Journal of Molecular Biology

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  • Uncovering the Role of Sgf73 in Maintaining SAGA Deubiquitinating Module Structure and Activity
    [Apr 2015]

    Publication date: 24 April 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 8

    Author(s): Ming Yan , Cynthia Wolberger

    The SAGA (Spt–Ada–Gcn5 acetyltransferase) complex performs multiple functions in transcription activation including deubiquitinating histone H2B, which is mediated by a subcomplex called the deubiquitinating module (DUBm). The yeast DUBm comprises a catalytic subunit, Ubp8, and three additional subunits, Sgf11, Sus1 and Sgf73, all of which are required for DUBm activity. A portion of the non-globular Sgf73 subunit lies between the Ubp8 catalytic domain and the ZnF-UBP domain and has been proposed to contribute to deubiquitinating activity by maintaining the catalytic domain in an active conformation. We report structural and solution studies of the DUBm containing two different Sgf73 point mutations that disrupt deubiquitinating activity. We find that the Sgf73 mutations abrogate deubiquitinating activity by impacting the Ubp8 ubiquitin-binding fingers region and they have an unexpected effect on the overall folding and stability of the DUBm complex. Taken together, our data suggest a role for Sgf73 in maintaining both the organization and the ubiquitin-binding conformation of Ubp8, thereby contributing to overall DUBm activity.
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    Categories: Journal Articles
  • Atomic resolution structures of discrete stages on the reaction coordinate of the [Fe4S4] enzyme IspG (GcpE)
    [Apr 2015]

    Publication date: Available online 11 April 2015
    Source:Journal of Molecular Biology

    Author(s): Felix Quitterer , Annika Frank , Ke Wang , Guodong Rao , Bing O'Dowd , Jikun Li , Francisco Guerra , Safwat Abdel-Azeim , Adelbert Bacher , Jörg Eppinger , Eric Oldfield , Michael Groll

    IspG is the penultimate enzyme in non-mevalonate biosynthesis of the universal terpene building blocks isopentenyl diphosphate and dimethylallyl diphosphate. Its mechanism of action has been the subject of numerous studies but remained unresolved due to difficulties in identifying distinct reaction intermediates. Using a moderate reducing agent as well as an epoxide substrate analogue, we were now able to trap and crystallographically characterize various stages in the IspG catalyzed conversion of 2-C-methyl-D-erythritol-2,4-cyclo-diphosphate (MEcPP) to (E)-1-hydroxy-2-methylbut-2-enyl-4-diphosphate (HMBPP). In addition, the enzyme’s structure was determined in complex with several inhibitors. These results, combined with recent electron paramagnetic resonance data, allowed us to deduce a detailed and complete IspG catalytic mechanism which describes all stages from initial ring opening to formation of HMBPP via discrete radical and carbanion intermediates. The data presented in this article provide a guide for the design of selective drugs against many pro- and eukaryotic pathogens to which the non-mevalonate pathway is essential for survival and virulence.
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    Categories: Journal Articles
  • Sallimus and the dynamics of sarcomere assembly in Drosophila flight muscles
    [Apr 2015]

    Publication date: Available online 11 April 2015
    Source:Journal of Molecular Biology

    Author(s): Zacharias Orfanos , Kevin Leonard , Chris Elliott , Anja Katzemich , Belinda Bullard , John Sparrow

    The Drosophila indirect flight muscles (IFM) can be used as a model for the study of sarcomere assembly. Here we use a transgenic line with a GFP exon inserted into the Z-disc-proximal portion of Sallimus (Sls), also known as Drosophila titin, to observe sarcomere assembly during IFM development. Firstly we confirm that Sls-GFP can be used in the heterozygote state without an obvious phenotype in IFM and other muscles. We then use Sls-GFP in the IFM to show that sarcomeres grow individually and uniformly throughout the fibre, growing in length and diameter linearly. Finally, we show that limiting the amounts of Sls in the IFM using RNAi leads to sarcomeres with smaller Z-discs in their core, while the thick/thin filament lattice can form peripherally without a Z-disc. Thick filament preparations from those muscles show that although the Z-disc-containing core has thick filaments of a regular length, filaments from the peripheral lattice are longer and asymmetrical around the bare zone. Therefore the Z-disc and Sls are required for thick filament length specification, but not for the assembly of the thin/thick filament lattice.
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    Categories: Journal Articles
  • Editorial Board
    [Apr 2015]

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









    Categories: Journal Articles
  • Contents List
    [Apr 2015]

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









    Categories: Journal Articles
  • A First Line of Stress Defense: Small Heat Shock Proteins and Their Function in Protein Homeostasis
    [Apr 2015]

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

    Author(s): Martin Haslbeck , Elizabeth Vierling

    Small heat shock proteins (sHsps) are virtually ubiquitous molecular chaperones that can prevent the irreversible aggregation of denaturing proteins. sHsps complex with a variety of non-native proteins in an ATP-independent manner and, in the context of the stress response, form a first line of defense against protein aggregation in order to maintain protein homeostasis. In vertebrates, they act to maintain the clarity of the eye lens, and in humans, sHsp mutations are linked to myopathies and neuropathies. Although found in all domains of life, sHsps are quite diverse and have evolved independently in metazoans, plants and fungi. sHsp monomers range in size from approximately 12 to 42kDa and are defined by a conserved β-sandwich α-crystallin domain, flanked by variable N- and C-terminal sequences. Most sHsps form large oligomeric ensembles with a broad distribution of different, sphere- or barrel-like oligomers, with the size and structure of the oligomers dictated by features of the N- and C-termini. The activity of sHsps is regulated by mechanisms that change the equilibrium distribution in tertiary features and/or quaternary structure of the sHsp ensembles. Cooperation and/or co-assembly between different sHsps in the same cellular compartment add an underexplored level of complexity to sHsp structure and function.
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  • Protein Quality Control under Oxidative Stress Conditions
    [Apr 2015]

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

    Author(s): Jan-Ulrik Dahl , Michael J. Gray , Ursula Jakob

    Accumulation of reactive oxygen and chlorine species (RO/CS) is generally regarded to be a toxic and highly undesirable event, which serves as contributing factor in aging and many age-related diseases. However, it is also put to excellent use during host defense, when high levels of RO/CS are produced to kill invading microorganisms and regulate bacterial colonization. Biochemical and cell biological studies of how bacteria and other microorganisms deal with RO/CS have now provided important new insights into the physiological consequences of oxidative stress, the major targets that need protection, and the cellular strategies employed by organisms to mitigate the damage. This review examines the redox-regulated mechanisms by which cells maintain a functional proteome during oxidative stress. We will discuss the well-characterized redox-regulated chaperone Hsp33, and we will review recent discoveries demonstrating that oxidative stress-specific activation of chaperone function is a much more widespread phenomenon than previously anticipated. New members of this group include the cytosolic ATPase Get3 in yeast, the Escherichia coli protein RidA, and the mammalian protein α2-macroglobulin. We will conclude our review with recent evidence showing that inorganic polyphosphate (polyP), whose accumulation significantly increases bacterial oxidative stress resistance, works by a protein-like chaperone mechanism. Understanding the relationship between oxidative and proteotoxic stresses will improve our understanding of both host–microbe interactions and how mammalian cells combat the damaging side effects of uncontrolled RO/CS production, a hallmark of inflammation.
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    Categories: Journal Articles
  • Spatially Organized Aggregation of Misfolded Proteins as Cellular Stress Defense Strategy
    [Apr 2015]

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

    Author(s): Stephanie B.M. Miller , Axel Mogk , Bernd Bukau

    An evolutionary conserved response of cells to proteotoxic stress is the organized sequestration of misfolded proteins into subcellular deposition sites. In Saccharomyces cerevisiae, three major sequestration sites for misfolded proteins exist, IPOD (insoluble protein deposit), INQ (intranuclear quality control compartment) [former JUNQ (juxtanuclear quality control compartment)] and CytoQ. IPOD is perivacuolar and predominantly sequesters amyloidogenic proteins. INQ and CytoQs are stress-induced deposits for misfolded proteins residing in the nucleus and the cytosol, respectively, and requiring cell-compartment-specific aggregases, nuclear Btn2 and cytosolic Hsp42 for formation. The organized aggregation of misfolded proteins is proposed to serve several purposes collectively increasing cellular fitness and survival under proteotoxic stress. These include (i) shielding of cellular processes from interference by toxic protein conformers, (ii) reducing the substrate burden for protein quality control systems upon immediate stress, (iii) orchestrating chaperone and protease functions for efficient repair or degradation of damaged proteins [this involves initial extraction of aggregated molecules via the Hsp70/Hsp104 bi-chaperone system followed by either refolding or proteasomal degradation or removal of entire aggregates by selective autophagy (aggrephagy) involving the adaptor protein Cue5] and (iv) enabling asymmetric retention of protein aggregates during cell division, thereby allowing for damage clearance in daughter cells. Regulated protein aggregation thus serves cytoprotective functions vital for the maintenance of cell integrity and survival even under adverse stress conditions and during aging.
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    Categories: Journal Articles
  • How Hsp70 Molecular Machines Interact with Their Substrates to Mediate Diverse Physiological Functions
    [Apr 2015]

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

    Author(s): Eugenia M. Clerico , Joseph M. Tilitsky , Wenli Meng , Lila M. Gierasch

    Hsp70 molecular chaperones are implicated in a wide variety of cellular processes, including protein biogenesis, protection of the proteome from stress, recovery of proteins from aggregates, facilitation of protein translocation across membranes, and more specialized roles such as disassembly of particular protein complexes. It is a fascinating question to ask how the mechanism of these deceptively simple molecular machines is matched to their roles in these wide-ranging processes. The key is a combination of the nature of the recognition and binding of Hsp70 substrates and the impact of Hsp70 action on their substrates. In many cases, the binding, which relies on interaction with an extended, accessible short hydrophobic sequence, favors more unfolded states of client proteins. The ATP-mediated dissociation of the substrate thus releases it in a relatively less folded state for downstream folding, membrane translocation, or hand-off to another chaperone. There are cases, such as regulation of the heat shock response or disassembly of clathrin coats, however, where binding of a short hydrophobic sequence selects conformational states of clients to favor their productive participation in a subsequent step. This Perspective discusses current understanding of how Hsp70 molecular chaperones recognize and act on their substrates and the relationships between these fundamental processes and the functional roles played by these molecular machines.
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  • BiP and Its Nucleotide Exchange Factors Grp170 and Sil1: Mechanisms of Action and Biological Functions
    [Apr 2015]

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

    Author(s): Julia Behnke , Matthias J. Feige , Linda M. Hendershot

    BiP (immunoglobulin heavy-chain binding protein) is the endoplasmic reticulum (ER) orthologue of the Hsp70 family of molecular chaperones and is intricately involved in most functions of this organelle through its interactions with a variety of substrates and regulatory proteins. Like all Hsp70 family members, the ability of BiP to bind and release unfolded proteins is tightly regulated by a cycle of ATP binding, hydrolysis, and nucleotide exchange. As a characteristic of the Hsp70 family, multiple DnaJ-like co-factors can target substrates to BiP and stimulate its ATPase activity to stabilize the binding of BiP to substrates. However, only in the past decade have nucleotide exchange factors for BiP been identified, which has shed light not only on the mechanism of BiP-assisted folding in the ER but also on Hsp70 family members that reside throughout the cell. We will review the current understanding of the ATPase cycle of BiP in the unique environment of the ER and how it is regulated by the nucleotide exchange factors, Grp170 (glucose-regulated protein of 170 kDa) and Sil1, both of which perform unanticipated roles in various biological functions and disease states.
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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