Publications
243 results found
Taglialegna A, Navarro S, Ventura S, et al., 2016, Staphylococcal Bap Proteins Build Amyloid Scaffold Biofilm Matrices in Response to Environmental Signals, PLOS Pathogens, Vol: 12, ISSN: 1553-7366
Biofilms are communities of bacteria that grow encased in an extracellular matrix that often contains proteins. The spatial organization and the molecular interactions between matrix scaffold proteins remain in most cases largely unknown. Here, we report that Bap protein of Staphylococcus aureus self-assembles into functional amyloid aggregates to build the biofilm matrix in response to environmental conditions. Specifically, Bap is processed and fragments containing at least the N-terminus of the protein become aggregation-prone and self-assemble into amyloid-like structures under acidic pHs and low concentrations of calcium. The molten globule-like state of Bap fragments is stabilized upon binding of the cation, hindering its self-assembly into amyloid fibers. These findings define a dual function for Bap, first as a sensor and then as a scaffold protein to promote biofilm development under specific environmental conditions. Since the pH-driven multicellular behavior mediated by Bap occurs in coagulase-negative staphylococci and many other bacteria exploit Bap-like proteins to build a biofilm matrix, the mechanism of amyloid-like aggregation described here may be widespread among pathogenic bacteria.
Berry J, Xu Y, Ward PN, et al., 2016, A comparative structure/function analysis of two type IV pilin DNA receptors defines a novel mode of DNA-binding, Structure, Vol: 24, Pages: 926-934, ISSN: 1878-4186
DNA transformation is a widespread process allowing bacteria to capture free DNAby using filamentous nano-machines composed of type IV pilins. These proteins canact as DNA receptors as demonstrated by the finding that Neisseria meningitidisComP minor pilin has intrinsic DNA-binding ability. ComP binds DNA better when itcontains the DNA uptake sequence (DUS) motif abundant in this species genome,playing a role in its trademark ability to selectively take up its own DNA. Here, wereport high-resolution structures for meningococcal ComP and Neisseria subflavaComPsub, which recognize different DUS motifs. We show that they are structurallyidentical type IV pilins that pack readily into filament models and display a uniqueDD-region delimited by two disulfide bonds. Functional analysis of ComPsub defines anew mode of DNA-binding involving the DD-region, adapted for exported DNAreceptors.
Taylor JD, Hawthorne WJ, Lo J, et al., 2016, Electrostatically-guided inhibition of Curli amyloid nucleation by the CsgC-like family of chaperones, Scientific Reports, Vol: 6, ISSN: 2045-2322
Polypeptide aggregation into amyloid is linked with several debilitating human diseases.Despite the inherent risk of aggregation-induced cytotoxicity, bacteria control the export ofamyloid-prone subunits and assemble adhesive amyloid fibres during biofilm formation. AnEscherichia protein, CsgC potently inhibits amyloid formation of curli amyloid proteins.Here we unlock its mechanism of action, and show that CsgC strongly inhibits primarynucleation via electrostatically-guided molecular encounters, which expands theconformational distribution of disordered curli subunits. This delays the formation of higherorder intermediates and maintains amyloidogenic subunits in a secretion-competent form.New structural insight also reveal that CsgC is part of diverse family of bacterial amyloidinhibitors. Curli assembly is therefore not only arrested in the periplasm, but the preservationof conformational flexibility also enables efficient secretion to the cellsurface. Understanding how bacteria safely handle amyloidogenic polypeptides contributetowards efforts to control aggregation in disease-causing amyloids and amyloid-based biotechnological applications.
Miliara X, Matthews S, 2016, Structural comparison of yeast and human intra-mitochondrial lipid transport systems, Biochemical Society Transactions, Vol: 44, Pages: 479-485, ISSN: 1470-8752
Mitochondria depend on a tightly regulated supply of phospholipids. The protein of relevant evolutionary and lymphoid interest (PRELI)/Ups1 family together with its mitochondrial chaperones [TP53-regulated inhibitor of apoptosis 1 (TRIAP1)/Mdm35] represents a unique heterodimeric lipid-transfer system that is evolutionary conserved from yeast to man. Recent X-ray crystal structures of the human and yeast systems are compared and discuss here and shed new insight into the mechanism of the PRELI/Ups1 system.
Leen EN, Sorgeloos F, Correia S, et al., 2016, Correction: A Conserved Interaction between a C-Terminal Motif in Norovirus VPg and the HEAT-1 Domain of eIF4G Is Essential for Translation Initiation., PLoS Pathog, Vol: 12
Taylor J, Taylor G, Hare S, et al., 2016, Structures of the DfsB protein family suggest a cationic, helical sibling-lethal factor peptide, Journal of Molecular Biology, Vol: 428, Pages: 554-560, ISSN: 1089-8638
Bacteria have developed a variety of mechanisms for survivingharsh environmental conditions, nutrient stress and overpopulation.Paenibacillus dendritiformis produces a lethal protein (Slf) that is ableto induce cell death in neighboring colonies and a phenotypic switch inmore distant ones. Slf is derived from the secreted precursor protein,DfsB, after proteolytic processing. Here, we present new crystalstructures of DfsB homologues from a variety of bacterial species and asurprising version present in the yeast Saccharomyces cerevisiae.Adopting a four-helix bundle decorated with a further three short heliceswithin intervening loops, DfsB belongs to a non-enzymatic class of theDinB fold. The structure suggests that the biologically-active Slffragment may possess a C-terminal helix rich in basic and aromaticresidues that suggest a functional mechanism akin to that for cationicantimicrobial peptides.
Leen EN, Sorgeloos F, Correia S, et al., 2016, A conserved interaction between a C-terminal motif in norovirus VPg and the HEAT-1 domain of eIF4G is essential for translation initiation, PLOS Pathogens, Vol: 12, ISSN: 1553-7366
Translation initiation is a critical early step in the replication cycle of the positive-sense, single-stranded RNA genome of noroviruses, a major cause of gastroenteritis in humans. Norovirus RNA, which has neither a 5´ m7G cap nor an internal ribosome entry site (IRES), adopts an unusual mechanism to initiate protein synthesis that relies on interactions between the VPg protein covalently attached to the 5´-end of the viral RNA and eukaryotic initiation factors (eIFs) in the host cell. For murine norovirus (MNV) we previously showed that VPg binds to the middle fragment of eIF4G (4GM; residues 652-1132). Here we have used pull-down assays, fluorescence anisotropy, and isothermal titration calorimetry (ITC) to demonstrate that a stretch of ~20 amino acids at the C terminus of MNV VPg mediates direct and specific binding to the HEAT-1 domain within the 4GM fragment of eIF4G. Our analysis further reveals that the MNV C terminus binds to eIF4G HEAT-1 via a motif that is conserved in all known noroviruses. Fine mutagenic mapping suggests that the MNV VPg C terminus may interact with eIF4G in a helical conformation. NMR spectroscopy was used to define the VPg binding site on eIF4G HEAT-1, which was confirmed by mutagenesis and binding assays. We have found that this site is non-overlapping with the binding site for eIF4A on eIF4G HEAT-1 by demonstrating that norovirus VPg can form ternary VPg-eIF4G-eIF4A complexes. The functional significance of the VPg-eIF4G interaction was shown by the ability of fusion proteins containing the C-terminal peptide of MNV VPg to inhibit in vitro translation of norovirus RNA but not cap- or IRES-dependent translation. These observations define important structural details of a functional interaction between norovirus VPg and eIF4G and reveal a binding interface that might be exploited as a target for antiviral therapy.
Wong CT, Xu Y, Gupta A, et al., 2015, Structural analysis of haemoglobin binding by HpuA from the Neisseriaceae family, Nature Communications, Vol: 6, ISSN: 2041-1723
The Neisseriaceae family of bacteria causes a range of diseases including meningitis, septicaemia, gonorrhoea and endocarditis, and extracts haem from haemoglobin as an important iron source within the iron-limited environment of its human host. Herein we report crystal structures of apo- and haemoglobin-bound HpuA, an essential component of this haem import system. The interface involves long loops on the bacterial receptor that present hydrophobic side chains for packing against the surface of haemoglobin. Interestingly, our structural and biochemical analyses of Kingella denitrificans and Neisseria gonorrhoeae HpuA mutants, although validating the interactions observed in the crystal structure, show how Neisseriaceae have the fascinating ability to diversify functional sequences and yet retain the haemoglobin binding function. Our results present the first description of HpuA’s role in direct binding of haemoglobin.
Pakharukova N, Garnett JA, Tuittila M, et al., 2015, Structural Insight into Archaic and Alternative Chaperone-Usher Pathways Reveals a Novel Mechanism of Pilus Biogenesis., PLOS Pathogens, Vol: 11, ISSN: 1553-7366
Gram-negative pathogens express fibrous adhesive organelles that mediate targeting to sites of infection. The major class of these organelles is assembled via the classical, alternative and archaic chaperone-usher pathways. Although non-classical systems share a wider phylogenetic distribution and are associated with a range of diseases, little is known about their assembly mechanisms. Here we report atomic-resolution insight into the structure and biogenesis of Acinetobacter baumannii Csu and Escherichia coli ECP biofilm-mediating pili. We show that the two non-classical systems are structurally related, but their assembly mechanism is strikingly different from the classical assembly pathway. Non-classical chaperones, unlike their classical counterparts, maintain subunits in a substantially disordered conformational state, akin to a molten globule. This is achieved by a unique binding mechanism involving the register-shifted donor strand complementation and a different subunit carboxylate anchor. The subunit lacks the classical pre-folded initiation site for donor strand exchange, suggesting that recognition of its exposed hydrophobic core starts the assembly process and provides fresh inspiration for the design of inhibitors targeting chaperone-usher systems.
Pinzan CF, Sardinha-Silva A, Almeida F, et al., 2015, Vaccination with Recombinant Microneme Proteins Confers Protection against Experimental Toxoplasmosis in Mice, PLOS One, Vol: 10, ISSN: 1932-6203
Toxoplasmosis, a zoonotic disease caused by Toxoplasma gondii, is an important publichealth problem and veterinary concern. Although there is no vaccine for human toxoplasmosis,many attempts have been made to develop one. Promising vaccine candidates utilizeproteins, or their genes, from microneme organelle of T. gondii that are involved in theinitial stages of host cell invasion by the parasite. In the present study, we used differentrecombinant microneme proteins (TgMIC1, TgMIC4, or TgMIC6) or combinations of theseproteins (TgMIC1-4 and TgMIC1-4-6) to evaluate the immune response and protectionagainst experimental toxoplasmosis in C57BL/6 mice. Vaccination with recombinantTgMIC1, TgMIC4, or TgMIC6 alone conferred partial protection, as demonstrated byreduced brain cyst burden and mortality rates after challenge. Immunization with TgMIC1-4or TgMIC1-4-6 vaccines provided the most effective protection, since 70% and 80% ofmice, respectively, survived to the acute phase of infection. In addition, these vaccinatedmice, in comparison to non-vaccinated ones, showed reduced parasite burden by 59% and68%, respectively. The protective effect was related to the cellular and humoral immuneresponses induced by vaccination and included the release of Th1 cytokines IFN-γ and IL-12, antigen-stimulated spleen cell proliferation, and production of antigen-specific serumantibodies. Our results demonstrate that microneme proteins are potential vaccines againstT. gondii, since their inoculation prevents or decreases the deleterious effects of theinfection.
Curry S, Leen EN, Sorgeloos F, et al., 2015, A conserved interaction between a C-terminal motif in Norovirus VPg and the HEAT-1 domain of eIF4G is essential for translation initiation, Publisher: bioRxiv
Translation initiation is a critical early step in the replication cycle of the positive-sense, single-stranded RNA genome of noroviruses, a major cause of gastroenteritis in humans. Norovirus RNA, which has neither a 5 ́ m7G cap nor an internal ribosome entry site (IRES), adopts an unusual mechanism to initiate protein synthesis that relies on interactions between the VPg protein covalently attached to the 5 ́-end of the viral RNA and eukaryotic initiation factors (eIFs) in the host cell. For murine norovirus (MNV) we previously showed that VPg binds to the middle fragment of eIF4G (4GM; residues 652-1132). Here we have used pull-down assays, fluorescence anisotropy, and isothermal titration calorimetry (ITC) to demonstrate that a stretch of ~20 amino acids at the C terminus of MNV VPg mediates direct and specific binding to the HEAT-1 domain within the 4GM fragment of eIF4G. Our analysis further reveals that the MNV C-terminus binds to eIF4G HEAT-1 via a motif that is conserved in all known noroviruses. Fine mutagenic mapping suggests that the MNV VPg C terminus may interact with eIF4G in a helical conformation. NMR spectroscopy was used to define the VPg binding site on eIF4G HEAT-1, which was confirmed by mutagenesis and binding assays. We have found that this site is non-overlapping with the binding site for eIF4A on eIF4G HEAT-1 by demonstrating that norovirus VPg can form ternary VPg-eIF4G-eIF4A complexes. The functional significance of the VPg-eIF4G interaction was shown by the ability of fusion proteins containing the C- terminal peptide of MNV VPg to inhibit translation of norovirus RNA but not cap- or IRES-dependent translation. These observations define important structural details of a functional interaction between norovirus VPg and eIF4G and reveal a binding interface that might be exploited as a target for antiviral therapy.
Polo LM, Grundy G, Rulten S, et al., 2015, New structural insights into PARP3 function, 40th Congress of the Federation-of-European-Biochemical-Societies (FEBS) - The Biochemical Basis of Life, Publisher: WILEY-BLACKWELL, Pages: 397-397, ISSN: 1742-464X
Matthews SJ, Miliara X, Garnett J, et al., 2015, Structural insight into the TRIAP1/PRELI-like domain family of mitochondrial phospholipid transfer complexes, EMBO Reports, Vol: 16, Pages: 824-835, ISSN: 1469-221X
The composition of the mitochondrial membrane is important for its architecture and proper function. Mitochondria depend on a tightly regulated supply of phospholipid via intra-mitochondrial synthesis and by direct import from the endoplasmic reticulum. The Ups1/PRELI-like family together with its mitochondrial chaperones (TRIAP1/Mdm35) represent a unique heterodimeric lipid transfer system that is evolutionary conserved from yeast to man. Work presented here provides new atomic resolution insight into the function of a human member of this system. Crystal structures of free TRIAP1 and the TRIAP1–SLMO1 complex reveal how the PRELI domain is chaperoned during import into the intermembrane mitochondrial space. The structural resemblance of PRELI-like domain of SLMO1 with that of mammalian phoshatidylinositol transfer proteins (PITPs) suggest that they share similar lipid transfer mechanisms, in which access to a buried phospholipid-binding cavity is regulated by conformationally adaptable loops.
Garnett JA, Diallo M, Matthews SJ, 2015, Purification, crystallization and preliminary X-ray diffraction analysis of the Escherichia coli common pilus chaperone EcpB, Acta Crystallographica Section F: Structural Biology Communications, Vol: 71, Pages: 676-679, ISSN: 2053-230X
Pili are key cell-surface components that allow the attachment of bacteria to both biological and abiotic solid surfaces, whilst also mediating interactions between themselves. In Escherichia coli, the common pilus (Ecp) belongs to an alternative chaperone-usher (CU) pathway that plays a major role in both early biofilm formation and host-cell adhesion. The chaperone EcpB is involved in the biogenesis of the filament, which is composed of EcpA and EcpD. Initial attempts at crystallizing EcpB using natively purified protein from the bacterial periplasm were not successful; however, after the isolation of EcpB under denaturing conditions and subsequent refolding, crystals were obtained at pH 8.0 using the sitting-drop method of vapour diffusion. Diffraction data have been processed to 2.4 Å resolution. These crystals belonged to the trigonal space group P3121 or P3221, with unit-cell parameters a = b = 62.65, c = 121.14 Å and one monomer in the asymmetric unit. Molecular replacement was unsuccessful, but selenomethionine-substituted protein and heavy-atom derivatives are being prepared for phasing. The three-dimensional structure of EcpB will provide invaluable information on the subtle mechanistic differences in biogenesis between the alternative and classical CU pathways. Furthermore, this is the first time that this refolding strategy has been used to purify CU chaperones, and it could be implemented in similar systems where it has not been possible to obtain highly ordered crystals.
Benjamin S, Williams F, Kerry L, et al., 2015, NMR assignment of the immune mapped protein 1 (IMP1) homologue from Plasmodium falciparum, Biomolecular NMR Assignments, Vol: 9, Pages: 393-395, ISSN: 1874-2718
Taylor JD, Matthews SJ, 2015, New insight into the molecular control of bacterial functional amyloids., Frontiers in Cellular and Infection Microbiology, Vol: 5, ISSN: 2235-2988
Amyloid protein structure has been discovered in a variety of functional or pathogenic contexts. What distinguishes the former from the latter is that functional amyloid systems possess dedicated molecular control systems that determine the timing, location, and structure of the fibers. Failure to guide this process can result in cytotoxicity, as observed in several pathologies like Alzheimer's and Parkinson's Disease. Many gram-negative bacteria produce an extracellular amyloid fiber known as curli via a multi-component secretion system. During this process, aggregation-prone, semi-folded curli subunits have to cross the periplasm and outer-membrane and self-assemble into surface-attached fibers. Two recent breakthroughs have provided molecular details regarding periplasmic chaperoning and subunit secretion. This review offers a combined perspective on these first mechanistic insights into the curli system.
Liu B, Zhu F, Wu H, et al., 2015, NMR assignment of the amylase-binding protein A from <i>Streptococcus parasanguinis</i>, BIOMOLECULAR NMR ASSIGNMENTS, Vol: 9, Pages: 173-175, ISSN: 1874-2718
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- Citations: 1
Garnett JA, Muhl D, Douse CH, et al., 2015, Structure-function analysis reveals that the Pseudomonas aeruginosa Tps4 two-partner secretion system is involved in CupB5 translocation, Protein Science, Vol: 24, Pages: 670-687, ISSN: 1469-896X
Pseudomonas aeruginosa is a Gram-negative opportunistic bacterium, synonymous withcystic fibrosis patients, which can cause chronic infection of the lungs. This pathogen is a modelorganism to study biofilms: a bacterial population embedded in an extracellular matrix that provideprotection from environmental pressures and lead to persistence. A number of Chaperone-UsherPathways, namely CupA-CupE, play key roles in these processes by assembling adhesive pili onthe bacterial surface. One of these, encoded by the cupB operon, is unique as it contains anonchaperone-usher gene product, CupB5. Two-partner secretion (TPS) systems are comprised ofa C-terminal integral membrane b-barrel pore with tandem N-terminal POTRA (POlypeptide TRansportAssociated) domains located in the periplasm (TpsB) and a secreted substrate (TpsA). UsingNMR we show that TpsB4 (LepB) interacts with CupB5 and its predicted cognate partner TpsA4(LepA), an extracellular protease. Moreover, using cellular studies we confirm that TpsB4 cantranslocate CupB5 across the P. aeruginosa outer membrane, which contrasts a previous observationthat suggested the CupB3 P-usher secretes CupB5. In support of our findings we also demonstratethat tps4/cupB operons are coregulated by the RocS1 sensor suggesting P. aeruginosa hasdeveloped synergy between these systems. Furthermore, we have determined the solutionstructureof the TpsB4-POTRA1 domain and together with restraints from NMR chemical shift mappingand in vivo mutational analysis we have calculated models for the entire TpsB4 periplasmic region in complex with both TpsA4 and CupB5 secretion motifs. The data highlight specific residuesfor TpsA4/CupB5 recognition by TpsB4 in the periplasm and suggest distinct roles for eachPOTRA domain.
Evans ML, Chorell E, Taylor JD, et al., 2015, The Bacterial Curli System Possesses a Potent and Selective Inhibitor of Amyloid Formation, MOLECULAR CELL, Vol: 57, Pages: 445-455, ISSN: 1097-2765
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- Citations: 138
Huynh M-H, Liu B, Henry M, et al., 2015, Structural Basis of <i>Toxoplasma gondii</i> MIC2-associated Protein Interaction with MIC2, JOURNAL OF BIOLOGICAL CHEMISTRY, Vol: 290, Pages: 1432-1441
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- Citations: 17
Young JC, Clements A, Lang AE, et al., 2014, The Escherichia coli effector EspJ blocks Src kinase activity via amidation and ADP ribosylation, Nature Communications, Vol: 5, ISSN: 2041-1723
The hallmark of enteropathogenic Escherichia coli (EPEC) infection is the formation of actin-rich pedestal-like structures, which are generated following phosphorylation of the bacterial effector Tir by cellular Src and Abl family tyrosine kinases. This leads to recruitment of the Nck–WIP–N-WASP complex that triggers Arp2/3-dependent actin polymerization in the host cell. The same phosphorylation-mediated signalling network is also assembled downstream of the Vaccinia virus protein A36 and the phagocytic Fc-gamma receptor FcγRIIa. Here we report that the EPEC type-III secretion system effector EspJ inhibits autophosphorylation of Src and phosphorylation of the Src substrates Tir and FcγRIIa. Consistent with this, EspJ inhibits actin polymerization downstream of EPEC, Vaccinia virus and opsonized red blood cells. We identify EspJ as a unique adenosine diphosphate (ADP) ribosyltransferase that directly inhibits Src kinase by simultaneous amidation and ADP ribosylation of the conserved kinase-domain residue, Src E310, resulting in glutamine-ADP ribose.
Matsuo E, Leon E, Matthews SJ, et al., 2014, Structure based modification of Bluetongue virus helicase protein VP6 to produce a viable VP6-truncated BTV, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, Vol: 451, Pages: 603-608, ISSN: 0006-291X
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- Citations: 7
Berry AA, Yang Y, Pakharukova N, et al., 2014, Structural Insight into Host Recognition by Aggregative Adherence Fimbriae of Enteroaggregative <i>Escherichia coli</i>, PLOS PATHOGENS, Vol: 10, ISSN: 1553-7366
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- Citations: 31
Mousnier A, Schroeder GN, Stoneham CA, et al., 2014, A New Method To Determine <i>In Vivo</i> Interactomes Reveals Binding of the <i>Legionella pneumophila</i> Effector PieE to Multiple Rab GTPases, MBIO, Vol: 5, ISSN: 2150-7511
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- Citations: 28
Lin J, Oh S-H, Jones R, et al., 2014, The peptide-binding cavity Is essential for Als3-mediated adhesion of Candida albicans to human cells, Journal of Biological Chemistry, Vol: 289, Pages: 18401-18412, ISSN: 1083-351X
Background: Of the eight cell surface glycoproteins in the C. albicans Als family, Als3 makes the largest contribution to adhesion to human cells.Results: Mutation of the Als3 peptide-binding cavity (PBC) results in loss of Als3 adhesive function.Conclusion: The PBC is required for Als3 adhesive function.Significance: Interfering with PBC function is a viable strategy for inhibiting C. albicans adhesion.
Lambert SM, Langley DR, Garnett JA, et al., 2014, The crystal structure of NS5A domain 1 from genotype 1a reveals new clues to the mechanism of action for dimeric HCV inhibitors, PROTEIN SCIENCE, Vol: 23, Pages: 723-734, ISSN: 0961-8368
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- Citations: 80
Xu Y, Plechanovova A, Simpson P, et al., 2014, Structural insight into SUMO chain recognition and manipulation by the ubiquitin ligase RNF4, NATURE COMMUNICATIONS, Vol: 5, ISSN: 2041-1723
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- Citations: 62
Liu B, Shadrin A, Sheppard C, et al., 2014, A bacteriophage transcription regulator inhibits bacterial transcription initiation by Sigma-factor displacement, Nucleic Acids Research, Vol: 42, Pages: 4294-4305, ISSN: 0305-1048
Bacteriophages (phages) appropriate essential processes of bacterial hosts to benefit their own development. The multisubunit bacterial RNA polymerase (RNAp) enzyme, which catalyses DNA transcription, is targeted by phage-encoded transcription regulators that selectively modulate its activity. Here, we describe the structural and mechanistic basis for the inhibition of bacterial RNAp by the transcription regulator P7 encoded by Xanthomonas oryzae phage Xp10. We reveal that P7 uses a two-step mechanism to simultaneously interact with the catalytic β and β’ subunits of the bacterial RNAp and inhibits transcription initiation by inducing the displacement of the σ70-factor on initial engagement of RNAp with promoter DNA. The new mode of interaction with and inhibition mechanism of bacterial RNAp by P7 underscore the remarkable variety of mechanisms evolved by phages to interfere with host transcription.
Liu B, Shadrin A, Sheppard C, et al., 2014, The sabotage of the bacterial transcription machinery by a small bacteriophage protein., Bacteriophage, Vol: 4, ISSN: 2159-7073
Many bacteriophages produce small proteins that specifically interfere with the bacterial host transcription machinery and thus contribute to the acquisition of the bacterial cell by the bacteriophage. We recently described how a small protein, called P7, produced by the Xp10 bacteriophage inhibits bacterial transcription initiation by causing the dissociation of the promoter specificity sigma factor subunit from the host RNA polymerase holoenzyme. In this addendum to the original publication, we present the highlights of that research.
Xu Y, Matthews S, 2013, TROSY NMR spectroscopy of large soluble proteins, Topics in Current Chemistry, Vol: 335, Pages: 97-119, ISSN: 0340-1022
Solution nuclear magnetic resonance spectroscopy is usually only used to study proteins with molecular weight not exceeding about 50 kDa. This size limit has been lifted significantly in recent years, thanks to the development of labelling methods and the application of transverse-relaxation optimized spectroscopy (TROSY). In particular, methyl-specific labelling and methyl-TROSY have been shown to be effective for supramolecular systems as large as about 1 MDa. In this chapter we review the available methods for labelling different kinds of methyl groups and the assignment strategies in very large protein systems. Several proteins are selected as examples to show how NMR helps to reveal the details of structure, interaction and dynamics of these proteins. © Springer-Verlag Berlin Heidelberg 2011.
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