Journal of Molecular Biology

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  • Prolyl Isomerization and Its Catalysis in Protein Folding and Protein Function
    [Apr 2015]

    Publication date: 10 April 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 7

    Author(s): Philipp A.M. Schmidpeter , Franz X. Schmid

    Prolyl isomerizations are intrinsically slow processes. They determine the rates of many protein folding reactions and control regulatory events in folded proteins. Prolyl isomerases are able to catalyze these isomerizations, and thus, they have the potential to assist protein folding and to modulate protein function. Here, we provide examples for how prolyl isomerizations limit protein folding and are accelerated by prolyl isomerases and how native-state prolyl isomerizations regulate protein functions. The roles of prolines in protein folding and protein function are closely interrelated because both of them depend on the coupling between cis/trans isomerization and conformational changes that can involve extended regions of a protein.
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    Categories: Journal Articles
  • Roles of Intramolecular and Intermolecular Interactions in Functional Regulation of the Hsp70 J-protein Co-Chaperone Sis1
    [Apr 2015]

    Publication date: 10 April 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 7

    Author(s): Hyun Young Yu , Thomas Ziegelhoffer , Jerzy Osipiuk , Szymon J. Ciesielski , Maciej Baranowski , Min Zhou , Andrzej Joachimiak , Elizabeth A. Craig

    Unlike other Hsp70 molecular chaperones, those of the eukaryotic cytosol have four residues, EEVD, at their C-termini. EEVD(Hsp70) binds adaptor proteins of the Hsp90 chaperone system and mitochondrial membrane preprotein receptors, thereby facilitating processing of Hsp70-bound clients through protein folding and translocation pathways. Among J-protein co-chaperones functioning in these pathways, Sis1 is unique, as it also binds the EEVD(Hsp70) motif. However, little is known about the role of the Sis1:EEVD(Hsp70) interaction. We found that deletion of EEVD(Hsp70) abolished the ability of Sis1, but not the ubiquitous J-protein Ydj1, to partner with Hsp70 in in vitro protein refolding. Sis1 co-chaperone activity with Hsp70∆EEVD was restored upon substitution of a glutamic acid of the J-domain. Structural analysis revealed that this key glutamic acid, which is not present in Ydj1, forms a salt bridge with an arginine of the immediately adjacent glycine-rich region. Thus, restoration of Sis1 in vitro activity suggests that intramolecular interactions between the J-domain and glycine-rich region control co-chaperone activity, which is optimal only when Sis1 interacts with the EEVD(Hsp70) motif. However, we found that disruption of the Sis1:EEVD(Hsp70) interaction enhances the ability of Sis1 to substitute for Ydj1 in vivo. Our results are consistent with the idea that interaction of Sis1 with EEVD(Hsp70) minimizes transfer of Sis1-bound clients to Hsp70s that are primed for client transfer to folding and translocation pathways by their preassociation with EEVD binding adaptor proteins. These interactions may be one means by which cells triage Ydj1- and Sis1-bound clients to productive and quality control pathways, respectively.
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    Categories: Journal Articles
  • Lysine Deacetylases Regulate the Heat Shock Response Including the Age-Associated Impairment of HSF1
    [Apr 2015]

    Publication date: 10 April 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 7

    Author(s): Elena Zelin , Brian C. Freeman

    Heat shock factor 1 (HSF1) is critical for defending cells from both acute and chronic stresses. In aging cells, the DNA binding activity of HSF1 deteriorates correlating with the onset of pathological events including neurodegeneration and heart disease. We find that DNA binding by HSF1 is controlled by lysine deacetylases with HDAC7, HDAC9, and SIRT1 distinctly increasing the magnitude and length of a heat shock response (HSR). In contrast, HDAC1 inhibits HSF1 in a deacetylase-independent manner. In aging cells, the levels of HDAC1 are elevated and the HSR is impaired, yet reduction of HDAC1 in aged cells restores the HSR. Our results provide a mechanistic basis for the age-associated regulation of the HSR. Besides HSF1, the deacetylases differentially modulate the activities of unrelated DNA binding proteins. Taken together, our data further support the model that lysine deacetylases are selective regulators of DNA binding proteins.
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    Categories: Journal Articles
  • Structural variations and solvent structure of r(UGGGGU) quadruplexes stabilized by Sr2+ ions
    [Apr 2015]

    Publication date: Available online 8 April 2015
    Source:Journal of Molecular Biology

    Author(s): Alastair C. Fyfe , Pete W. Dunten , Monika M. Martick , William G. Scott

    Guanine-rich sequences can, under appropriate conditions, adopt a distinctive, four-stranded, helical fold known as a G-quadruplex. Interest in quadruplex folds has grown in recent years as evidence of their biological relevance has accumulated from both sequence analysis and function-specific assays. The folds are unusually stable and their formation appears to require close management to maintain cell health; regulatory failure correlates with genomic instability and a number of cancer phenotypes. Biologically relevant quadruplex folds are anticipated to form transiently in mRNA and in single-stranded, unwound DNA. To elucidate factors, including bound solvent, that contribute to the stability of RNA quadruplexes, we examine, by x-ray crystallography and SAXS, the structure of a previously-reported tetramolecular quadruplex, UGGGGU stabilized by Sr2+ ions. Crystal forms of the octameric assembly formed by this sequence exhibit unusually strong diffraction and anomalous signal enabling the construction of reliable models to a resolution of 0.88 Å. The solvent structure confirms hydration patterns reported for other nucleic acid helical conformations and provides support for the greater stability of RNA quadruplexes relative to DNA. Novel features detected in the octameric RNA assembly include a new crystal form, evidence of multiple conformations and structural variations in the 3’ U-tetrad, including one that leads to the formation of a hydrated internal cavity.
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    Categories: Journal Articles
  • SimC7 is a novel NAD(P)H-dependent ketoreductase essential for the antibiotic activity of the DNA gyrase inhibitor simocyclinone
    [Apr 2015]

    Publication date: Available online 8 April 2015
    Source:Journal of Molecular Biology

    Author(s): Martin Schäfer , Tung B.K. Le , Stephen J. Hearnshaw , Anthony Maxwell , Gregory L. Challis , Barrie Wilkinson , Mark J. Buttner

    Simocyclinone D8 (SD8) is a potent DNA gyrase inhibitor produced by Streptomyces antibioticus Tü6040. The simocyclinone (sim) biosynthetic gene cluster has been sequenced and a hypothetical biosynthetic pathway proposed. The tetraene linker in SD8 was suggested to be the product of a modular type I polyketide synthase (PKS) working in trans with two monofunctional enzymes. One of these monofunctional enzymes, SimC7, was proposed to supply a dehydratase activity missing from two modules of the PKS. In this study, we report the function of SimC7. We isolated the entire ~72 kb sim cluster on a single phage artificial chromosome (PAC) clone and produced simocyclinone heterologously in a Streptomyces coelicolor strain engineered for improved antibiotic production. Deletion of simC7 resulted in the production of a novel simocyclinone, 7-oxo-SD8, which unexpectedly carried a normal tetraene linker but was altered in the angucyclinone moiety. We demonstrate that SimC7 is an NAD(P)H-dependent ketoreductase that catalyses the conversion of 7-oxo-SD8 into SD8. 7-oxo-SD8 was essentially inactive as a DNA gyrase inhibitor, and the reduction of the keto group by SimC7 was shown to be crucial for high affinity binding to the enzyme. Thus SimC7 is an angucyclinone ketoreductase that is essential for the biological activity of simocyclinone.
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    Categories: Journal Articles
  • Immunoaffinity enrichment coupled to quantitative mass spectrometry reveals ubiquitin mediated signaling events
    [Apr 2015]

    Publication date: Available online 8 April 2015
    Source:Journal of Molecular Biology

    Author(s): Kebing Yu , Lilian Phu , Eugene Varfolomeev , Daisy Bustos , Domagoj Vucic , Donald S. Kirkpatrick

    Ubiquitination is one of the most prevalent posttranslational modifications in eukaryotic cells, with functional importance in protein degradation, subcellular localization and signal transduction pathways. Immunoaffinity enrichment coupled with quantitative mass spectrometry enables the in depth characterization of protein ubiquitination events at the site specific level. We have applied this strategy to investigate cellular response triggered by two distinct types agents: small molecule inhibitors of the tumor-associated kinases MEK and PI3K or the pro-inflammatory cytokine IL-17. Temporal profiling of protein ubiquitination events across a series of time points covering the biological response permits interrogation of signaling through thousands of quantified proteins, of which only a subset display significant and physiologically meaningful regulation. Distinctive clusters of residues within proteins can display distinct temporal patterns attributable to diverse molecular functions, although the majority of differential ubiquitination appears as a coordinated response across the modifiable residues present within an individual substrate. In cells treated with a combination of MEK and PI3K inhibitors we found differential ubiquitination of MEK within the first hour after treatment and a series of mitochondria proteins at later time points. In the IL-17 signaling pathway, ubiquitination events on several signaling proteins including HOIL-1 and TOLLIP were observed. The functional relevance of these putative IL-17 mediators was subsequently validated by knockdown of HOIL-1, HOIP and TOLIP, each of which decreased IL-17 stimulated cytokine production. Together, these data validate proteomic profiling of protein ubiquitination as a viable approach for identifying dynamic signaling components in response to intracellular and extracellular perturbations.





    Categories: Journal Articles
  • Structure of a single-chain Fv bound to the 17N-terminal amino acids of huntingtin provides insights into pathogenic amyloid formation and suppression
    [Apr 2015]

    Publication date: Available online 8 April 2015
    Source:Journal of Molecular Biology

    Author(s): Erwin De Genst , Dimitri Y. Chirgadze , Fabrice A.C. Klein , David C. Butler , Dijana Matak-Vinković , Yvon Trottier , James S. Huston , Anne Messer , Christopher M. Dobson

    Huntington’s disease is triggered by misfolding of fragments of mutant forms of the huntingtin protein (mHTT) with aberrant polyglutamine expansions. The C4 single-chain Fv antibody (scFv), binds to the first 17 residues of huntingtin (HTT(1-17)) and generates substantial protection against multiple phenotypic pathologies in situ and in vivo. We show in this paper how C4 scFv inhibits amyloid formation by exon1 fragments of huntingtin in vitro. To elucidate the structural basis for this inhibition and protection, we have determined the crystal structure of the complex of C4 scFv and HTT(1-17). The peptide binds with residues 3-11 forming an amphipathic helix that makes contact with the antibody fragment in such a way that the hydrophobic face of this helix is shielded from the solvent. Residues 12-17 of the peptide are in an extended conformation and interact with the same region of another C4 scFv:HTT(1-17) complex in the asymmetric unit, resulting in a β-sheet interface within a dimeric C4 scFv:HTT(1-17) complex. The nature of this scFv-peptide complex was further explored in solution by high resolution NMR and physicochemical analysis of species in solution. This structure provides insights into the manner in which C4 scFv inhibits the aggregation of HTT and its therapeutic potential, and may form a structural basis for the possible nature of initial interactions that underlie the formation of disease-associated amyloid fibrils by HTT.
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    Categories: Journal Articles
  • The response of Greek key proteins to changes in connectivity depends on the nature of their secondary structure
    [Apr 2015]

    Publication date: Available online 7 April 2015
    Source:Journal of Molecular Biology

    Author(s): Katherine R. Kemplen , David De Sancho , Jane Clarke

    What governs the balance between connectivity and topology in regulating the mechanism of protein folding? We use circular permutation to vary the order of the helices in all-α Greek key protein FADD to investigate this question. Unlike all-β Greek key proteins, where changes in the order of secondary structure cause a shift in the folding nucleus, the position of the nucleus in FADD is unchanged, even when permutation reduces the complexity significantly. We suggest that this is because local helical contacts are so dominant that permutation has little effect on the entropic cost of forming the folding nucleus whereas, in all-β Greek key proteins, all interactions in the nucleus are long-range. Thus the type of secondary structure modulates the sensitivity of proteins to changes in connectivity.
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    Categories: Journal Articles
  • Mechanistic Asymmetry in Hsp90 Dimers
    [Apr 2015]

    Publication date: Available online 3 April 2015
    Source:Journal of Molecular Biology

    Author(s): Julia M. Flynn , Parul Mishra , Daniel N.A. Bolon

    Hsp90 is a molecular chaperone that facilitates the maturation of signaling proteins including many kinases and steroid hormone receptors. Through these client proteins, Hsp90 is a key mediator of many physiological processes and has emerged as a promising drug target in cancer. Additionally, Hsp90 can mask or potentiate the impact of mutations in clients with remarkable influence on evolutionary adaptations. The influential roles of Hsp90 in biology and disease have stimulated extensive research into the molecular mechanism of this chaperone. These studies have shown that Hsp90 is a homodimeric protein that requires ATP hydrolysis and a host of accessory proteins termed co-chaperones to facilitate the maturation of clients to their active states. Flexible hinge regions between its three structured domains enable Hsp90 to sample dramatically distinct conformations that are influenced by nucleotide, client, and co-chaperone binding. While it is clear that Hsp90 can exist in symmetrical conformations, recent studies have indicated that this homodimeric chaperone can also assume a variety of asymmetric conformations and complexes that are important for client maturation. The visualization of Hsp90-client complexes at high resolution together with tools to independently manipulate each subunit in the Hsp90 dimer are providing new insights into the asymmetric function of each subunit during client maturation.
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    Categories: Journal Articles
  • Small molecule transport by CarO, an abundant eight-stranded β-barrel outer membrane protein from Acinetobacter baumannii
    [Apr 2015]

    Publication date: Available online 3 April 2015
    Source:Journal of Molecular Biology

    Author(s): Michael Zahn , Tommaso D’Agostino , Elif Eren , Arnaud Baslé , Matteo Ceccarelli , Bert van den Berg

    Outer membrane (OM) β-barrel proteins composed of 12-18 β-strands mediate cellular entry of small molecules in Gram-negative bacteria. Small OM proteins with barrels of ten strands or less are not known to transport small molecules. CarO from Acinetobacter baumannii is a small OM protein that has been implicated in the uptake of ornithine and carbapenem antibiotics. Here we report crystal structures of three isoforms of CarO. The structures are very similar and show a monomeric 8-stranded barrel lacking an open channel. CarO has a substantial extracellular domain resembling a glove that contains all the divergent residues between the different isoforms. Liposome swelling experiments demonstrate that full-length CarO as well as a "loop-less" truncation mutant mediate small-molecule uptake at low levels, but that they are unlikely to mediate passage of carbapenem antibiotics. These results are confirmed by biased Molecular Dynamics simulations which allowed us to quantitatively model the transport of selected small molecules.
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    Categories: Journal Articles
  • Integrating –omics: systems biology as explored through C. elegans research
    [Apr 2015]

    Publication date: Available online 1 April 2015
    Source:Journal of Molecular Biology

    Author(s): Roel Van Assche , Valérie Broeckx , Kurt Boonen , Evelyne Maes , Wouter De Haes , Liliane Schoofs , Liesbet Temmerman

    -Omics data have become indispensable to systems biology, which aims to describe the full complexity of functional cells, tissues, organs and organisms. Generating vast amounts of data via such methods, researchers have invested in ways of handling and interpreting these. From the large volumes of -omics data that have been gathered over the years, it is clear that the information derived from one -ome is usually far from complete. Now, individual techniques and methods for integration are maturing to the point that researchers can focus on network-based integration, rather than simply interpreting single-ome studies. This review evaluates the application of integrated -omics approaches with a focus on C. elegans studies, intending to direct researchers in this field to useful databases and inspiring examples.
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    Categories: Journal Articles
  • Unwinding the Mechanisms of a DEAD-Box RNA Helicase in Cancer
    [Apr 2015]

    Publication date: Available online 30 March 2015
    Source:Journal of Molecular Biology

    Author(s): Rick Russell







    Categories: Journal Articles
  • Functional Characterization of a Conserved Archaeal Viral Operon Revealing Single-Stranded DNA Binding, Annealing and Nuclease Activities
    [Apr 2015]

    Publication date: Available online 29 March 2015
    Source:Journal of Molecular Biology

    Author(s): Yang Guo , Birthe B. Kragelund , Malcolm F. White , Xu Peng

    The majority of archaeal viral genes are of unknown function hindering our understanding of the virus life cycle and viral interactions with their host. Here, we first describe functional characterization of ORF131b (gp17) and ORF436 (gp18) of Sulfolobus islandicus rod-shaped virus 2 (SIRV2), both encoding proteins of unknown function and forming an operon with ORF207 (gp19). SIRV2 gp17 was found to be a single-stranded DNA (ssDNA) binding protein different in structure from all previously characterized ssDNA binding proteins. Mutagenesis of a few conserved basic residues suggested a U-shaped binding path for ssDNA. The recombinant gp18 showed an ssDNA annealing activity often associated with helicases and recombinases. To gain insight into the biological role of the entire operon, we characterized SIRV2 gp19 and showed it to possess a 5′→3′ ssDNA exonuclease activity, in addition to the previously demonstrated ssDNA endonuclease activity. Further, in vitro pull-down assay demonstrated interactions between gp17 and gp18 and between gp18 and gp19 with the former being mediated by the intrinsically disordered C-terminus of gp17. The strand-displacement replication mode proposed previously for rudiviruses and the close interaction among the ssDNA binding, annealing and nuclease proteins strongly point to a role of the gene operon in genome maturation and/or DNA recombination that may function in viral DNA replication/repair.
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    Categories: Journal Articles
  • An ATPase-Deficient Variant of the SNF2 Family Member HELLS Shows Altered Dynamics at Pericentromeric Heterochromatin
    [Apr 2015]

    Publication date: Available online 28 March 2015
    Source:Journal of Molecular Biology

    Author(s): Cristiana Lungu , Kathrin Muegge , Albert Jeltsch , Renata Z. Jurkowska

    The HELLS (helicase, lymphoid specific, also known as lymphoid-specific helicase) protein is related to the SNF2 (sucrose non-fermentable 2) family of chromatin remodeling ATPases. It is required for efficient DNA methylation in mammals, particularly at heterochromatin-located repetitive sequences. In this study, we investigated the interaction of HELLS with chromatin and used an ATPase-deficient HELLS variant to address the role of ATP hydrolysis in this process. Chromatin fractionation experiments demonstrated that, in the absence of the ATPase activity, HELLS is retained at the nuclear matrix compartment, defined in part by lamin B1. Microscopy studies revealed a stronger association of the ATPase-deficient mutant with heterochromatin. These results were further supported by fluorescence recovery after photobleaching measurements, which showed that, at heterochromatic sites, wild-type HELLS is very dynamic, with a recovery half-time of 0.8s and a mobile protein fraction of 61%. In contrast, the ATPase-deficient mutant displayed 4.5-s recovery half-time and a reduction in the mobile fraction to 30%. We also present evidence suggesting that, in addition to the ATPase activity, a functional H3K9me3 signaling pathway contributes to an efficient release of HELLS from pericentromeric chromatin. Overall, our results show that a functional ATPase activity is not required for the recruitment of HELLS to heterochromatin, but it is important for the release of the enzyme from these sites.
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    Categories: Journal Articles
  • Editorial Board
    [Apr 2015]

    Publication date: 27 March 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 6, Part A









    Categories: Journal Articles
  • Editorial Board
    [Apr 2015]

    Publication date: 27 March 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 6, Part B









    Categories: Journal Articles
  • Contents List
    [Apr 2015]

    Publication date: 27 March 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 6, Part B









    Categories: Journal Articles
  • Contents List
    [Apr 2015]

    Publication date: 27 March 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 6, Part A









    Categories: Journal Articles
  • By Any Means Necessary: Discovering a Bcl-xL Specific Domain
    [Apr 2015]

    Publication date: 27 March 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 6, Part B

    Author(s): Kurt D. Deshayes







    Categories: Journal Articles
  • Editorial Overview: Elucidation of Protein Translocation Pathways, Part II
    [Apr 2015]

    Publication date: 27 March 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 6, Part A

    Author(s): Dejana Mokranjac , Stephen High







    Categories: Journal Articles