología , Proteínas Luminiscentes/genética , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/genética , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Levaduras/metabolismoRESUMEN
Hedgehog signaling is critical for correct embryogenesis and tissue development. However, on maturation, signaling is also found to be aberrantly activated in many cancers. Palmitoylation of the secreted signaling protein sonic hedgehog (Shh) by the enzyme hedgehog acyltransferase (Hhat) is required for functional signaling. To quantify this important posttranslational modification, many in vitro Shh palmitoylation assays employ radiolabeled fatty acids, which have limitations in terms of cost and safety. Here we present a click chemistry armed enzyme-linked immunosorbent assay (click-ELISA) for assessment of Hhat activity through acylation of biotinylated Shh peptide with an alkyne-tagged palmitoyl-CoA (coenzyme A) analogue. Click chemistry functionalization of the alkyne tag with azido-FLAG peptide allows analysis through an ELISA protocol and colorimetric readout. This assay format identified the detergent n-dodecyl ß-d-maltopyranoside as an improved solubilizing agent for Hhat activity. Quantification of the potency of RU-SKI small molecule Hhat inhibitors by click-ELISA indicated IC50 values in the low- or sub-micromolar range. A stopped assay format was also employed that allows measurement of Hhat kinetic parameters where saturating substrate concentrations exceed the binding capacity of the streptavidin-coated plate. Therefore, click-ELISA represents a nonradioactive method for assessing protein palmitoylation in vitro that is readily expandable to other classes of protein lipidation.
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Aciltransferasas/metabolismo , Proteínas Hedgehog/metabolismo , Procesamiento Proteico-Postraduccional , Aciltransferasas/antagonistas & inhibidores , Aciltransferasas/química , Aciltransferasas/genética , Biotinilación , Química Clic , Detergentes/química , Inhibidores Enzimáticos/farmacología , Ensayo de Inmunoadsorción Enzimática , Ácidos Grasos Insaturados/farmacología , Células HEK293 , Proteínas Hedgehog/química , Humanos , Proteínas Inmovilizadas/química , Proteínas Inmovilizadas/metabolismo , Lipoilación/efectos de los fármacos , Maltosa/análogos & derivados , Maltosa/química , Oligopéptidos/química , Oligopéptidos/metabolismo , Palmitoil Coenzima A/análogos & derivados , Palmitoil Coenzima A/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Solubilidad , Estreptavidina/química , Estreptavidina/metabolismo , Especificidad por SustratoRESUMEN
Heparan sulfate (HS) polysaccharides are major constituents of the extracellular matrix, which are involved in myriad structural and signaling processes. Mature HS polysaccharides contain complex, non-templated patterns of sulfation and epimerization, which mediate interactions with diverse protein partners. Complex HS modifications form around initial clusters of glucosamine-N-sulfate (GlcNS) on nascent polysaccharide chains, but the mechanistic basis underpinning incorporation of GlcNS itself into HS remains unclear. Here, we determine cryo-electron microscopy structures of human N-deacetylase-N-sulfotransferase (NDST)1, the bifunctional enzyme primarily responsible for initial GlcNS modification of HS. Our structures reveal the architecture of both NDST1 deacetylase and sulfotransferase catalytic domains, alongside a non-catalytic N-terminal domain. The two catalytic domains of NDST1 adopt a distinct back-to-back topology that limits direct cooperativity. Binding analyses, aided by activity-modulating nanobodies, suggest that anchoring of the substrate at the sulfotransferase domain initiates the NDST1 catalytic cycle, providing a plausible mechanism for cooperativity despite spatial domain separation. Our data shed light on key determinants of NDST1 activity, and describe tools to probe NDST1 function in vitro and in vivo.
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Heparitina Sulfato , Sulfotransferasas , Humanos , Microscopía por Crioelectrón , Heparitina Sulfato/metabolismo , Dominio Catalítico , Sulfotransferasas/metabolismo , Matriz Extracelular/metabolismoRESUMEN
Nanobodies are recombinant antigen-specific single domain antibodies (VHHs) derived from the heavy chain-only subset of camelid immunoglobulins. Their small molecular size, facile expression, high affinity, and stability have combined to make them unique targeting reagents with numerous applications in the biomedical sciences. From our work in producing nanobodies to over sixty different proteins, we present a standardised workflow for nanobody discovery from llama immunisation, library building, panning, and small-scale expression for prioritisation of binding clones. In addition, we introduce our suites of mammalian and bacterial vectors, which can be used to functionalise selected nanobodies for various applications such as in imaging and purification. Key features ⢠Standardise the process of building nanobody libraries and finding nanobody binders so that it can be repeated in any lab with reasonable equipment. ⢠Introduce two suites of vectors to functionalise nanobodies for production in either bacterial or mammalian cells.
RESUMEN
The third complementary-determining regions of the heavy-chain (CDR3H) variable regions (VH) of some cattle antibodies are highly extended, consisting of 48 or more residues. These `ultralong' CDR3Hs form ß-ribbon stalks that protrude from the surface of the antibody with a disulfide cross-linked knob region at their apex that dominates antigen interactions over the other CDR loops. The structure of the Fab fragment of a naturally paired bovine ultralong antibody (D08), identified by single B-cell sequencing, has been determined to 1.6â Å resolution. By swapping the D08 native light chain with that of an unrelated antigen-unknown ultralong antibody, it is shown that interactions between the CDR3s of the variable domains potentially affect the fine positioning of the ultralong CDR3H; however, comparison with other crystallographic structures shows that crystalline packing is also a major contributor. It is concluded that, on balance, the exact positioning of ultralong CDR3H loops is most likely to be due to the constraints of crystal packing.
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Regiones Determinantes de Complementariedad , Fragmentos Fab de Inmunoglobulinas , Cadenas Pesadas de Inmunoglobulina , Cadenas Ligeras de Inmunoglobulina , Modelos Moleculares , Animales , Bovinos , Cadenas Pesadas de Inmunoglobulina/química , Cristalografía por Rayos X , Cadenas Ligeras de Inmunoglobulina/química , Cadenas Ligeras de Inmunoglobulina/genética , Regiones Determinantes de Complementariedad/química , Fragmentos Fab de Inmunoglobulinas/química , Secuencia de Aminoácidos , Conformación ProteicaRESUMEN
Potassium channels of the Two-Pore Domain (K2P) subfamily, KCNK1-KCNK18, play crucial roles in controlling the electrical activity of many different cell types and represent attractive therapeutic targets. However, the identification of highly selective small molecule drugs against these channels has been challenging due to the high degree of structural and functional conservation that exists not only between K2P channels, but across the whole K+ channel superfamily. To address the issue of selectivity, here we generate camelid antibody fragments (nanobodies) against the TREK-2 (KCNK10) K2P K+ channel and identify selective binders including several that directly modulate channel activity. X-ray crystallography and CryoEM data of these nanobodies in complex with TREK-2 also reveal insights into their mechanisms of activation and inhibition via binding to the extracellular loops and Cap domain, as well as their suitability for immunodetection. These structures facilitate design of a biparatropic inhibitory nanobody with markedly improved sensitivity. Together, these results provide important insights into TREK channel gating and provide an alternative, more selective approach to modulation of K2P channel activity via their extracellular domains.
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Canales de Potasio de Dominio Poro en Tándem , Anticuerpos de Dominio Único , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Anticuerpos de Dominio Único/metabolismo , Anticuerpos de Dominio Único/inmunología , Anticuerpos de Dominio Único/química , Humanos , Cristalografía por Rayos X , Animales , Microscopía por Crioelectrón , Células HEK293 , Modelos MolecularesRESUMEN
Schistosomiasis is caused by parasites of the genus Schistosoma, which infect more than 200 million people. Praziquantel (PZQ) has been the main drug for controlling schistosomiasis for over four decades, but despite that it is ineffective against juvenile worms and size and taste issues with its pharmaceutical forms impose challenges for treating school-aged children. It is also important to note that PZQ resistant strains can be generated in laboratory conditions and observed in the field, hence its extensive use in mass drug administration programs raises concerns about resistance, highlighting the need to search for new schistosomicidal drugs. Schistosomes survival relies on the redox enzyme thioredoxin glutathione reductase (TGR), a validated target for the development of new anti-schistosomal drugs. Here we report a high-throughput fragment screening campaign of 768 compounds against S. mansoni TGR (SmTGR) using X-ray crystallography. We observed 49 binding events involving 35 distinct molecular fragments which were found to be distributed across 16 binding sites. Most sites are described for the first time within SmTGR, a noteworthy exception being the "doorstop pocket" near the NADPH binding site. We have compared results from hotspots and pocket druggability analysis of SmTGR with the experimental binding sites found in this work, with our results indicating only limited coincidence between experimental and computational results. Finally, we discuss that binding sites at the doorstop/NADPH binding site and in the SmTGR dimer interface, should be prioritized for developing SmTGR inhibitors as new antischistosomal drugs.
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Complejos Multienzimáticos , NADH NADPH Oxidorreductasas , Esquistosomiasis mansoni , Esquistosomiasis , Animales , Niño , Humanos , Schistosoma mansoni , Cristalografía por Rayos X , NADP/metabolismo , Esquistosomiasis/tratamiento farmacológico , Sitios de Unión , Esquistosomiasis mansoni/parasitologíaRESUMEN
The Omicron strains of SARS-CoV-2 pose a significant challenge to the development of effective antibody-based treatments as immune evasion has compromised most available immune therapeutics. Therefore, in the 'arms race' with the virus, there is a continuing need to identify new biologics for the prevention or treatment of SARS-CoV-2 infections. Here, we report the isolation of nanobodies that bind to the Omicron BA.1 spike protein by screening nanobody phage display libraries previously generated from llamas immunized with either the Wuhan or Beta spike proteins. The structure and binding properties of three of these nanobodies (A8, H6 and B5-5) have been characterized in detail providing insight into their binding epitopes on the Omicron spike protein. Trimeric versions of H6 and B5-5 neutralized the SARS-CoV-2 variant of concern BA.5 both in vitro and in the hamster model of COVID-19 following nasal administration. Thus, either alone or in combination could serve as starting points for the development of new anti-viral immunotherapeutics.