Binding of the TRF2 iDDR motif to RAD50 highlights a convergent evolutionary strategy to inactivate MRN at telomeres.

Telomeres protect chromosome ends from unscheduled DNA repair, including from the MRN (MRE11, RAD50, NBS1) complex, which processes double-stranded DNA breaks (DSBs) via activation of the ATM kinase, promotes DNA end-tethering aiding the non-homologous end-joining (NHEJ) pathway, and initiates DSB resection through the MRE11 nuclease. A protein motif (MIN, for MRN inhibitor) inhibits MRN at budding yeast telomeres by binding to RAD50 and evolved at least twice, in unrelated telomeric proteins Rif2 and Taz1. We identify the iDDR motif of human shelterin protein TRF2 as a third example of convergent evolution for this telomeric mechanism for binding MRN, despite the iDDR lacking sequence homology to the MIN motif. CtIP is required for activation of MRE11 nuclease action, and we provide evidence for binding of a short C-terminal region of CtIP to a RAD50 interface that partly overlaps with the iDDR binding site, indicating that the interaction is mutually exclusive. In addition, we show that the iDDR impairs the DNA binding activity of RAD50. These results highlight direct inhibition of MRN action as a crucial role of telomeric proteins across organisms and point to multiple mechanisms enforced by the iDDR to disable the many activities of the MRN complex.

The physiological effect of rimI/rimJ silencing by CRISPR interference in Mycobacterium smegmatis mc2155.

N-terminal acetylation of proteins is an important post-translational modification (PTM) found in eukaryotes and prokaryotes. In bacteria, N-terminal acetylation is suggested to play various regulatory roles related to protein stability, gene expression, stress response, and virulence; however, the mechanism of such response remains unclear. The proteins, namely RimI/RimJ, are involved in N-terminal acetylation in mycobacteria. In this study, we used CRISPR interference (CRISPRi) to silence rimI/rimJ in Mycobacterium smegmatis mc2155 to investigate the physiological effects of N-terminal acetylation in cell survival and stress response. Repeat analysis of growth curves in rich media and biofilm analysis in minimal media of various mutant strains and wild-type bacteria did not show significant differences that could be attributed to the rimI/rimJ silencing. However, total proteome and acetylome profiles varied significantly across mutants and wild-type strains, highlighting the role of RimI/RimJ in modulating levels of proprotein acetylation in the cellular milieu. Further, we observed a significant increase in the minimum inhibitory concentration (MIC) (from 64 to 1024 µg ml-1) for the drug isoniazid in rimI mutant strains. The increase in MIC value for the drug isoniazid in the mutant strains suggests the link between N-terminal acetylation and antibiotic resistance. The study highlights the utility of CRISPRi as a convenient tool to study the role of PTMs, such as acetylation in mycobacteria. It also identifies rimI/rimJ genes as necessary for managing cellular response against antibiotic stress. Further research would be required to decipher the potential of targeting acetylation to enhance the efficacy of existing antibiotics.

Molecular insights into effector binding by DgoR, a GntR/FadR family transcriptional repressor of D-galactonate metabolism in Escherichia coli.

Several GntR/FadR transcriptional regulators govern sugar acid metabolism in bacteria. Although effectors have been identified for a few sugar acid regulators, the mode of effector binding is unknown. Even in the overall FadR subfamily, there are limited details on effector-regulator interactions. Here, we identified the effector-binding cavity in Escherichia coli DgoR, a FadR subfamily transcriptional repressor of D-galactonate metabolism that employs D-galactonate as its effector. Using a genetic screen, we isolated several dgoR superrepressor alleles. Blind docking suggested eight amino acids corresponding to these alleles to form a part of the effector-binding cavity. In vivo and in vitro assays showed that these mutations compromise the inducibility of DgoR without affecting its oligomeric status or affinity for target DNA. Taking Bacillus subtilis GntR as a representative, we demonstrated that the effector-binding cavity is similar among FadR subfamily sugar acid regulators. Finally, a comparison of sugar acid regulators with other FadR members suggested conserved features of effector-regulator recognition within the FadR subfamily. Sugar acid metabolism is widely implicated in bacterial colonization and virulence. The present study sets the basis to investigate the influence of natural genetic variations in FadR subfamily regulators on their sensitivity to sugar acids and ultimately on host-bacterial interactions.

Modeling of a 14 kDa RUVBL2-Binding Domain with Medium Resolution Cryo-EM Density.

The number of high-resolution structures of protein complexes obtained using cryo-electron microscopy (cryo-EM) is increasing rapidly. Cryo-EM maps of large macromolecular complexes frequently contain regions resolved at different resolution levels, and modeling atomic structures de novo can be difficult for domains determined at worse than 5 Å in the absence of atomic information from other structures. Here we describe the details and step-by-step decisions in the strategy we followed to model the RUVBL2-binding domain (RBD), a 14 kDa domain at the C-terminus of RNA Polymerase II associated protein 3 (RPAP3) for which atomic information was not available. Modeling was performed on a cryo-EM map at 4.0-5.5 Å resolution, integrating information from secondary structure predictions, homology modeling, restraints from cross-linked mass spectrometry, and molecular dynamics (MD) in AMBER. Here, we compare our model with the structure of RBD determined by NMR to evaluate our strategy. We also perform new MD simulations to describe important residues mediating the interaction of RBD with RUVBL2 and analyze their conservation in RBD homologous domains. Our approach and its evaluation can serve as an example to address the analysis of medium resolution regions in cryo-EM maps.

Chryseobacterium glaciei sp. nov., isolated from the surface of a glacier in the Indian trans-Himalayas.

A novel bacterial strain, IHBB 10212T, of the genus Chryseobacterium was isolated from a glacier near the Kunzum Pass located in the Lahaul-Spiti in the North-Western Himalayas of India. The cells were Gram-negative, aerobic, non-sporulating, single rods, lacked flagella, and formed yellow to orange pigmented colonies. The strain utilized maltose, trehalose, sucrose, gentibiose, glucose, mannose, fructose, mannitol, arabitol and salicin for growth. Flexirubin-type pigments were produced by strain IHBB 10212T. The 16S rRNA gene sequence analysis showed relatedness of strain IHBB 10212T to Chryseobacterium polytrichastri DSM 26899T (97.43 %), Chryseobacterium greenlandense CIP 110007T (97.29 %) and Chryseobacterium aquaticum KCTC 12483T (96.80 %). Iso-C15 : 0 and summed feature 3 (C16 : 1ω7c/C16 : 1ω6c) constituted the major cellular fatty acids. The polar lipids present were six unidentified aminolipids, one unidentified phospholipid and three unidentified lipids. MK-6 was identified as the major quinone. The DNA G+C content was 34.08  mol%. Digital DNA-DNA hybridization of strain IHBB 10212T with C. polytrichastri, C. greenlandense and C. aquaticum showed values far below the prescribed thresholds of 95 % for average nucleotide identity and 70 % for the Genome-to-Genome Distance Calculator for species delineation. Based on its differences from validly published Chryseobacterium species, strain IHBB 10212T is identified as a new species, for which the proposed name is Chryseobacterium glaciei sp. nov., with IHBB 10212T as the type strain (=MTCC 12457T=JCM 31156T=KACC 19170T).

Psychromicrobium lacuslunae sp. nov., isolated from a high altitude lake.

The novel strain IHBB 11108T was a psychrotolerant and alkaliphilic bacterium isolated from the subsurface water of Chandra Tal Lake in the Lahaul-Spiti valley located in the Indian trans-Himalayas. Cells were Gram-stain-positive, aerobic, non-motile, catalase-positive and oxidase-negative. The strain grew at 5-37 °C (optimum 28 °C), pH 5.0-12.0 (optimum pH 7.0) and with up to 8 % NaCl (optimum 1 %). The 16S rRNA gene sequence analysis revealed the highest relatedness of strain IHBB 11108T with Psychromicrobium silvestre DSM 102047T (97.5 %), Arthrobacter russicus DSM 14555T (97.4 %) and Renibacterium salmoninarum ATCC 33209T (97.4 %). The strain contained a quinone system with 57.2 % MK-9(H2), 39.1 % MK-10(H2), 3.0 % MK-8(H2) and 0.7 % MK-7(H2). The polar lipids detected were diphosphatidylglycerol, dimannosylglyceride, phosphatidylinositol, phosphatidylglycerol, monogalactosyldiacylglycerol, one unidentified glycolipid and four unidentified lipids. The cell-wall peptidoglycan structure type was A3α l-Lys-l-Thr-l-Ala with substitution of the α-carboxyl group of d-Glu by alanine amide. Anteiso-C15 : 0, iso-C16 : 0 and anteiso-C17 : 0 were the predominant fatty acids. The genomic DNA G+C content was 59.0 mol%. The DNA-DNA relatedness of strain IHBB 11108T was 46.7±2.2, 43.1±2.5 and 19.1±2.4 % with P. silvestre DSM 102047T, A. russicus DSM 14555T and R. salmoninarum ATCC 33209T, respectively. On the basis of the results of the phenotypic, chemotaxonomic and phylogenetic analyses, IHBB 11108T is considered to represent a novel species of the genus Psychromicrobium for which the name Psychromicrobium lacuslunae sp. nov. is proposed. The type strain is IHBB 11108T (=MTCC 12460T=MCC 2780T=JCM 31143T=KACC 19070T).

Advances on the Structure of the R2TP/Prefoldin-like Complex.

Cellular stability, assembly and activation of a growing list of macromolecular complexes require the action of HSP90 working in concert with the R2TP/Prefoldin-like (R2TP/PFDL) co-chaperone. RNA polymerase II, snoRNPs and complexes of PI3-kinase-like kinases, a family that includes the ATM, ATR, DNA-PKcs, TRAPP, SMG1 and mTOR proteins, are among the clients of the HSP90-R2TP system. Evidence links the R2TP/PFDL pathway with cancer, most likely because of the essential role in pathways commonly deregulated in cancer. R2TP forms the core of the co-cochaperone and orchestrates the recruitment of HSP90 and clients, whereas prefoldin and additional prefoldin-like proteins, including URI, associate with R2TP, but their function is still unclear. The mechanism by which R2TP/PFLD facilitates assembly and activation of such a variety of macromolecular complexes is poorly understood. Recent efforts in the structural characterization of R2TP have started to provide some mechanistic insights. We summarize recent structural findings, particularly how cryo-electron microscopy (cryo-EM) is contributing to our understanding of the architecture of the R2TP core complex. Structural differences discovered between yeast and human R2TP reveal unanticipated complexities of the metazoan R2TP complex, and opens new and interesting questions about how R2TP/PFLD works.

Tah1 helix-swap dimerization prevents mixed Hsp90 co-chaperone complexes.

Specific co-chaperone adaptors facilitate the recruitment of client proteins to the Hsp90 system. Tah1 binds the C-terminal conserved MEEVD motif of Hsp90, thus linking an eclectic set of client proteins to the R2TP complex for their assembly and regulation by Hsp90. Rather than the normal complement of seven α-helices seen in other tetratricopeptide repeat (TPR) domains, Tah1 unusually consists of the first five only. Consequently, the methionine of the MEEVD peptide remains exposed to solvent when bound by Tah1. In solution Tah1 appears to be predominantly monomeric, and recent structures have failed to explain how Tah1 appears to prevent the formation of mixed TPR domain-containing complexes such as Cpr6-(Hsp90)2-Tah1. To understand this further, the crystal structure of Tah1 in complex with the MEEVD peptide of Hsp90 was determined, which shows a helix swap involving the fifth α-helix between two adjacently bound Tah1 molecules. Dimerization of Tah1 restores the normal binding environment of the bound Hsp90 methionine residue by reconstituting a TPR binding site similar to that in seven-helix-containing TPR domain proteins. Dimerization also explains how other monomeric TPR-domain proteins are excluded from forming inappropriate mixed co-chaperone complexes.

In vitro effects of alginate lyase SG4 + produced by Paenibacillus lautus alone and combined with antibiotics on biofilm formation by mucoid Pseudomonas aeruginosa.

Alginate is a major extra polymeric substance in the biofilm formed by mucoid Pseudomonas aeruginosa. It is the main proven perpetrator of lung infections in patients suffering from cystic fibrosis. Alginate lyases are very important in the treatment of cystic fibrosis. This study evaluated the role of standalone and in conjugation, effect of alginate lyase of SG4 + isolated from Paenibacillus lautus in enhancing in vitro bactericidal activity of gentamicin and amikacin on mucoid P. aeruginosa. Using Response Surface Methodology (RSM) alginate lyase SG4 + production was optimized in shake flask and there 8.49-fold enhancement in enzyme production. In fermenter, maximum growth (10.15 mg/ml) and alginate lyase (1.46 International Units) production, 1.71-fold was increased using Central Composite Design (CCD). Further, fermentation time was reduced from 48 to 20 h. To the best of our knowledge this is the first report in which CCD was used for fermenter studies to optimize alginate lyase production. The Km and Vmax of purified enzyme were found to be 2.7 mg/ml and 0.84 mol/ml-min, respectively. The half-life (t 1/2) of purified alginate lyase SG4 + at 37 °C was 180 min. Alginate lyase SG4 + in combination with gentamicin and amikacin eradiated 48.4- 52.3% and 58- 64.6%, alginate biofilm formed by P. aeruginosa strains, respectively. The study proves that alginate lyase SG4 + has excellent exopolysaccharide disintegrating ability and may be useful in development of potent therapeutic agent to treat P. aeruginosa biofilms.

A snapshot of the aryl hydrocarbon receptor complex.

In this issue of Structure, Wen et al. present the cryo-EM structure of the aryl hydrocarbon receptor (AhR) and show how it is recruited and stabilized by the HSP90 molecular chaperone and its co-chaperones XAP2 and p23.

Structure of the TELO2-TTI1-TTI2 complex and its function in TOR recruitment to the R2TP chaperone.

The R2TP (RUVBL1-RUVBL2-RPAP3-PIH1D1) complex, in collaboration with heat shock protein 90 (HSP90), functions as a chaperone for the assembly and stability of protein complexes, including RNA polymerases, small nuclear ribonucleoprotein particles (snRNPs), and phosphatidylinositol 3-kinase (PI3K)-like kinases (PIKKs) such as TOR and SMG1. PIKK stabilization depends on an additional complex of TELO2, TTI1, and TTI2 (TTT), whose structure and function are poorly understood. The cryoelectron microscopy (cryo-EM) structure of the human R2TP-TTT complex, together with biochemical experiments, reveals the mechanism of TOR recruitment to the R2TP-TTT chaperone. The HEAT-repeat TTT complex binds the kinase domain of TOR, without blocking its activity, and delivers TOR to the R2TP chaperone. In addition, TTT regulates the R2TP chaperone by inhibiting RUVBL1-RUVBL2 ATPase activity and by modulating the conformation and interactions of the PIH1D1 and RPAP3 components of R2TP. Taken together, our results show how TTT couples the recruitment of TOR to R2TP with the regulation of this chaperone system.

Structural mechanism for regulation of the AAA-ATPases RUVBL1-RUVBL2 in the R2TP co-chaperone revealed by cryo-EM.

The human R2TP complex (RUVBL1-RUVBL2-RPAP3-PIH1D1) is an HSP90 co-chaperone required for the maturation of several essential multiprotein complexes, including RNA polymerase II, small nucleolar ribonucleoproteins, and PIKK complexes such as mTORC1 and ATR-ATRIP. RUVBL1-RUVBL2 AAA-ATPases are also primary components of other essential complexes such as INO80 and Tip60 remodelers. Despite recent efforts, the molecular mechanisms regulating RUVBL1-RUVBL2 in these complexes remain elusive. Here, we report cryo-EM structures of R2TP and show how access to the nucleotide-binding site of RUVBL2 is coupled to binding of the client recruitment component of R2TP (PIH1D1) to its DII domain. This interaction induces conformational rearrangements that lead to the destabilization of an N-terminal segment of RUVBL2 that acts as a gatekeeper to nucleotide exchange. This mechanism couples protein-induced motions of the DII domains with accessibility of the nucleotide-binding site in RUVBL1-RUVBL2, and it is likely a general mechanism shared with other RUVBL1-RUVBL2-containing complexes.

Heterologous expression and structure-function relationship of low-temperature and alkaline active protease from Acinetobacter sp. IHB B 5011(MN12).

The gene encoding protease from Acinetobacter sp. IHB B 5011(MN12) was cloned and expressed in Escherichia coli BL21(DE3). The nucleotide sequence revealed 1323bp ORF encoding 441 amino acids protein with molecular weight 47.2kDa. The phylogenetic analysis showed clustering of Alp protease with subtilisin-like serine proteases of S8 family. The amino acid sequence was comprised of N-terminal signal peptide 1-21 amino acids, pre-peptide 22-143 amino acids, peptidase S8 domain 144-434 amino acids, and pro-peptide 435-441 amino acids at C-terminus. Three constructs with signal peptide pET-Alp, without signal peptide pET-Alp1 and peptidase S8 domain pET-Alp2 were prepared for expression in E. coli BL21(DE3). The recombinant proteins Alp1 and Alp2 expressed as inclusion bodies showed ∼50kDa and ∼40kDa bands, respectively. The pre-propeptide ∼11kDa removed from Alp1 resulted in mature protein of ∼35kDa with 1738Umg-1 specific activity. The recombinant protease was optimally active at 40°C and pH 9, and stable over 10-70°C and 6-12pH. The activity at low-temperature and alkaline pH was supported by high R/(R+K) ratio, more glycine, less proline, negatively charged amino acids, less salt bridges and longer loops. These properties suggested the suitability of Alp as additive in the laundry.

Author Correction: RPAP3 provides a flexible scaffold for coupling HSP90 to the human R2TP co-chaperone complex.

In the originally published version of this article, the affiliation details for Hugo Muñoz-Hernández, Carlos F. Rodríguez and Oscar Llorca incorrectly omitted 'Centro de Investigaciones Biológicas (CIB), Spanish National Research Council (CSIC), Ramiro de Maeztu 9, 28040 Madrid, Spain'. This has now been corrected in both the PDF and HTML versions of the Article.

RPAP3 provides a flexible scaffold for coupling HSP90 to the human R2TP co-chaperone complex.

The R2TP/Prefoldin-like co-chaperone, in concert with HSP90, facilitates assembly and cellular stability of RNA polymerase II, and complexes of PI3-kinase-like kinases such as mTOR. However, the mechanism by which this occurs is poorly understood. Here we use cryo-EM and biochemical studies on the human R2TP core (RUVBL1-RUVBL2-RPAP3-PIH1D1) which reveal the distinctive role of RPAP3, distinguishing metazoan R2TP from the smaller yeast equivalent. RPAP3 spans both faces of a single RUVBL ring, providing an extended scaffold that recruits clients and provides a flexible tether for HSP90. A 3.6 Å cryo-EM structure reveals direct interaction of a C-terminal domain of RPAP3 and the ATPase domain of RUVBL2, necessary for human R2TP assembly but absent from yeast. The mobile TPR domains of RPAP3 map to the opposite face of the ring, associating with PIH1D1, which mediates client protein recruitment. Thus, RPAP3 provides a flexible platform for bringing HSP90 into proximity with diverse client proteins.

Genome assembly of Chryseobacterium sp. strain IHBB 10212 from glacier top-surface soil in the Indian trans-Himalayas with potential for hydrolytic enzymes.

The cold-active esterases are gaining importance due to their catalytic activities finding applications in chemical industry, food processes and detergent industry as additives, and organic synthesis of unstable compounds as catalysts. In the present study, the complete genome sequence of 4,843,645 bp with an average 34.08% G + C content and 4260 protein-coding genes are reported for the low temperature-active esterase-producing novel strain of Chrysobacterium isolated from the top-surface soil of a glacier in the cold deserts of the Indian trans-Himalayas. The genome contained two plasmids of 16,553 and 11,450 bp with 40.54 and 40.37% G + C contents, respectively. Several genes encoding the hydrolysis of ester linkages of triglycerides into fatty acids and glycerol were predicted in the genome. The annotation also predicted the genes encoding proteases, lipases, amylases, ß-glucosidases, endoglucanases and xylanases involved in biotechnological processes. The complete genome sequence of Chryseobacterium sp. strain IHBB 10212 and two plasmids have been deposited vide accession numbers CP015199, CP015200 and CP015201 at DDBJ/EMBL/GenBank.

The structure of FKBP38 in complex with the MEEVD tetratricopeptide binding-motif of Hsp90.

Tetratricopeptide (TPR) domains are known protein interaction domains. We show that the TPR domain of FKBP8 selectively binds Hsp90, and interactions upstream of the conserved MEEVD motif are critical for tight binding. In contrast FKBP8 failed to bind intact Hsp70. The PPIase domain was not essential for the interaction with Hsp90 and binding was completely encompassed by the TPR domain alone. The conformation adopted by Hsp90 peptides, containing the conserved MEEVD motif, in the crystal structure were similar to that seen for the TPR domains of CHIP, AIP and Tah1. The carboxylate clamp interactions with bound Hsp90 peptide were a critical component of the interaction and mutation of Lys 307, involved in the carboxylate clamp, completely disrupted the interaction with Hsp90. FKBP8 binding to Hsp90 did not substantially influence its ATPase activity.

The Structure of the R2TP Complex Defines a Platform for Recruiting Diverse Client Proteins to the HSP90 Molecular Chaperone System.

The R2TP complex, comprising the Rvb1p-Rvb2p AAA-ATPases, Tah1p, and Pih1p in yeast, is a specialized Hsp90 co-chaperone required for the assembly and maturation of multi-subunit complexes. These include the small nucleolar ribonucleoproteins, RNA polymerase II, and complexes containing phosphatidylinositol-3-kinase-like kinases. The structure and stoichiometry of yeast R2TP and how it couples to Hsp90 are currently unknown. Here, we determine the 3D organization of yeast R2TP using sedimentation velocity analysis and cryo-electron microscopy. The 359-kDa complex comprises one Rvb1p/Rvb2p hetero-hexamer with domains II (DIIs) forming an open basket that accommodates a single copy of Tah1p-Pih1p. Tah1p-Pih1p binding to multiple DII domains regulates Rvb1p/Rvb2p ATPase activity. Using domain dissection and cross-linking mass spectrometry, we identified a unique region of Pih1p that is essential for interaction with Rvb1p/Rvb2p. These data provide a structural basis for understanding how R2TP couples an Hsp90 dimer to a diverse set of client proteins and complexes.

Complete Genome Sequencing of Protease-Producing Novel Arthrobacter sp. Strain IHBB 11108 Using PacBio Single-Molecule Real-Time Sequencing Technology.

A previously uncharacterized species of the genus Arthrobacter, strain IHBB 11108 (MCC 2780), is a Gram-positive, strictly aerobic, nonmotile, cold-adapted, and protease-producing alkaliphilic actinobacterium, isolated from shallow undersurface water from Chandra Tal Lake, Lahaul-Spiti, India. The complete genome of the strain is 3.6 Mb in size with an average 58.97% G+C content.

Complete Genome Sequence of Paenibacillus sp. Strain IHBB 10380 Using PacBio Single-Molecule Real-Time Sequencing Technology.

The complete genome sequence of 5.77 Mb is reported for Paenibacillus sp. strain IHBB 10380, isolated from the cold desert area of the northwestern Himalayas and exhibiting amylase and cellulase activities. The gene-coding clusters predicted the presence of genes for hydrolytic enzymes in the genome.