RESUMEN
Genome segregation is a fundamental process that preserves the genetic integrity of all organisms, but the mechanisms driving genome segregation in archaea remain enigmatic. This study delved into the unknown function of SegC (SSO0033), a novel protein thought to be involved in chromosome segregation in archaea. Using fluorescence polarization DNA binding assays, we discovered the ability of SegC to bind DNA without any sequence preference. Furthermore, we determined the crystal structure of SegC at 2.8 Å resolution, revealing the multimeric configuration and forming a large positively charged surface that can bind DNA. SegC has a tertiary structure folding similar to those of the ThDP-binding fold superfamily, but SegC shares only 5-15% sequence identity with those proteins. Unexpectedly, we found that SegC has nucleotide triphosphatase (NTPase) activity. We also determined the SegC-ADP complex structure, identifying the NTP binding pocket and relative SegC residues involved in the interaction. Interestingly, images from negative-stain electron microscopy revealed that SegC forms filamentous structures in the presence of DNA and NTPs. Further, more uniform and larger SegC-filaments are observed, when SegA-ATP was added. Notably, the introduction of SegB disrupts these oligomers, with ATP being essential for regulating filament formation. These findings provide insights into the functional and structural role of SegC in archaeal chromosome segregation.
RESUMEN
The ParABS system, composed of ParA (an ATPase), ParB (a DNA binding protein), and parS (a centromere-like DNA), regulates bacterial chromosome partition. The ParB-parS partition complex interacts with the nucleoid-bound ParA to form the nucleoid-adaptor complex (NAC). In Helicobacter pylori, ParA and ParB homologs are encoded as HpSoj and HpSpo0J (HpParA and HpParB), respectively. We determined the crystal structures of the ATP hydrolysis deficient mutant, HpParAD41A, and the HpParAD41A-DNA complex. We assayed the CTPase activity of HpParB and identified two potential DNA binding modes of HpParB regulated by CTP, one is the specific DNA binding by the DNA binding domain and the other is the non-specific DNA binding through the C-terminal domain under the regulation of CTP. We observed an interaction between HpParAD41A and the N-terminus fragment of HpParB (residue 1-10, HpParBN10) and determined the crystal structure of the ternary complex, HpParAD41A-DNA-HpParBN10 complex which mimics the NAC formation. HpParBN10 binds near the HpParAD41A dimer interface and is clamped by flexible loops, L23 and L34, through a specific cation-π interaction between Arg9 of HpParBN10 and Phe52 of HpParAD41A. We propose a molecular mechanism model of the ParABS system providing insight into chromosome partition in bacteria.
Asunto(s)
Proteínas Bacterianas , Cromosomas Bacterianos , Proteínas de Unión al ADN , Helicobacter pylori , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Helicobacter pylori/genética , Helicobacter pylori/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Cromosomas Bacterianos/metabolismo , Cromosomas Bacterianos/química , Cromosomas Bacterianos/genética , Modelos Moleculares , Cristalografía por Rayos X , Unión Proteica , ADN Bacteriano/metabolismo , ADN Bacteriano/química , ADN Bacteriano/genética , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/genética , Segregación Cromosómica , Adenosina Trifosfato/metabolismo , Sitios de UniónRESUMEN
BACKGROUND: Most tail-anchored (TA) membrane proteins are delivered to the endoplasmic reticulum through a conserved posttranslational pathway. Although core mechanisms underlying the targeting and insertion of TA proteins are well established in eukaryotes, their role in mediating TA protein biogenesis in plants remains unclear. We reported the crystal structures of algal arsenite transporter 1 (ArsA1), which possesses an approximately 80-kDa monomeric architecture and carries chloroplast-localized TA proteins. However, the mechanistic basis of ArsA2, a Get3 (guided entry of TA proteins 3) homolog in plants, for TA recognition remains unknown. RESULTS: Here, for the first time, we present the crystal structures of the diatom Pt-Get3a that forms a distinct ellipsoid-shaped tetramer in the open (nucleotide-bound) state through crystal packing. Pulldown assay results revealed that only tetrameric Pt-Get3a can bind to TA proteins. The lack of the conserved zinc-coordination CXXC motif in Pt-Get3a potentially leads to the spontaneous formation of a distinct parallelogram-shaped dimeric conformation in solution, suggesting a new dimer state for subsequent tetramerization upon TA targeting. Pt-Get3a nonspecifically binds to different subsets of TA substrates due to the lower hydrophobicity of its α-helical subdomain, which is implicated in TA recognition. CONCLUSIONS: Our study provides new insights into the mechanisms underlying TA protein shielding by tetrameric Get3 during targeting to the diatom's cell membrane.
Asunto(s)
Diatomeas , Diatomeas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Multimerización de ProteínaRESUMEN
Alanyl-tRNA synthetase (AlaRS) retains a conserved prototype structure throughout its biology, consisting of catalytic, tRNA-recognition, editing, and C-Ala domains. The catalytic and tRNA-recognition domains catalyze aminoacylation, the editing domain hydrolyzes mischarged tRNAAla, and C-Ala-the major tRNA-binding module-targets the elbow of the L-shaped tRNAAla. Interestingly, a mini-AlaRS lacking the editing and C-Ala domains is recovered from the Tupanvirus of the amoeba Acanthamoeba castellanii. Here we show that Tupanvirus AlaRS (TuAlaRS) is phylogenetically related to its host's AlaRS. Despite lacking the conserved amino acid residues responsible for recognition of the identity element of tRNAAla (G3:U70), TuAlaRS still specifically recognized G3:U70-containing tRNAAla. In addition, despite lacking C-Ala, TuAlaRS robustly binds and charges microAla (an RNA substrate corresponding to the acceptor stem of tRNAAla) as well as tRNAAla, indicating that TuAlaRS exclusively targets the acceptor stem. Moreover, this mini-AlaRS could functionally substitute for yeast AlaRS in vivo. This study suggests that TuAlaRS has developed a new tRNA-binding mode to compensate for the loss of C-Ala.
Asunto(s)
Alanina-ARNt Ligasa , Alanina-ARNt Ligasa/genética , Alanina-ARNt Ligasa/química , Alanina-ARNt Ligasa/metabolismo , ARN de Transferencia de Alanina/química , ARN de Transferencia de Alanina/genética , ARN de Transferencia de Alanina/metabolismo , Escherichia coli/genética , ARN de Transferencia/metabolismoRESUMEN
BACKGROUND: Human traits, diseases susceptibility, and clinical outcomes vary hugely among individuals. Despite a fundamental understanding of genetic (or environmental) contributions, the detailed mechanisms of how genetic variation impacts molecular or cellular behaviours of a gene, and subsequently leads to such variability remain poorly understood. METHODS: Here, in addition to phenome-wide correlations, we leveraged multiomics to exploit mechanistic links, from genetic polymorphism to protein structural or functional changes and a cross-omics perturbation landscape of a germline variant. RESULTS: We identified a missense cis-acting expression quantitative trait locus in CLEC18A (rs75776403) in which the altered residue (T151âM151) disrupts the lipid-binding ability of the protein domain. The altered allele carriage led to a metabolic and proliferative shift, as well as immune deactivation, therefore determines human anthropometrics (body height), kidney, and hematological traits. CONCLUSIONS: Collectively, we uncovered genetic pleiotropy in human complex traits and diseases via CLEC18A rs75776403-regulated pathways.
Asunto(s)
Pleiotropía Genética , Polimorfismo Genético , Alelos , Estudio de Asociación del Genoma Completo , Humanos , Lectinas Tipo C/genética , Fenotipo , Polimorfismo de Nucleótido SimpleRESUMEN
Antimicrobial peptides (AMPs) have been developed for the treatment of bacterial infections, but their applications are limited to topical infections since they are sequestered and inhibited in serum. Here we have discovered that the inhibition of AMPs by human serum was mediated through high-density lipoproteins (HDL) which are known to remove cholesterol from peripheral tissues. The susceptibility of AMPs to HDL varied depending on the degree of hydrophobicity of AMPs and their binding affinities to HDL. The phospholipids, such as phosphatidylcholine, of HDL were essential for AMP-binding. The dynamic binding interactions between AMPs and HDL were mediated through the hydrophobic interactions rather than by ionic strength. Interestingly, some AMPs, such as SMAP29, dissociated from the AMP-HDL complex and translocated to bacteria upon contact, while some AMPs, such as LL37, remained in complex with HDL. These results suggest that HDL binds AMPs and facilitates the translocation of them to the bacteria.
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Antibacterianos/metabolismo , Péptidos Antimicrobianos/metabolismo , Bacterias/metabolismo , Proteínas Sanguíneas/metabolismo , Lípidos/química , Lipoproteínas HDL/metabolismo , Suero/metabolismo , Humanos , Interacciones Hidrofóbicas e HidrofílicasRESUMEN
Genome segregation is a vital process in all organisms. Chromosome partitioning remains obscure in Archaea, the third domain of life. Here, we investigated the SegAB system from Sulfolobus solfataricus. SegA is a ParA Walker-type ATPase and SegB is a site-specific DNA-binding protein. We determined the structures of both proteins and those of SegA-DNA and SegB-DNA complexes. The SegA structure revealed an atypical, novel non-sandwich dimer that binds DNA either in the presence or in the absence of ATP. The SegB structure disclosed a ribbon-helix-helix motif through which the protein binds DNA site specifically. The association of multiple interacting SegB dimers with the DNA results in a higher order chromatin-like structure. The unstructured SegB N-terminus plays an essential catalytic role in stimulating SegA ATPase activity and an architectural regulatory role in segrosome (SegA-SegB-DNA) formation. Electron microscopy results also provide a compact ring-like segrosome structure related to chromosome organization. These findings contribute a novel mechanistic perspective on archaeal chromosome segregation.
Asunto(s)
Proteínas Arqueales/genética , Segregación Cromosómica , Cromosomas de Archaea/genética , ADN de Archaea/genética , Sulfolobus solfataricus/genética , Adenosina Difosfato/metabolismo , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Proteínas Arqueales/química , Proteínas Arqueales/metabolismo , Cromatina/genética , Cromatina/metabolismo , Cromatina/ultraestructura , Cristalografía por Rayos X , ADN de Archaea/química , ADN de Archaea/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Microscopía Electrónica , Modelos Moleculares , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Complejos Multiproteicos/ultraestructura , Mutación , Conformación de Ácido Nucleico , Unión Proteica , Conformación Proteica , Sulfolobus solfataricus/metabolismoRESUMEN
DNA damage leads to stalled or collapsed replication forks. Replication restart primosomes re-initiate DNA synthesis at these stalled or collapsed DNA replication forks, which is important for bacterial survival. Primosomal protein PriA specifically recognizes the DNA fork structure and recruits other primosomal proteins to load the replicative helicase, in order to re-establish the replication fork. PriA binding on DNA is the first step to restart replication forks for proper DNA repair. Using a single-molecule fluorescence colocalization experiment, we measured the thermodynamic and real-time kinetic properties of fluorescence-labeled Gram-positive bacteria Geobacillus stearothermophilus PriA binding on DNA forks. We showed that PriA preferentially binds to a DNA fork structure with a fully duplexed leading strand at sub-nanomolar affinity (Kd = 268 ± 99 pM). PriA binds dynamically, and its association and dissociation rate constants can be determined using the appearance and disappearance of the fluorescence signal. In addition, we showed that PriA binds to DNA forks as a monomer using photobleaching step counting. This information offers a molecular basis essential for understanding the mechanism of replication restart.
Asunto(s)
Proteínas Bacterianas/química , ADN Bacteriano/química , Proteínas de Unión al ADN/química , Geobacillus stearothermophilus/química , Sitios de Unión , Replicación del ADN , Imagen ÓpticaRESUMEN
Chronic hepatitis B (CHB) infection is rarely eradicated by current antiviral nucleos(t)ide analogues. We found that α2,6-biantennary sialoglycans of HBV surface antigen (HBsAg) bound human SIGLEC-3 (CD33) by IP and ELISA, and the binding affinity between SIGLEC-3 and α2,6-biantennary sialoglycans was determined by biolayer interferometry (equilibrium dissociation constant [KD]: 1.95 × 10-10 ± 0.21 × 10-10 M). Moreover, HBV activated SIGLEC-3 on myeloid cells and induced immunosuppression by stimulating immunoreceptor tyrosine-based inhibitory motif phosphorylation and SHP-1/-2 recruitment via α2,6-biantennary sialoglycans on HBsAg. An antagonistic anti-SIGLEC-3 mAb reversed this effect and enhanced cytokine production in response to TLR-7 agonist GS-9620 in PBMCs from CHB patients. Moreover, anti-SIGLEC-3 mAb alone was able to upregulate the expression of molecules involved in antigen presentation, such as CD80, CD86, CD40, MHC-I, MHC-II, and PD-L1 in CD14+ cells. Furthermore, SIGLEC-3 SNP rs12459419 C, which expressed a higher amount of SIGLEC-3, was associated with increased risk of hepatocellular carcinoma (HCC) in CHB patients (HR: 1.256, 95% CI: 1.027-1.535, P = 0.0266). Thus, blockade of SIGLEC-3 is a promising strategy to reactivate host immunity to HBV and lower the incidence of HCC in the CHB patient population.
Asunto(s)
Presentación de Antígeno , Carcinoma Hepatocelular/inmunología , Antígenos de Superficie de la Hepatitis B/inmunología , Virus de la Hepatitis B/inmunología , Hepatitis B Crónica/inmunología , Neoplasias Hepáticas/inmunología , Células Mieloides/inmunología , Proteínas de Neoplasias/inmunología , Lectina 3 Similar a Ig de Unión al Ácido Siálico/inmunología , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/virología , Femenino , Virus de la Hepatitis B/genética , Hepatitis B Crónica/genética , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/virología , Masculino , Proteínas de Neoplasias/genética , Polimorfismo de Nucleótido Simple , Lectina 3 Similar a Ig de Unión al Ácido Siálico/genéticaRESUMEN
DNA replication forks often encounter template DNA lesions that can stall their progression. The PriA-dependent pathway is the major replication restart mechanism in Gram-positive bacteria, and it requires several primosome proteins. Among them, PriA protein - a 3' to 5' superfamily-2 DNA helicase - is the key factor in recognizing DNA lesions and it also recruits other proteins. Here, we investigated the ATPase and helicase activities of Streptococcus pneumoniae PriA (SpPriA) through biochemical and kinetic analyses. By comparing various DNA substrates, we observed that SpPriA is unable to unwind duplex DNA with high GC content. We constructed a deletion mutant protein (SpPriAdeloop) from which the loop area of the DNA-binding domain of PriA had been removed. Functional assays on SpPriAdeloop revealed that the loop area is important in endowing DNA-binding properties on the helicase. We also show that the presence of DnaD loader protein is important for enhancing SpPriA ATPase and DNA unwinding activities.
Asunto(s)
Proteínas Bacterianas/metabolismo , ADN Helicasas/metabolismo , Replicación del ADN , ADN Bacteriano/metabolismo , Streptococcus pneumoniae/metabolismo , Proteínas Bacterianas/genética , ADN Helicasas/genética , ADN Bacteriano/genética , Streptococcus pneumoniae/genéticaRESUMEN
Inorganic phosphate (Pi) is a fundamental and essential element for nucleotide biosynthesis, energy supply, and cellular signaling in living organisms. Human phosphate transporter (hPiT) dysfunction causes numerous diseases, but the molecular mechanism underlying transporters remains elusive. We report the structure of the sodium-dependent phosphate transporter from Thermotoga maritima (TmPiT) in complex with sodium and phosphate (TmPiT-Na/Pi) at 2.3-angstrom resolution. We reveal that one phosphate and two sodium ions (Pi-2Na) are located at the core of TmPiT and that the third sodium ion (Nafore) is located near the inner membrane boundary. We propose an elevator-like mechanism for sodium and phosphate transport by TmPiT, with the TmPiT-Na/Pi complex adopting an inward occluded conformation. We found that disease-related hPiT variants carry mutations in the corresponding sodium- and phosphate-binding residues identified in TmPiT. Our three-dimensional structure of TmPiT provides a framework for understanding PiT dysfunction and for future structure-based drug design.
RESUMEN
In mammals and yeast, tail-anchored (TA) membrane proteins destined for the post-translational pathway are safely delivered to the endoplasmic reticulum (ER) membrane by a well-known targeting factor, TRC40/Get3. In contrast, the underlying mechanism for translocation of TA proteins in plants remains obscure. How this unique eukaryotic membrane-trafficking system correctly distinguishes different subsets of TA proteins destined for various organelles, including mitochondria, chloroplasts and the ER, is a key question of long standing. Here, we present crystal structures of algal ArsA1 (the Get3 homolog) in a distinct nucleotide-free open state and bound to adenylyl-imidodiphosphate. This approximately 80-kDa protein possesses a monomeric architecture, with two ATPase domains in a single polypeptide chain. It is capable of binding chloroplast (TOC34 and TOC159) and mitochondrial (TOM7) TA proteins based on features of its transmembrane domain as well as the regions immediately before and after the transmembrane domain. Several helices located above the TA-binding groove comprise the interlocking hook-like motif implicated by mutational analyses in TA substrate recognition. Our data provide insights into the molecular basis of the highly specific selectivity of interactions of algal ArsA1 with the correct sets of TA substrates before membrane targeting in plant cells.
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Cloroplastos/metabolismo , Proteínas de la Membrana/metabolismo , Retículo Endoplásmico/metabolismo , Unión Proteica , Transporte de ProteínasRESUMEN
ParABS, an important DNA partitioning process in chromosome segregation, includes ParA (an ATPase), ParB (a parS binding protein) and parS (a centromere-like DNA). The homologous proteins of ParA and ParB in Helicobacter pylori are HpSoj and HpSpo0J, respectively. We analyzed the ATPase activity of HpSoj and found that it is enhanced by both DNA and HpSpo0J. Crystal structures of HpSoj and its DNA complexes revealed a typical ATPase fold and that it is dimeric. DNA binding by HpSoj is promoted by ATP. The HpSoj-ATP-DNA complex non-specifically binds DNA through a continuous basic binding patch formed by lysine residues, with a single DNA-binding site. This complex exhibits a DNA-binding adept state with an active ATP-bound conformation, whereas the HpSoj-ADP-DNA complex may represent a transient DNA-bound state. Based on structural comparisons, HpSoj exhibits a similar DNA binding surface to the bacterial ParA superfamily, but the archaeal ParA superfamily exhibits distinct non-specific DNA-binding via two DNA-binding sites. We detected the HpSpo0J-HpSoj-DNA complex by electron microscopy and show that this nucleoid-adaptor complex (NAC) is formed through HpSoj and HpSpo0J interaction and parS DNA binding. NAC formation is promoted by HpSoj participation and specific parS DNA facilitation.
Asunto(s)
Adenosina Trifosfatasas/química , Proteínas Bacterianas/química , Segregación Cromosómica/genética , Helicobacter pylori/genética , Adenosina Trifosfatasas/genética , Proteínas Bacterianas/genética , Sitios de Unión , Centrómero/genética , Cromosomas Bacterianos/genética , Cristalografía por Rayos X , ADN/química , ADN/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Helicobacter pylori/química , Helicobacter pylori/patogenicidadRESUMEN
Prodigiosin is an intensely red pigment comprising three pyrroles. The biosynthetic pathway includes a two-step proline oxidation catalyzed by phosphatidylinositol N-acetylglucosaminyltransferase subunitâ A (PigA), with flavin adenine dinucleotide (FAD) as its cofactor. The enzyme is crystallized in the apo form and in complex with FAD and proline. As an acyl coenzymeâ A dehydrogenase (ACAD) family member, the protein folds into a ß-sheet flanked by two α-helical domains. PigA forms a tetramer, which is consistent with analytical ultracentrifugation results. FAD binds to PigA in a similar way to that in the other enzymes of the ACAD family. The variable conformations of loop ß4-ß5 and helix αG correlate well with the structural flexibility required for substrate entrance to the Re side of FAD. Modeling with PigG, the acyl carrier protein, suggests a reasonable mode of interaction with PigA. The structure helps to explain the proline oxidation mechanism, in which Glu244 plays a central role by abstracting the substrate protons. It also reveals a plausible pocket for oxygen binding to the Si side of FAD.
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Ésteres/metabolismo , N-Acetilglucosaminiltransferasas/metabolismo , Prodigiosina/biosíntesis , Compuestos de Azufre/metabolismo , Cristalografía por Rayos X , Ésteres/química , Modelos Moleculares , Estructura Molecular , N-Acetilglucosaminiltransferasas/química , Oxidación-Reducción , Prodigiosina/química , Compuestos de Azufre/químicaRESUMEN
The antimicrobial peptide, epinecidin-1 (Epi), was identified from Epinephelus coioides and may have clinical application for treating sepsis. Epi has been shown to ameliorate antibiotic-resistant bacteria-induced sepsis in mice, but further evaluation in mixed-flora models and a description of the protective mechanisms are essential to establish this peptide as a potential therapeutic. Therefore, we first tested the protective effects of Epi against polymicrobial sepsis-induced bactericidal infection, inflammation and lung injury that result from cecal ligation and puncture in mice. Furthermore, since lipopolysaccharide (LPS) is a key inducer of inflammation during bacterial infection and sepsis, we also tested the LPS-antagonizing activity and related mechanisms of Epi-mediated protection in mice with LPS-induced endotoxemia and LPS-treated Raw264.7 mouse macrophage cells. Epi rescued mice from both polymicrobial sepsis and endotoxemia after delayed administration and suppressed both lung and systemic inflammatory responses, while attenuating lung injury and diminishing bacterial load. In vitro studies revealed that Epi suppressed LPS-induced inflammatory cytokine production. Mechanistically, Epi disrupted the interaction between LPS and LPS binding protein, competed with LPS for binding on the cell surface, and inhibited Toll-like receptor 4 endocytosis, resulting in inhibition of LPS-induced reactive oxygen species/p38/Akt/NF-κB signaling and subsequent cytokine production. Overall, our results demonstrate that Epi is a promising therapeutic agent for endotoxemia and polymicrobial sepsis.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/farmacología , Endotoxemia/tratamiento farmacológico , Proteínas de Peces/farmacología , Sustancias Protectoras/farmacología , Animales , Antiinfecciosos/farmacología , Carga Bacteriana , Ciego/microbiología , Ciego/cirugía , Línea Celular , Citocinas/metabolismo , Modelos Animales de Enfermedad , Endotoxemia/etiología , Femenino , Ligadura , Receptores de Lipopolisacáridos/efectos de los fármacos , Lipopolisacáridos/farmacología , Sistema de Señalización de MAP Quinasas , Ratones , Ratones Endogámicos C57BL , FN-kappa B/metabolismo , Células RAW 264.7 , Especies Reactivas de Oxígeno/metabolismo , Receptor Toll-Like 4/metabolismoRESUMEN
The DnaB primosomal protein from Gram-positive bacteria plays a key role in DNA replication and restart as a loader protein for the recruitment of replisome cascade proteins. Previous investigations have established that DnaB is composed of an N-terminal domain, a middle domain, and a C-terminal domain. However, structural evidence for how DnaB functions at the atomic level is lacking. Here, we report the crystal structure of DnaB, encompassing the N-terminal and middle domains (residues 1-300), from Geobacillus stearothermophilus (GstDnaB1-300) at 2.8 Å resolution. Our structure revealed that GstDnaB1-300 forms a tetramer with two basket-like architectures, a finding consistent with those from solution studies using analytical ultracentrifugation. Furthermore, our results from both GST pulldown assays and analytical ultracentrifugation show that GstDnaB1-300 is sufficient to form a complex with PriA, the primosomal reinitiation protein. Moreover, with the aid of small angle X-ray scattering experiments, we also determined the structural envelope of full-length DnaB (GstDnaBFL) in solution. These small angle X-ray scattering studies indicated that GstDnaBFL has an elongated conformation and that the protruding density envelopes originating from GstDnaB1-300 could completely accommodate the GstDnaB C-terminal domain (residues 301-461). Taken together with biochemical assays, our results suggest that GstDnaB uses different domains to distinguish the PriA interaction and single-stranded DNA binding. These findings can further extend our understanding of primosomal assembly in replication restart.
Asunto(s)
Proteínas Bacterianas/metabolismo , AdnB Helicasas/química , AdnB Helicasas/metabolismo , Multimerización de Proteína , ADN de Cadena Simple/metabolismo , Geobacillus stearothermophilus/enzimología , Modelos Moleculares , Unión Proteica , Dominios Proteicos , Estructura Cuaternaria de Proteína , Alineación de SecuenciaRESUMEN
Heparin-binding hemagglutinin (HBHA) is a 199 amino acid virulence factor at the envelope of Mycobacterium tuberculosis that contributes to latent tuberculosis. The binding of HBHA to respiratory epithelial cells, which leads to extrapulmonary dissemination of the pathogen, is mediated by cell-surface heparan sulfate (HS). We report the structural characterization of the HBHA/HS complex by NMR spectroscopy. To develop a model for the molecular recognition, the first chemically synthesized uniformly 13 C- and 15 N-labeled HS octasaccharide and a uniformly 13 C- and 15 N-labeled form of HBHA were prepared. Residues 180-195 at the C-terminal region of HBHA show large chemical shift perturbation upon association with the octasaccharide. Molecular dynamics simulations conforming to the multidimensional NMR data revealed key electrostatic and even hydrophobic interactions between the binding partners that may aid in the development of agents targeting the binding event.
Asunto(s)
Heparitina Sulfato/química , Lectinas/química , Mycobacterium tuberculosis/química , Oligosacáridos/química , Modelos Moleculares , Estructura MolecularRESUMEN
Most chloroplast proteins are synthesized in the cytosol as higher molecular weight preproteins and imported via the translocons in the outer (TOC) and inner (TIC) envelope membranes of chloroplasts. Toc159 functions as a primary receptor and directly binds preproteins through its dimeric GTPase domain. As a first step toward a molecular understanding of how Toc159 mediates preprotein import, we mapped the preprotein-binding regions on the Toc159 GTPase domain (Toc159G) of pea (Pisum sativum) using cleavage by bound preproteins conjugated with the artificial protease FeBABE and cysteine-cysteine cross-linking. Our results show that residues at the dimer interface and the switch II region of Toc159G are in close proximity to preproteins. The mature portion of preproteins was observed preferentially at the dimer interface, whereas the transit peptide was found at both regions equally. Chloroplasts from transgenic plants expressing engineered Toc159 with a cysteine placed at the dimer interface showed increased cross-linking to bound preproteins. Our data suggest that, during preprotein import, the Toc159G dimer disengages and the dimer interface contacts translocating preproteins, which is consistent with a model in which conformational changes induced by dimer-monomer conversion in Toc159 play a direct role in facilitating preprotein import.
Asunto(s)
Proteínas de Cloroplastos/metabolismo , Cloroplastos/metabolismo , Proteínas de Plantas/metabolismo , Precursores de Proteínas/metabolismo , Secuencia de Aminoácidos , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Sitios de Unión/genética , Proteínas de Cloroplastos/genética , Electroforesis en Gel de Poliacrilamida , GTP Fosfohidrolasas/química , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Mutación , Pisum sativum/genética , Pisum sativum/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente , Unión Proteica , Multimerización de Proteína , Precursores de Proteínas/genética , Estructura Terciaria de Proteína , Transporte de Proteínas , Homología de Secuencia de AminoácidoRESUMEN
Bacterial ribonucleotide reductases (RNRs) play an important role in the synthesis of dNTPs and their expression is regulated by the transcription factors, NrdR and Fur. Recent transcriptomic studies using deletion mutants have indicated a role for NrdR in bacterial chemotaxis and in the maintenance of topoisomerase levels. However, NrdR deletion alone has no effect on bacterial growth or virulence in infected flies or in human blood cells. Furthermore, transcriptomic studies are limited to the deletion strain alone, and so are inadequate for drawing biological implications when the NrdR repressor is active or abundant. Therefore, further examination is warranted of changes in the cellular proteome in response to both NrdR overexpression, as well as deletion, to better understand its functional relevance as a bacterial transcription repressor. Here, we profile bacterial fate under conditions of overexpression and deletion of NrdR in E. coli. Biochemical assays show auxiliary zinc enhances the DNA binding activity of NrdR. We also demonstrate at the physiological level that increased nrdR expression causes a significant reduction in bacterial growth and fitness even at normal temperatures, and causes lethality at elevated temperatures. Corroborating these direct effects, global proteome analysis following NrdR overexpression showed a significant decrease in global protein expression. In parallel, studies on complementary expression of downregulated essential genes polA, eno and thiL showed partial rescue of the fitness defect caused by NrdR overexpression. Deletion of downregulated non-essential genes ygfK and trxA upon NrdR overexpression resulted in diminished bacterial growth and fitness suggesting an additional role for NrdR in regulating other genes. Moreover, in comparison with NrdR deletion, E. coli cells overexpressing NrdR showed significantly diminished adherence to human epithelial cells, reflecting decreased bacterial virulence. These results suggest that elevated expression of NrdR could be a suitable means to retard bacterial growth and virulence, as its elevated expression reduces bacterial fitness and impairs host cell adhesion.
Asunto(s)
Infecciones por Escherichia coli , Proteínas de Escherichia coli , Escherichia coli , Proteoma , Proteínas Represoras , Transcripción Genética , Adhesión Bacteriana/genética , Células CACO-2 , Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/patogenicidad , Infecciones por Escherichia coli/genética , Infecciones por Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Eliminación de Gen , Regulación Bacteriana de la Expresión Génica/genética , Humanos , Viabilidad Microbiana/genética , Proteoma/biosíntesis , Proteínas Represoras/genética , Proteínas Represoras/metabolismoRESUMEN
Cellulases from Bacillus and Geobacillus bacteria are potentially useful in the biofuel and animal feed industries. One of the unique characteristics of these enzymes is that they are usually quite thermostable. We previously identified a cellulase, GsCelA, from thermophilic Geobacillus sp. 70PC53, which is much more thermostable than its Bacillus homolog, BsCel5A. Thus, these two cellulases provide a pair of structures ideal for investigating the mechanism regarding how these cellulases can retain activity at high temperature. In the present study, we applied the SCHEMA non-contiguous recombination algorithm as a novel tool, which assigns protein sequences into blocks for domain swapping in a way that lessens structural disruption, to generate a set of chimeric proteins derived from the recombination of GsCelA and BsCel5A. Analyzing the activity and thermostability of this designed library set, which requires only a limited number of chimeras by SCHEMA calculations, revealed that one of the blocks may contribute to the higher thermostability of GsCelA. When tested against swollen Avicel, the highly thermostable chimeric cellulase C10 containing this block showed significantly higher activity (22%-43%) and higher thermostability compared to the parental enzymes. With further structural determinations and mutagenesis analyses, a 310 helix was identified as being responsible for the improved thermostability of this block. Furthermore, in the presence of ionic calcium and crown ether (CR), the chimeric C10 was found to retain 40% residual activity even after heat treatment at 90°C. Combining crystal structure determinations and structure-guided SCHEMA recombination, we have determined the mechanism responsible for the high thermostability of GsCelA, and generated a novel recombinant enzyme with significantly higher activity.