RESUMEN
The bacterial flagellar motor is a supramolecular protein machine that drives rotation of the flagellum for motility, which is essential for bacterial survival in different environments and a key determinant of pathogenicity. The detailed structure of the flagellar motor remains unknown. Here we present an atomic-resolution cryoelectron microscopy (cryo-EM) structure of the bacterial flagellar motor complexed with the hook, consisting of 175 subunits with a molecular mass of approximately 6.3 MDa. The structure reveals that 10 peptides protruding from the MS ring with the FlgB and FliE subunits mediate torque transmission from the MS ring to the rod and overcome the symmetry mismatch between the rotational and helical structures in the motor. The LP ring contacts the distal rod and applies electrostatic forces to support its rotation and torque transmission to the hook. This work provides detailed molecular insights into the structure, assembly, and torque transmission mechanisms of the flagellar motor.
Asunto(s)
Flagelos/fisiología , Flagelos/ultraestructura , Salmonella typhimurium/fisiología , Microscopía por Crioelectrón , Conformación Proteica , TorqueRESUMEN
AMPylation is a post-translational modification in which AMP is added to the amino acid side chains of proteins1,2. Here we show that, with ATP as the ligand and actin as the host activator, the effector protein LnaB of Legionella pneumophila exhibits AMPylase activity towards the phosphoryl group of phosphoribose on PRR42-Ub that is generated by the SidE family of effectors, and deubiquitinases DupA and DupB in an E1- and E2-independent ubiquitination process3-7. The product of LnaB is further hydrolysed by an ADP-ribosylhydrolase, MavL, to Ub, thereby preventing the accumulation of PRR42-Ub and ADPRR42-Ub and protecting canonical ubiquitination in host cells. LnaB represents a large family of AMPylases that adopt a common structural fold, distinct from those of the previously known AMPylases, and LnaB homologues are found in more than 20 species of bacterial pathogens. Moreover, LnaB also exhibits robust phosphoryl AMPylase activity towards phosphorylated residues and produces unique ADPylation modifications in proteins. During infection, LnaB AMPylates the conserved phosphorylated tyrosine residues in the activation loop of the Src family of kinases8,9, which dampens downstream phosphorylation signalling in the host. Structural studies reveal the actin-dependent activation and catalytic mechanisms of the LnaB family of AMPylases. This study identifies, to our knowledge, an unprecedented molecular regulation mechanism in bacterial pathogenesis and protein phosphorylation.
Asunto(s)
Adenosina Monofosfato , Proteínas Bacterianas , Legionella pneumophila , Fosfotirosina , Transducción de Señal , Humanos , Actinas/metabolismo , Adenosina Monofosfato/metabolismo , Adenosina Trifosfato/metabolismo , ADP-Ribosilación , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Hidrólisis , Legionella pneumophila/enzimología , Legionella pneumophila/metabolismo , Legionella pneumophila/patogenicidad , Ligandos , Modelos Moleculares , N-Glicosil Hidrolasas/metabolismo , Fosforilación , Procesamiento Proteico-Postraduccional , Tirosina/metabolismo , Tirosina/química , Ubiquitina/metabolismo , Ubiquitinación , Enzimas Desubicuitinizantes/metabolismo , Pliegue de Proteína , Fosfotirosina/química , Fosfotirosina/metabolismoRESUMEN
Ubiquitination is essential for numerous eukaryotic cellular processes. Here, we show that the type III effector CteC from Chromobacterium violaceum functions as an adenosine diphosphate (ADP)-ribosyltransferase that specifically modifies ubiquitin via threonine ADP-ribosylation on residue T66. The covalent modification prevents the transfer of ubiquitin from ubiquitin-activating enzyme E1 to ubiquitin-conjugating enzyme E2, which inhibits subsequent ubiquitin activation by E2 and E3 enzymes in the ubiquitination cascade and leads to the shutdown of polyubiquitin synthesis in host cells. This unique modification also causes dysfunction of polyubiquitin chains in cells, thereby blocking host ubiquitin signaling. The disruption of host ubiquitination by CteC plays a crucial role in C. violaceum colonization in mice during infection. CteC represents a family of effector proteins in pathogens of hosts from different kingdoms. All the members of this family specifically ADP-ribosylate ubiquitin. The action of CteC reveals a new mechanism for interfering with host ubiquitination by pathogens.
Asunto(s)
ADP-Ribosilación , Proteínas Bacterianas/metabolismo , Chromobacterium/metabolismo , Poliubiquitina/metabolismo , Treonina/metabolismo , Enzimas Activadoras de Ubiquitina/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Animales , Proteínas Bacterianas/genética , Chromobacterium/genética , Femenino , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Procesamiento Proteico-Postraduccional , Treonina/genética , Enzimas Activadoras de Ubiquitina/genética , Enzimas Ubiquitina-Conjugadoras/genética , UbiquitinaciónRESUMEN
Rab GTPases are frequent targets of vacuole-living bacterial pathogens for appropriate trafficking of the vacuole. Here we discover that bacterial effectors including VirA from nonvacuole Shigella flexneri and EspG from extracellular Enteropathogenic Escherichia coli (EPEC) harbor TBC-like dual-finger motifs and exhibits potent RabGAP activities. Specific inactivation of Rab1 by VirA/EspG disrupts ER-to-Golgi trafficking. S. flexneri intracellular persistence requires VirA TBC-like GAP activity that mediates bacterial escape from autophagy-mediated host defense. Rab1 inactivation by EspG severely blocks host secretory pathway, resulting in inhibited interleukin-8 secretion from infected cells. Crystal structures of VirA/EspG-Rab1-GDP-aluminum fluoride complexes highlight TBC-like catalytic role for the arginine and glutamine finger residues and reveal a 3D architecture distinct from that of the TBC domain. Structure of Arf6-EspG-Rab1 ternary complex illustrates a pathogenic signaling complex that rewires host Arf signaling to Rab1 inactivation. Structural distinctions of VirA/EspG further predict a possible extensive presence of TBC-like RabGAP effectors in counteracting various host defenses.
Asunto(s)
Factores de Ribosilacion-ADP/metabolismo , Escherichia coli Enteropatógena/patogenicidad , Proteínas de Escherichia coli/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Shigella flexneri/patogenicidad , Factores de Virulencia/metabolismo , Secuencia de Aminoácidos , Animales , Autofagia , Disentería Bacilar/inmunología , Disentería Bacilar/microbiología , Escherichia coli Enteropatógena/metabolismo , Infecciones por Escherichia coli/inmunología , Infecciones por Escherichia coli/microbiología , Proteínas de Escherichia coli/química , Fibroblastos/metabolismo , Interleucina-8/inmunología , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Alineación de Secuencia , Shigella flexneri/metabolismo , Virulencia , Factores de Virulencia/químicaRESUMEN
Ubiquitination plays essential roles in eukaryotic cellular processes. The effector protein CteC from Chromobacterium violaceum blocks host ubiquitination by mono-ADP-ribosylation of ubiquitin (Ub) at residue T66. However, the structural basis for this modification is unknown. Here we report three crystal structures of CteC in complexes with Ub, NAD+ or ADP-ribosylated Ub, which represent different catalytic states of CteC in the modification. CteC adopts a special 'D-E' catalytic motif for catalysis and binds NAD+ in a half-ligand binding mode. The specific recognition of Ub by CteC is determined by a relatively separate Ub-targeting domain and a long loop L6, not the classic ADP-ribosylating turn-turn loop. Structural analyses with biochemical results reveal that CteC represents a large family of poly (ADP-ribose) polymerase (PARP)-like ADP-ribosyltransferases, which harbors chimeric features from the R-S-E and H-Y-E classes of ADP-ribosyltransferases. The family of CteC-like ADP-ribosyltransferases has a common 'D-E' catalytic consensus and exists extensively in bacteria and eukaryotic microorganisms.
Asunto(s)
Treonina , Ubiquitina , Ubiquitina/química , Treonina/metabolismo , NAD/metabolismo , ADP-Ribosilación , ADP Ribosa Transferasas/química , Poli(ADP-Ribosa) Polimerasas/química , Bacterias/metabolismo , Adenosina Difosfato RibosaRESUMEN
Archaeal ribosomes have many domain-specific features; however, our understanding of these structures is limited. We present 10 cryo-electron microscopy (cryo-EM) structures of the archaeal ribosome from crenarchaeota Sulfolobus acidocaldarius (Sac) at 2.7-5.7 Å resolution. We observed unstable conformations of H68 and h44 of ribosomal RNA (rRNA) in the subunit structures, which may interfere with subunit association. These subunit structures provided models for 12 rRNA expansion segments and 3 novel r-proteins. Furthermore, the 50S-aRF1 complex structure showed the unique domain orientation of aRF1, possibly explaining P-site transfer RNA (tRNA) release after translation termination. Sac 70S complexes were captured in seven distinct steps of the tRNA translocation reaction, confirming conserved structural features during archaeal ribosome translocation. In aEF2-engaged 70S ribosome complexes, 3D classification of cryo-EM data based on 30S head domain identified two new translocation intermediates with 30S head domain tilted 5-6° enabling its disengagement from the translocated tRNA and its release post-translocation. Additionally, we observed conformational changes to aEF2 during ribosome binding and switching from three different states. Our structural and biochemical data provide new insights into archaeal translation and ribosome translocation.
Archaeal ribosomes display variations in their ribosomal proteins and ribosomal RNA (rRNA) expansion segments (ESs). Protein translation in archaea combines features in both bacterial and eukaryotic translation. In this study, we present 10 cryo-electron microscopy structures of the archaeal ribosome from crenarchaeota Sulfolobus acidocaldarius (Sac). The 50S and 30S subunit structures present 3 novel ribosomal proteins and 12 rRNA ESs. The 70S Sac ribosome structures were captured in seven distinct functional states, including pre-, intermediate- and post-translocation states. Specifically, we identified two novel translocation intermediates, in which the 30S subunit head domain tilts outward to release the translocated P-site transfer RNA. The structures of archaeal ribosomes provide insights into the archaeal translation and ribosome translocation.
Asunto(s)
Ribosomas , Sulfolobus acidocaldarius , Microscopía por Crioelectrón , Proteínas Ribosómicas/metabolismo , Ribosomas/metabolismo , ARN Ribosómico/metabolismo , ARN de Transferencia/metabolismo , Sulfolobus acidocaldarius/citología , Sulfolobus acidocaldarius/metabolismoRESUMEN
The effector MavC of the bacterial pathogen Legionella pneumophila catalyzes a noncanonical ubiquitination of the host ubiquitin-conjugating E2 enzyme UBE2N by crosslinking a glutamine residue of ubiquitin to UBE2N lysine residues via its transglutaminase activity. A new study by Gan et al (2020) reveals that L. pneumophila reverses this noncanonical ubiquitination via its ubiquitin deamidase effector MvcA to allow precise temporal regulation of host signaling during infection.
Asunto(s)
Legionella pneumophila , Ubiquitina , Enzimas Ubiquitina-Conjugadoras , UbiquitinaciónRESUMEN
NAC is one of the largest family of plant-specific transcription factors, and it plays important roles in plant development and stress responses. The study identified 72 LpNACs genes from the perennial ryegrass genome database. Gene length, MW and pI of NAC family transcription factors varied, but the gene structure and motifs were relatively conserved in bioinformatics analysis. Phylogenetic analyses of perennial ryegrass, rice and Arabidopsis were performed to study the evolutionary and functional relationships in various species. The expression of LpNAC genes that respond to various abiotic stresses including high salinity, ABA, high temperature, polyethylene glycol (PEG) and heavy metal was comprehensively analyzed. The present study provides a basic understanding of the NAC gene family in perennial ryegrass for further abiotic stress studies and improvements in breeding.
Asunto(s)
Lolium/genética , Familia de Multigenes , Proteínas de Plantas/genética , Estrés Fisiológico/genética , Factores de Transcripción/genética , Secuencias de Aminoácidos , Genes de Plantas , Genoma de Planta , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/clasificación , Proteínas de Plantas/metabolismo , Alineación de Secuencia , Factores de Transcripción/química , Factores de Transcripción/clasificación , Factores de Transcripción/metabolismo , TranscriptomaRESUMEN
Chitosan (CTS) is a deacetylated derivative of chitin that is involved in adaptive response to abiotic stresses. However, the regulatory role of CTS in heat tolerance is still not fully understood in plants, especially in grass species. The aim of this study was to investigate whether the CTS could reduce heat-induced senescence and damage to creeping bentgrass associated with alterations in antioxidant defense, chlorophyll (Chl) metabolism, and the heat shock pathway. Plants were pretreated exogenously with or without CTS (0.1 g L-1) before being exposed to normal (23/18 °C) or high-temperature (38/33 °C) conditions for 15 days. Heat stress induced detrimental effects, including declines in leaf relative water content and photochemical efficiency, but significantly increased reactive oxygen species (ROS) accumulation, membrane lipid peroxidation, and Chl loss in leaves. The exogenous application of CTS significantly alleviated heat-induced damage in creeping bentgrass leaves by ameliorating water balance, ROS scavenging, the maintenance of Chl metabolism, and photosynthesis. Compared to untreated plants under heat stress, CTS-treated creeping bentgrass exhibited a significantly higher transcription level of genes involved in Chl biosynthesis (AsPBGD and AsCHLH), as well as a lower expression level of Chl degradation-related gene (AsPPH) and senescence-associated genes (AsSAG12, AsSAG39, Asl20, and Ash36), thus reducing leaf senescence and enhancing photosynthetic performance under heat stress. In addition, the foliar application of CTS significantly improved antioxidant enzyme activities (SOD, CAT, POD, and APX), thereby effectively reducing heat-induced oxidative damage. Furthermore, heat tolerance regulated by the CTS in creeping bentgrass was also associated with the heat shock pathway, since AsHSFA-6a and AsHSP82 were significantly up-regulated by the CTS during heat stress. The potential mechanisms of CTS-regulated thermotolerance associated with other metabolic pathways still need to be further studied in grass species.
Asunto(s)
Agrostis/efectos de los fármacos , Antioxidantes/farmacología , Quitosano/farmacología , Clorofila/metabolismo , Calor , Hojas de la Planta/efectos de los fármacos , Agrostis/metabolismo , Respuesta al Choque Térmico/efectos de los fármacos , Hojas de la Planta/metabolismoRESUMEN
The type IVb secretion system (T4BSS) of Legionella pneumophila is a multiple-component apparatus that delivers â¼300 virulent effector proteins into host cells. The injected effectors modulate host cellular processes to promote bacterial infection and proliferation. IcmS and IcmW are two conserved small, acidic adaptor proteins that form a binary complex to interact with many effectors and facilitate their translocation. IcmS and IcmW can also interact with DotL, an ATPase of the type IV coupling protein complex (T4CP). However, how IcmS-IcmW recognizes effectors, and what the roles of IcmS-IcmW are in T4BSSs are unclear. In this study, we found that IcmS and IcmW form a 1:1 heterodimeric complex to bind effector substrates. Both IcmS and IcmW adopt new structural folds and have no structural similarities with known effector chaperones. IcmS has a compact global structure with an α/ß fold, while IcmW adopts a fully α-folded, relatively loose architecture. IcmS stabilizes IcmW by binding to its two C-terminal α-helices. Photocrosslinking assays revealed that the IcmS-IcmW complex binds its cognate effectors via an extended hydrophobic surface, which can also interact with the C terminus of DotL. A crystal structure of the DotL-IcmS-IcmW complex reveals extensive and highly stable interactions between DotL and IcmS-IcmW. Moreover, IcmS-IcmW recruits LvgA to DotL and assembles a unique T4CP. These data suggest that IcmS-IcmW also functions as an inseparable integral component of the DotL-T4CP complex in the bacterial inner membrane. This study provides molecular insights into the dual roles of the IcmS-IcmW complex in T4BSSs.
Asunto(s)
Chaperonas Moleculares/química , Sistemas de Secreción Tipo IV/química , Proteínas Bacterianas/metabolismo , Sitios de Unión , Legionella pneumophila/química , Legionella pneumophila/metabolismo , Chaperonas Moleculares/metabolismo , Unión Proteica , Sistemas de Secreción Tipo IV/metabolismo , Factores de Virulencia/metabolismoRESUMEN
The common vetch (Vicia sativa) is often used as feed for livestock because of its high nutritional value. However, drought stress reduces forage production through plant damage. Here, we studied the transcriptional profiles of common vetch exposed to drought in order to understand the molecular mechanisms of drought tolerance in this species. The genome of the common vetch has not been sequenced, therefore we used Illumina sequencing to generate de novo transcriptomes. Nearly 500 million clean reads were used to generate 174,636 transcripts, including 122,299 unigenes. In addition, 5313 transcription factors were identified and these transcription factors were classified into 79 different gene families. We also identified 11,181 SSR loci from di- to hexa-nucleotides whose repeat number was greater than five. On the basis of differentially expressed genes, Gene Ontology analysis identified many drought-relevant categories, including "oxidation-reduction process", "lipid metabolic process" and "oxidoreductase activity". In addition to these, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis identified pathways, such as "Plant hormone signal transduction", "Glycolysis/Gluconeogenesis" and "Phenylpropanoid biosynthesis", as differentially expressed in the plants exposed to drought. The expression results in this study will be useful for further extending our knowledge on the drought tolerance of common vetch.
Asunto(s)
Adaptación Fisiológica/genética , Sequías , Genes de Plantas , Estudios de Asociación Genética , Análisis de Secuencia de ADN/métodos , Vicia sativa/genética , Vicia sativa/fisiología , Análisis por Conglomerados , Regulación de la Expresión Génica de las Plantas , Ontología de Genes , Repeticiones de Microsatélite/genética , Anotación de Secuencia Molecular , Reproducibilidad de los Resultados , Estrés Fisiológico/genética , Transcriptoma/genéticaRESUMEN
Macrophages represent one of the first lines of host immune defenses against the invasion of pathogenic bacteria. Many receptors, immune signaling pathways and cellular processes in macrophages, including Toll-like receptors, Nod-like receptors, phagocytosis, autophagy and programmed cell death, are involved in combating the infection of bacterial pathogens. For efficient colonization in the host, bacterial pathogens have evolved diverse mechanisms to interfere with macrophage functions to evade host defenses. The major weapons utilized by bacterial pathogens are protein toxins and effectors secreted via specific bacterial secretion systems, including type I-VII secretion apparatuses. In recent years, great advances have been achieved in understanding how bacterial toxins and effectors subvert immune signaling and cellular processes of macrophages. In this review, we focus on the toxins and effectors that modulate the phagocytosis, intracellular immune signaling pathways, autophagy and programmed cell death processes of macrophages from the bacterium Legionella pneumophila, Shigella flexneri, Listeria monocytogenes, Salmonella spp., Yersinia spp., enteropathogenic E. coli and Mycobacterium tuberculosis.
Asunto(s)
Sistemas de Secreción Bacterianos/inmunología , Toxinas Bacterianas/biosíntesis , Bacterias Gramnegativas/inmunología , Bacterias Grampositivas/inmunología , Evasión Inmune , Macrófagos/inmunología , Animales , Apoptosis , Autofagia , Sistemas de Secreción Bacterianos/genética , Toxinas Bacterianas/genética , Regulación de la Expresión Génica , Bacterias Gramnegativas/crecimiento & desarrollo , Bacterias Grampositivas/crecimiento & desarrollo , Humanos , Inmunidad Innata , Macrófagos/microbiología , Macrófagos/patología , Proteínas NLR/genética , Proteínas NLR/inmunología , Fagocitosis , Transducción de Señal , Receptores Toll-Like/genética , Receptores Toll-Like/inmunologíaRESUMEN
Nipah virus (NiV) is a paramyxovirus that infects host cells through the coordinated efforts of two envelope glycoproteins. The G glycoprotein attaches to cell receptors, triggering the fusion (F) glycoprotein to execute membrane fusion. Here we report the first crystal structure of the pre-fusion form of the NiV-F glycoprotein ectodomain. Interestingly this structure also revealed a hexamer-of-trimers encircling a central axis. Electron tomography of Nipah virus-like particles supported the hexameric pre-fusion model, and biochemical analyses supported the hexamer-of-trimers F assembly in solution. Importantly, structure-assisted site-directed mutagenesis of the interfaces between F trimers highlighted the functional relevance of the hexameric assembly. Shown here, in both cell-cell fusion and virus-cell fusion systems, our results suggested that this hexamer-of-trimers assembly was important during fusion pore formation. We propose that this assembly would stabilize the pre-fusion F conformation prior to cell attachment and facilitate the coordinated transition to a post-fusion conformation of all six F trimers upon triggering of a single trimer. Together, our data reveal a novel and functional pre-fusion architecture of a paramyxoviral fusion glycoprotein.
Asunto(s)
Infecciones por Henipavirus/metabolismo , Virus Nipah/química , Proteínas del Envoltorio Viral/química , Internalización del Virus , Cristalografía por Rayos X , Electroforesis en Gel de Poliacrilamida , Células HEK293 , Humanos , Mutagénesis Sitio-Dirigida , Virus Nipah/metabolismo , Conformación Proteica , Proteínas del Envoltorio Viral/metabolismoRESUMEN
Leucine-rich repeat G-protein-coupled receptors (LGRs) are a unique class of G-protein-coupled receptors characterized by a large extracellular domain to recognize ligands and regulate many important developmental processes. Among the three groups of LGRs, group B members (LGR4-6) recognize R-spondin family proteins (Rspo1-4) to stimulate Wnt signaling. In this study, we successfully utilized the "hybrid leucine-rich repeat technique," which fused LGR4 with the hagfish VLR protein, to obtain two recombinant human LGR4 proteins, LGR415 and LGR49. We determined the crystal structures of ligand-free LGR415 and the LGR49-Rspo1 complex. LGR4 exhibits a twisted horseshoe-like structure. Rspo1 adopts a flat and ß-fold architecture and is bound in the concave surface of LGR4 in the complex through electrostatic and hydrophobic interactions. All the Rspo1-binding residues are conserved in LGR4-6, suggesting that LGR4-6 bind R-spondins through an identical surface. Structural analysis of our LGR4-Rspo1 complex with the previously determined LGR4 and LGR5 structures revealed that the concave surface of LGR4 is the sole binding site for R-spondins, suggesting a one-site binding model of LGR4-6 in ligand recognition. The molecular mechanism of LGR4-6 is distinct from the two-step mechanism of group A receptors LGR1-3 and the multiple-interface binding model of group C receptors LGR7-8, suggesting LGRs utilize the divergent mechanisms for ligand recognition. Our structures, together with previous reports, provide a comprehensive understanding of the ligand recognition by LGRs.
Asunto(s)
Receptores Acoplados a Proteínas G/química , Trombospondinas/química , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Diseño de Fármacos , Regulación del Desarrollo de la Expresión Génica , Humanos , Leucina/química , Ligandos , Datos de Secuencia Molecular , Mutagénesis , Plásmidos , Unión Proteica , Multimerización de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Homología de Secuencia de Aminoácido , Células Madre/citología , Proteínas Wnt/metabolismoRESUMEN
Ubiquitination is generally considered as a eukaryotic protein modification, which is catalysed by a three-enzyme cascade and is reversed by deubiquitinating enzymes. Ubiquitination directs protein degradation and regulates cell signalling, thereby plays key roles in many cellular processes including immune response, vesicle trafficking and cell cycle. Bacterial pathogens inject a series of virulent proteins, named effectors, into the host cells. Increasing evidence suggests that many effectors hijack the host ubiquitin pathways to benefit bacterial infection. This review summarizes the known functions and mechanisms of effectors from human bacterial pathogens including enteropathogenic Escherichia coli, Salmonella, Shigella, Chlamydia and Legionella, highlighting the diversity in their mechanisms for manipulating the host ubiquitin pathways. Many effectors adopt the molecular mimicry strategy to harbour similar structures or functional motifs with those of the host E3 ligases and deubiquitinases. On the other hand, a few of effectors evolve novel structures or new enzymatic activities to modulate various steps of the host ubiquitin pathways. The diversity in the mechanisms enhances the efficient exploitation of the host ubiquitination signalling by bacteria.
Asunto(s)
Bacterias/metabolismo , Proteínas Bacterianas/metabolismo , Interacciones Huésped-Patógeno , Complejo de la Endopetidasa Proteasomal/metabolismo , Ubiquitina/metabolismo , Factores de Virulencia/metabolismo , Humanos , Procesamiento Proteico-PostraduccionalRESUMEN
Ubc13 is an important ubiquitin-conjugating (E2) enzyme in the NF-κB signaling pathway. The Shigella effector OspI targets Ubc13 and deamidates Gln100 of Ubc13 to a glutamic acid residue, leading to the inhibition of host inflammatory responses. Here we report the crystal structure of the OspI-Ubc13 complex at 2.3 Å resolution. The structure reveals that OspI uses two differently charged regions to extensively interact with the α1 helix, L1 loop and L2 loop of Ubc13. The Gln100 residue is bound within the hydrophilic catalytic pocket of OspI. A comparison between Ubc13-bound and wild-type free OspI structures revealed that Ubc13 binding induces notable structural reassembly of the catalytic pocket, suggesting that substrate binding might be involved in the catalysis of OspI. The OspI-binding sites in Ubc13 largely overlap with the binding residues for host ubiquitin E3 ligases and a deubiquitinating enzyme, which suggests that the bacterial effector and host proteins exploit the same surface on Ubc13 for specific recognition. Biochemical results indicate that both of the differently charged regions in OspI are important for the interaction with Ubc13, and the specificity determinants in Ubc13 for OspI recognition reside in the distinct residues in the α1 helix and L2 region. Our study reveals the molecular basis of Ubc13 deamidation by OspI, as well as a convergence of E2 recognition by bacterial and host proteins.
Asunto(s)
Amidohidrolasas/química , Amidohidrolasas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Shigella flexneri/metabolismo , Enzimas Ubiquitina-Conjugadoras/química , Enzimas Ubiquitina-Conjugadoras/metabolismo , Secuencia de Aminoácidos , Sistemas de Secreción Bacterianos , Sitios de Unión/genética , Cristalografía por Rayos X , Células HEK293 , Humanos , Inflamación/inmunología , Inflamación/microbiología , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , FN-kappa B/metabolismo , Unión Proteica/genética , Alineación de Secuencia , Shigella flexneri/inmunología , Factor 6 Asociado a Receptor de TNF/metabolismo , UbiquitinaciónRESUMEN
The bacterial flagellar motor is a huge bidirectional rotary nanomachine that drives rotation of the flagellum for bacterial motility. The cytoplasmic C ring of the flagellar motor functions as the switch complex for the rotational direction switching from counterclockwise to clockwise. However, the structural basis of the rotational switching and how the C ring is assembled have long remained elusive. Here, we present two high-resolution cryo-electron microscopy structures of the C ring-containing flagellar basal body-hook complex from Salmonella Typhimurium, which are in the default counterclockwise state and in a constitutively active CheY mutant-induced clockwise state, respectively. In both complexes, the C ring consists of four subrings, but is in two different conformations. The CheY proteins are bound into an open groove between two adjacent protomers on the surface of the middle subring of the C ring and interact with the FliG and FliM subunits. The binding of the CheY protein induces a significant upward shift of the C ring towards the MS ring and inward movements of its protomers towards the motor center, which eventually remodels the structures of the FliG subunits and reverses the orientations and surface electrostatic potential of the αtorque helices to trigger the counterclockwise-to-clockwise rotational switching. The conformational changes of the FliG subunits reveal that the stator units on the motor require a relocation process in the inner membrane during the rotational switching. This study provides unprecedented molecular insights into the rotational switching mechanism and a detailed overall structural view of the bacterial flagellar motors.
RESUMEN
Quantitative reverse transcription PCR (qRT-PCR) can screen applicable reference genes of species, and reference genes can be used to reduce experimental errors. Sudan grass (Sorghum sudanense (Piper) Stapf) is a high-yield, abiotic-tolerant annual high-quality forage with a wide range of uses. However, no studies have reported reference genes suitable for Sudan grass. Therefore, we found eight candidate reference genes, including UBQ10, HIS3, UBQ9, Isoform0012931, PP2A, ACP2, eIF4α, and Actin, under salt stress (NaCl), drought stress (DR), acid aluminum stress (AlCl3), and methyl jasmonate treatment (MeJA). By using geNorm, NormFinder, BestKeeper, and RefFinder, we ranked eight reference genes on the basis of their expression stabilities. The results indicated that the best reference gene was PP2A under all treatments. eIF4α can be used in CK, MeJA, NaCl, and DR. HIS3 can serve as the best reference gene in AlCl3. Two target genes (Isoform0007606 and Isoform0002387) belong to drought-stress-response genes, and they are highly expressed in Sudan grass according to transcriptome data. They were used to verify eight candidate reference genes under drought stress. The expression trends of the two most stable reference genes were similar, but the trend in expression for Actin showed a significant difference. The reference genes we screened provided valuable guidance for future research on Sudan grass.
Asunto(s)
Piper , Sorghum , Estrés Fisiológico/genética , Transcripción Reversa , Sorghum/genética , Genes de Plantas , Piper/genética , Actinas/genética , Cloruro de Sodio/farmacología , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Regulación de la Expresión Génica de las PlantasRESUMEN
Bacterial pathogens deliver effector proteins with diverse biochemical activities into host cells, thereby modulating various host functions. Legionella pneumophila hijacks host vesicle trafficking to avoid phagosome-lysosome fusion, a mechanism that is dependent on the Legionella Dot/Icm type IV secretion system. SidM/DrrA, a Legionella type IV effector, is important for the interactions of Legionella-containing vacuoles with host endoplasmic reticulum-derived vesicles. SidM is the only known protein that catalyzes both the exchange of GDP for GTP and GDI displacement from small GTPase Rab1. We determined the crystal structures of SidM alone (residues 317-647) and SidM (residues 193-550) in complex with nucleotide-free WT Rab1. The SidM structure contains an N-terminal helical domain with a potential new function, a Rab1-activation domain, and a C-terminal phosphatidylinositol 4-phosphate-binding P4M domain. The Rab1-activation domain has extensive strong interactions mainly with Rab1 switch I and II regions that undergo substantial conformational changes on SidM binding. Mutations of switch-contacting residues in SidM attenuate both the nucleotide exchange and GDI displacement activities. Structural comparisons of Rab1 in the SidM complex with Rab1-GDP and Ypt1-GDP in the GDI complex identify key conformational changes that disrupt the nucleotide and GDI binding of Rab1. Further biochemical and structural analyses reveal a unique mechanism of coupled GDP release and GDI displacement likely triggered by the SidM-induced drastic displacement of switch I of Rab1.