RESUMEN
The bacterial pathogen Francisella tularensis possesses a noncanonical type VI secretion system (T6SS) that is required for phagosomal escape in infected macrophages. KCl stimulation has been previously used to trigger assembly and secretion of the T6SS in culture. By differential proteomics, we found here that the amounts of the T6SS proteins remained unchanged upon KCl stimulation, suggesting involvement of post-translational modifications in T6SS assembly. A phosphoproteomic analysis indeed identified a unique phosphorylation site on IglB, a key component of the T6SS sheath. Substitutions of Y139 with alanine or phosphomimetics prevented T6SS formation and abolished phagosomal escape whereas substitution with phenylalanine delayed but did not abolish phagosomal escape in J774-1 macrophages. Altogether our data demonstrated that the Y139 site of IglB plays a critical role in T6SS biogenesis, suggesting that sheath phosphorylation could participate to T6SS dynamics.Data are available via ProteomeXchange with identifier PXD013619; and on MS-Viewer, key lkaqkllxwx.
Asunto(s)
Francisella tularensis/metabolismo , Sistemas de Secreción Tipo VI/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Línea Celular , Procesamiento Automatizado de Datos , Francisella tularensis/genética , Francisella tularensis/ultraestructura , Cromatografía de Gases y Espectrometría de Masas , Humanos , Macrófagos/microbiología , Estructura Molecular , Mutagénesis Sitio-Dirigida , Fosforilación , Cloruro de Potasio/farmacología , Procesamiento Proteico-Postraduccional , Proteómica , Espectrometría de Masas en Tándem , Sistemas de Secreción Tipo VI/química , Sistemas de Secreción Tipo VI/efectos de los fármacos , Sistemas de Secreción Tipo VI/genéticaRESUMEN
Norcoclaurine synthase (NCS) is a Pictet-Spenglerase that catalyzes the first key step in plant benzylisoquinoline alkaloid metabolism, a compound family that includes bioactive natural products such as morphine. The enzyme has also shown great potential as a biocatalyst for the formation of chiral isoquinolines. Here we present new high-resolution X-ray crystallography data describing Thalictrum flavum NCS bound to a mechanism-inspired ligand. The structure supports two key features of the NCS "dopamine-first" mechanism: the binding of dopamine catechol to Lys-122 and the position of the carbonyl substrate binding site at the active site entrance. The catalytically vital residue Glu-110 occupies a previously unobserved ligand-bound conformation that may be catalytically significant. The potential roles of inhibitory binding and alternative amino acid conformations in the mechanism have also been revealed. This work significantly advances our understanding of the NCS mechanism and will aid future efforts to engineer the substrate scope and catalytic properties of this useful biocatalyst.
Asunto(s)
Ligasas de Carbono-Nitrógeno/química , Ligasas de Carbono-Nitrógeno/metabolismo , Dopamina/metabolismo , Dominio Catalítico , Cristalografía por Rayos X , Modelos Moleculares , Thalictrum/enzimologíaRESUMEN
BACKGROUND: Stationary phase survival proteins (Sps) were found in Firmicutes as having analogous domain compositions, and in some cases genome context, as the resuscitation promoting factors of Actinobacteria, but with a different putative peptidoglycan cleaving domain. RESULTS: The first structure of a Firmicute Sps protein YuiC from B. subtilis, is found to be a stripped down version of the cell-wall peptidoglycan hydrolase MltA. The YuiC structures are of a domain swapped dimer, although some monomer is also found in solution. The protein crystallised in the presence of pentasaccharide shows a 1,6-anhydrodisaccharide sugar product, indicating that YuiC cleaves the sugar backbone to form an anhydro product at least on lengthy incubation during crystallisation. CONCLUSIONS: The structural simplification of MltA in Sps proteins is analogous to that of the resuscitation promoting factor domains of Actinobacteria, which are stripped down versions of lysozyme and soluble lytic transglycosylase proteins.
Asunto(s)
Fosfatasa Ácida/química , Bacillus subtilis/metabolismo , Proteínas Bacterianas/química , Bacillus subtilis/química , Dominio Catalítico , Cristalografía por Rayos X , Glicosiltransferasas/química , Modelos Moleculares , Oligosacáridos/metabolismo , Estructura Secundaria de ProteínaRESUMEN
BACKGROUND: Mutations in dysferlin, the first protein linked with the cell membrane repair mechanism, causes a group of muscular dystrophies called dysferlinopathies. Dysferlin is a type two-anchored membrane protein, with a single C terminal trans-membrane helix, and most of the protein lying in cytoplasm. Dysferlin contains several C2 domains and two DysF domains which are nested one inside the other. Many pathogenic point mutations fall in the DysF domain region. RESULTS: We describe the crystal structure of the human dysferlin inner DysF domain with a resolution of 1.9 Ångstroms. Most of the pathogenic mutations are part of aromatic/arginine stacks that hold the domain in a folded conformation. The high resolution of the structure show that these interactions are a mixture of parallel ring/guanadinium stacking, perpendicular H bond stacking and aliphatic chain packing. CONCLUSIONS: The high resolution structure of the Dysferlin DysF domain gives a template on which to interpret in detail the pathogenic mutations that lead to disease.
Asunto(s)
Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Proteínas Musculares/química , Proteínas Musculares/metabolismo , Arginina/metabolismo , Cristalografía por Rayos X , Disferlina , Humanos , Enlace de Hidrógeno , Modelos Moleculares , Distrofia Muscular de Cinturas/genética , Mutación Missense , Conformación Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Alineación de Secuencia , Triptófano/metabolismoRESUMEN
[This corrects the article DOI: 10.1371/journal.pone.0060143.].
RESUMEN
This paper presents the structure of MsAcg (MSMEG_5246), a Mycobacterium smegmatis homologue of Mycobacterium tuberculosis Acg (Rv2032) in its reduced form at 1.6 Å resolution using x-ray crystallography. Rv2032 is one of the most induced genes under the hypoxic model of tuberculosis dormancy. The Acg family turns out to be unusual flavin mononucleotide (FMN)-binding proteins that have probably arisen by gene duplication and fusion from a classical homodimeric nitroreductase such that the monomeric protein resembles a classical nitroreductase dimer but with one active site deleted and the other active site covered by a unique lid. The FMN cofactor is not reduced by either NADH or NADPH, but the chemically reduced enzyme is capable of reduction of nitro substrates, albeit at no kinetic advantage over free FMN. The reduced enzyme is rapidly oxidized by oxygen but without any evidence for a radical state commonly seen in oxygen-sensitive nitroreductases. The presence of the unique lid domain, the lack of reduction by NAD(P)H, and the slow rate of reaction of the chemically reduced protein raises a possible alternative function of Acg proteins in FMN storage or sequestration from other biochemical pathways as part of the bacteria's adaptation to a dormancy state.
Asunto(s)
Mycobacterium smegmatis/enzimología , Mycobacterium tuberculosis/enzimología , Nitrorreductasas/química , Sitios de Unión , Cristalografía por Rayos X , Mononucleótido de Flavina/metabolismo , Modelos Moleculares , Mycobacterium smegmatis/química , Mycobacterium smegmatis/genética , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/genética , NAD/metabolismo , NADP/metabolismo , Nitrorreductasas/genética , Nitrorreductasas/metabolismoRESUMEN
The Collaborative Computational Project No. 4 (CCP4) is a UK-led international collective with a mission to develop, test, distribute and promote software for macromolecular crystallography. The CCP4 suite is a multiplatform collection of programs brought together by familiar execution routines, a set of common libraries and graphical interfaces. The CCP4 suite has experienced several considerable changes since its last reference article, involving new infrastructure, original programs and graphical interfaces. This article, which is intended as a general literature citation for the use of the CCP4 software suite in structure determination, will guide the reader through such transformations, offering a general overview of the new features and outlining future developments. As such, it aims to highlight the individual programs that comprise the suite and to provide the latest references to them for perusal by crystallographers around the world.
Asunto(s)
Proteínas , Programas Informáticos , Proteínas/química , Cristalografía por Rayos X , Sustancias MacromolecularesRESUMEN
Tetrahydroprotoberberine and protoberberine alkaloids are a group of biologically active natural products with complex molecular scaffolds. Isolation from plants is challenging and stereoselective synthetic routes, particularly of methylated compounds are limited, reducing the potential use of these compounds. In this work, we describe chemoenzymatic cascades toward various 13-methyl-tetrahydroprotoberberbine scaffolds using a stereoselective Pictet-Spenglerase, regioselective catechol O-methyltransferases and selective chemical Pictet-Spengler reactions. All reactions could be performed sequentially, without the workup or purification of any synthetic intermediates. Moreover, the naturally occurring alkaloids have the (+)-configuration and importantly here, a strategy to the (-)-isomers was developed. A methyl group at C-8 was also introduced with some stereocontrol, influenced by the stereochemistry at C-13. Furthermore, a single step reaction was found to convert tetrahydroprotoberberine alkaloids into the analogous protoberberine scaffold, avoiding the use of harsh oxidizing conditions or a selective oxidase. This work provides facile, selective routes toward novel analogues of bioactive alkaloids.
Asunto(s)
Alcaloides/química , Alcaloides de Berberina/farmacología , Alcaloides/aislamiento & purificación , Alcaloides de Berberina/química , Alcaloides de Berberina/aislamiento & purificación , Productos Biológicos , Estructura MolecularRESUMEN
OBJECTIVES: Identification and validation of novel therapeutic targets is imperative to tackle the rise of drug resistance in tuberculosis. An essential Mur ligase-like gene (Rv3712), expected to be involved in cell-wall peptidoglycan (PG) biogenesis and conserved across mycobacteria, including the genetically depleted Mycobacterium leprae, was the primary focus of this study. METHODS: Biochemical analysis of Rv3712 was performed using inorganic phosphate release assays. The operon structure was identified using reverse-transcriptase PCR and a transcription/translation fusion vector. In vivo mycobacterial protein fragment complementation assays helped generate the interactome. RESULTS: Rv3712 was found to be an ATPase. Characterization of its operon revealed a mycobacteria-specific promoter driving the co-transcription of Rv3712 and Rv3713. The two gene products were found to interact with each other in vivo. Sequence-based functional assignments reveal that Rv3712 and Rv3713 are likely to be the mycobacterial PG precursor-modifying enzymes MurT and GatD, respectively. An in vivo network involving Mtb-MurT, regulatory proteins and cell division proteins was also identified. CONCLUSIONS: Understanding the role of the enzyme complex in the context of PG metabolism and cell division, and the implications for antimicrobial resistance and host immune responses will facilitate the design of therapeutics that are targeted specifically to M. tuberculosis.
RESUMEN
Resuscitation-promoting factor (RPF) proteins reactivate stationary-phase cultures of (G+C)-rich Gram-positive bacteria including the causative agent of tuberculosis, Mycobacterium tuberculosis. We report the solution structure of the RPF domain from M. tuberculosis Rv1009 (RpfB) solved by heteronuclear multidimensional NMR. Structural homology with various glycoside hydrolases suggested that RpfB cleaved oligosaccharides. Biochemical studies indicate that a conserved active site glutamate is important for resuscitation activity. These data, as well as the presence of a clear binding pocket for a large molecule, indicate that oligosaccharide cleavage is probably the signal for revival from dormancy.
Asunto(s)
Proteínas Bacterianas/química , Citocinas/química , Glicósido Hidrolasas/química , Mycobacterium tuberculosis/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/fisiología , Sitios de Unión , Citocinas/metabolismo , Citocinas/fisiología , Ácido Glutámico/química , Espectroscopía de Resonancia Magnética , Muramidasa/química , Estructura Terciaria de Proteína , Soluciones/química , Homología Estructural de Proteína , Trisacáridos/química , Trisacáridos/metabolismoRESUMEN
Pictet-Spenglerases provide a key role in the biosynthesis of many biologically active alkaloids. There is increasing use of these biocatalysts as an alternative to traditional organic synthetic methods as they provide stereoselective and regioselective control under mild conditions. Products from these enzymes also contain privileged drug scaffolds (such as tetrahydroisoquinoline or ß-carboline moieties), so there is interest in the characterization and use of these enzymes as versatile biocatalysts to synthesize analogs of the corresponding natural products for drug discovery. This review discusses all known Pictet-Spenglerase enzymes and their applications as biocatalysts.
Asunto(s)
Alcaloides/biosíntesis , Enzimas/metabolismo , Aldehídos/química , Bacterias/enzimología , Biocatálisis , Productos Biológicos/química , Carbolinas/química , Ciclización , Descubrimiento de Drogas , Etilaminas/química , Humanos , Cetonas/química , Conformación Molecular , Plantas/enzimología , Estereoisomerismo , Tetrahidroisoquinolinas/químicaRESUMEN
The 1-aryl-tetrahydroisoquinoline (1-aryl-THIQ) moiety is found in many biologically active molecules. Single enantiomer chemical syntheses are challenging and although some biocatalytic routes have been reported, the substrate scope is limited to certain structural motifs. The enzyme norcoclaurine synthase (NCS), involved in plant alkaloid biosynthesis, has been shown to perform stereoselective Pictet-Spengler reactions between dopamine and several carbonyl substrates. Here, benzaldehydes are explored as substrates and found to be accepted by both wild-type and mutant constructs of NCS. In particular, the variant M97V gives a range of (1 S)-aryl-THIQs in high yields (48-99%) and e.e.s (79-95%). A co-crystallised structure of the M97V variant with an active site reaction intermediate analogue is also obtained with the ligand in a pre-cyclisation conformation, consistent with (1 S)-THIQs formation. Selected THIQs are then used with catechol O-methyltransferases with exceptional regioselectivity. This work demonstrates valuable biocatalytic approaches to a range of (1 S)-THIQs.
RESUMEN
ROCK-I (Rho-associated kinase 1) is a serine/threonine kinase that can be activated by RhoA and inhibited by RhoE. ROCK-I has an N-terminal kinase domain, a central coiled-coil region and a RhoA-binding domain near the C-terminus. We have previously shown that RhoE binds to the N-terminal 420 amino acids of ROCK-I, which includes the kinase domain as well as N-terminal and C-terminal extensions. In the present study, we show that N-terminus-mediated dimerization of ROCK-I is required for RhoE binding. The central coiled-coil domain can also dimerize ROCK-I in cells, but this is insufficient in the absence of the N-terminus to allow RhoE binding. The kinase activity of ROCK-I(1-420) is required for dimerization and RhoE binding; however, inclusion of part of the coiled-coil domain compensates for lack of kinase activity, allowing RhoE to bind. N-terminus-mediated dimerization is also required for ROCK-I to induce the formation of stellate actin stress fibres in cells. These results indicate that dimerization via the N-terminus is critical for ROCK-I function in cells and for its regulation by RhoE.
Asunto(s)
Actinas/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Quinasas Asociadas a rho/química , Quinasas Asociadas a rho/metabolismo , Animales , Células COS , Chlorocebus aethiops , Citoesqueleto/metabolismo , Dimerización , Fosforilación , Unión Proteica , Quinasas Asociadas a rho/genéticaRESUMEN
The novel bacterial cytokine family--resuscitation-promoting factors (Rpfs)--share a conserved domain of uncharacterized function. Predicting the structure of this domain suggests that Rpfs possess a lysozyme-like domain. The model highlights the good conservation of residues involved in catalysis and substrate binding. A lysozyme-like function makes sense for this domain in the light of experimental characterization of the biological function of Rpfs.
Asunto(s)
Proteínas Bacterianas , Citocinas/química , Citocinas/genética , Muramidasa/genética , Secuencia de Aminoácidos , Animales , Modelos Moleculares , Datos de Secuencia Molecular , Muramidasa/química , Estructura Terciaria de Proteína , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
Tuberculosis (TB), caused by the intracellular pathogen Mycobacterium tuberculosis, remains one of the leading causes of mortality across the world. There is an urgent requirement to build a robust arsenal of effective antimicrobials, targeting novel molecular mechanisms to overcome the challenges posed by the increase of antibiotic resistance in TB. Mycobacterium tuberculosis has a unique cell envelope structure and composition, containing a peptidoglycan layer that is essential for maintaining cellular integrity and for virulence. The enzymes involved in the biosynthesis, degradation, remodelling and recycling of peptidoglycan have resurfaced as attractive targets for anti-infective drug discovery. Here, we review the importance of peptidoglycan, including the structure, function and regulation of key enzymes involved in its metabolism. We also discuss known inhibitors of ATP-dependent Mur ligases, and discuss the potential for the development of pan-enzyme inhibitors targeting multiple Mur ligases.
Asunto(s)
Pared Celular/química , Mycobacterium tuberculosis/enzimología , Peptidoglicano/química , Tuberculosis/microbiología , Animales , Antituberculosos , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/metabolismo , Vías Biosintéticas/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Humanos , Ligasas/genética , Ligasas/metabolismo , Ratones , Mycobacterium tuberculosis/efectos de los fármacos , VirulenciaRESUMEN
Heterogeneity in small heat shock proteins (sHsps) spans multiple spatiotemporal regimes-from fast fluctuations of part of the protein, to conformational variability of tertiary structure, plasticity of the interfaces, and polydispersity of the inter-converting, and co-assembling oligomers. This heterogeneity and dynamic nature of sHsps has significantly hindered their structural characterization. Atomic coordinates are particularly lacking for vertebrate sHsps, where most available structures are of extensively truncated homomers. sHsps play important roles in maintaining protein levels in the cell and therefore in organismal health and disease. HspB2 and HspB3 are vertebrate sHsps that are found co-assembled in neuromuscular cells, and variants thereof are associated with disease. Here, we present the structure of human HspB2/B3, which crystallized as a hetero-tetramer in a 3:1 ratio. In the HspB2/B3 tetramer, the four α-crystallin domains (ACDs) assemble into a flattened tetrahedron which is pierced by two non-intersecting approximate dyads. Assembly is mediated by flexible "nuts and bolts" involving IXI/V motifs from terminal regions filling ACD pockets. Parts of the N-terminal region bind in an unfolded conformation into the anti-parallel shared ACD dimer grooves. Tracts of the terminal regions are not resolved, most likely due to their disorder in the crystal lattice. This first structure of a full-length human sHsp heteromer reveals the heterogeneous interactions of the terminal regions and suggests a plasticity that is important for the cytoprotective functions of sHsps.
Asunto(s)
Proteínas de Choque Térmico HSP27/química , Proteínas de Choque Térmico/química , Modelos Moleculares , Conformación Proteica , Multimerización de Proteína , Secuencia de Aminoácidos , Proteínas de Choque Térmico HSP27/genética , Proteínas de Choque Térmico HSP27/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Espectroscopía de Resonancia Magnética , Unión Proteica , Dominios y Motivos de Interacción de ProteínasRESUMEN
When stressed, bacteria can enter various non-dividing states, which are medically important. For example, dormancy is used by Mycobacterium tuberculosis to evade host responses. A major breakthrough has been the discovery of resuscitation-promoting factor (Rpf) from Micrococcus luteus, which is an extremely potent anti-dormancy factor. Mycobacteria have multiple proteins that contain this domain. Surprisingly, the highly conserved resuscitation-promoting factor domain has strong structural similarities to lysozyme and soluble lytic transglycosylases, and it has been demonstrated that resuscitation-promoting factors cleave peptidoglycan. This suggests that the activation of dormant cells requires peptidoglycan hydrolysis, which either alters the mechanical properties of the cell wall to facilitate cell division or releases lysis products that function as anti-dormancy signals.
Asunto(s)
Proteínas Bacterianas/metabolismo , Citocinas/metabolismo , Viabilidad Microbiana , Peptidoglicano/metabolismo , Proteínas Bacterianas/química , Citocinas/química , N-Acetil Muramoil-L-Alanina Amidasa/metabolismo , Peptidoglicano Glicosiltransferasa/metabolismo , Esporas Bacterianas/metabolismoRESUMEN
Aphids use chemical cues to locate hosts and find mates. The vetch aphid Megoura viciae feeds exclusively on the Fabaceae, whereas the currant-lettuce aphid Nasonovia ribisnigri alternates hosts between the Grossulariaceae and Asteraceae. Both species use alarm pheromones to warn of dangers. For N. ribisnigri this pheromone is a single component (E)-ß-farnesene but M. viciae uses a mixture of (E)-ß-farnesene, (-)-α-pinene, ß-pinene, and limonene. Odorant-binding proteins (OBP) are believed to capture and transport such semiochemicals to their receptors. Here, we report the first aphid OBP crystal structures and examine their molecular interactions with the alarm pheromone components. Our study reveals some unique structural features: 1) the lack of an internal ligand binding site; 2) a striking groove in the surface of the proteins as a putative binding site; 3) the N-terminus rather than the C-terminus occupies the site closing off the conventional OBP pocket. The results from fluorescent binding assays, molecular docking and dynamics demonstrate that OBP3 from M. viciae can bind to all four alarm pheromone components and the differential ligand binding between these very similar OBP3s from the two aphid species is determined mainly by the direct π-π interactions between ligands and the aromatic residues of OBP3s in the binding pocket.
Asunto(s)
Áfidos , Proteínas de Insectos/química , Proteínas de Insectos/metabolismo , Feromonas/metabolismo , Receptores Odorantes/química , Receptores Odorantes/metabolismo , Animales , Fluorometría , Cinética , Modelos Moleculares , Simulación del Acoplamiento Molecular , Unión Proteica , Conformación ProteicaRESUMEN
The opposing activities of 53BP1 and BRCA1 influence pathway choice in DNA double-strand-break repair. How BRCA1 counteracts the inhibitory effect of 53BP1 on DNA resection and homologous recombination is unknown. Here we identify the site of BRCA1-BARD1 required for priming ubiquitin transfer from E2â¼ubiquitin and demonstrate that BRCA1-BARD1's ubiquitin ligase activity is required for repositioning 53BP1 on damaged chromatin. We confirm H2A ubiquitination by BRCA1-BARD1 and show that an H2A-ubiquitin fusion protein promotes DNA resection and repair in BARD1-deficient cells. BRCA1-BARD1's function in homologous recombination requires the chromatin remodeler SMARCAD1. SMARCAD1 binding to H2A-ubiquitin and optimal localization to sites of damage and activity in DNA repair requires its ubiquitin-binding CUE domains. SMARCAD1 is required for 53BP1 repositioning, and the need for SMARCAD1 in olaparib or camptothecin resistance is alleviated by 53BP1 loss. Thus, BRCA1-BARD1 ligase activity and subsequent SMARCAD1-dependent chromatin remodeling are critical regulators of DNA repair.