Journal of Molecular Biology

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  • Editorial Board
    [May 2014]

    Publication date: 29 May 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 11









    Categories: Journal Articles
  • Contents List
    [May 2014]

    Publication date: 29 May 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 11









    Categories: Journal Articles
  • Separate Molecular Determinants in Amyloidogenic and Antimicrobial Peptides
    [May 2014]

    Publication date: 29 May 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 11

    Author(s): Michael Landreh , Jan Johansson , Hans Jörnvall

    Several amyloid-forming and antimicrobial peptides (AMYs and AMPs) have the ability to bind to and damage cell membranes. In addition, some AMYs possess antimicrobial activity and some AMPs form amyloid-like fibrils, relating the two peptide types and their properties. However, a comparison of their sequence characteristics reveals important differences. The high β-strand and aggregation propensities typical of AMYs are largely absent in α-helix-forming AMPs, which are instead marked by a strong amphipathic moment not generally found in AMYs. Although a few peptides, for example, islet amyloid polypeptide and dermaseptin S9, combine some determinants of both groups, the structural distinctions suggest that antimicrobial activity and amyloid formation are separate features not generally associated.
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    Categories: Journal Articles
  • Identification of the Active Sites in the Methyltransferases of a Transcribing dsRNA Virus
    [May 2014]

    Publication date: 29 May 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 11

    Author(s): Bin Zhu , Chongwen Yang , Hongrong Liu , Lingpeng Cheng , Feng Song , Songjun Zeng , Xiaojun Huang , Gang Ji , Ping Zhu

    Many double-stranded RNA (dsRNA) viruses are capable of transcribing and capping RNA within a stable icosahedral viral capsid. The turret of turreted dsRNA viruses belonging to the family Reoviridae is formed by five copies of the turret protein, which contains domains with both 7-N-methyltransferase and 2′-O-methyltransferase activities, and serves to catalyze the methylation reactions during RNA capping. Cypovirus of the family Reoviridae provides a good model system for studying the methylation reactions in dsRNA viruses. Here, we present the structure of a transcribing cypovirus to a resolution of ~3.8Å by cryo-electron microscopy. The binding sites for both S-adenosyl-l-methionine and RNA in the two methyltransferases of the turret were identified. Structural analysis of the turret in complex with RNA revealed a pathway through which the RNA molecule reaches the active sites of the two methyltransferases before it is released into the cytoplasm. The pathway shows that RNA capping reactions occur in the active sites of different turret protein monomers, suggesting that RNA capping requires concerted efforts by at least three turret protein monomers. Thus, the turret structure provides novel insights into the precise mechanisms of RNA methylation.
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    Categories: Journal Articles
  • Post-Translational Regulation of CD133 by ATase1/ATase2-Mediated Lysine Acetylation
    [May 2014]

    Publication date: 29 May 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 11

    Author(s): Anthony B. Mak , Mariana Pehar , Allison M.L. Nixon , Rashida A. Williams , Andrea C. Uetrecht , Luigi Puglielli , Jason Moffat

    The CD133 cell-surface protein expresses the AC133 epitope that is associated with cancer progenitor cells and cancer resistance to traditional anticancer therapies. We report that the endoplasmic reticulum Golgi intermediate compartment residing acetyltransferases, ATase1 (NAT8B) and ATase2 (NAT8), can physically interact with CD133 to acetylate the protein on three lysine residues predicted to reside on the first extracellular loop of CD133. Site-directed mutagenesis of these residues mimicking a loss of acetylation and downregulation or inhibition of ATase1/ATase2 resulted in near-complete abolishment of CD133 protein expression. We also demonstrate that targeting ATase1/ATase2 results in apoptosis of CD133 expressing acute lymphoblastic leukemia cells. Taken together, we suggest that lysine acetylation on predicted extracellular residues plays a key role in expression and trafficking of CD133 protein to the cell surface and can be targeted to disrupt CD133 regulation and function.
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  • APE1 Incision Activity at Abasic Sites in Tandem Repeat Sequences
    [May 2014]

    Publication date: 29 May 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 11

    Author(s): Mengxia Li , Jens Völker , Kenneth J. Breslauer , David M. Wilson III

    Repetitive DNA sequences, such as those present in microsatellites and minisatellites, telomeres, and trinucleotide repeats (linked to fragile X syndrome, Huntington disease, etc.), account for nearly 30% of the human genome. These domains exhibit enhanced susceptibility to oxidative attack to yield base modifications, strand breaks, and abasic sites; have a propensity to adopt non-canonical DNA forms modulated by the positions of the lesions; and, when not properly processed, can contribute to genome instability that underlies aging and disease development. Knowledge on the repair efficiencies of DNA damage within such repetitive sequences is therefore crucial for understanding the impact of such domains on genomic integrity. In the present study, using strategically designed oligonucleotide substrates, we determined the ability of human apurinic/apyrimidinic endonuclease 1 (APE1) to cleave at apurinic/apyrimidinic (AP) sites in a collection of tandem DNA repeat landscapes involving telomeric and CAG/CTG repeat sequences. Our studies reveal the differential influence of domain sequence, conformation, and AP site location/relative positioning on the efficiency of APE1 binding and strand incision. Intriguingly, our data demonstrate that APE1 endonuclease efficiency correlates with the thermodynamic stability of the DNA substrate. We discuss how these results have both predictive and mechanistic consequences for understanding the success and failure of repair protein activity associated with such oxidatively sensitive, conformationally plastic/dynamic repetitive DNA domains.
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    Categories: Journal Articles
  • Mitochondrial DNA Variant in COX1 Subunit Significantly Alters Energy Metabolism of Geographically Divergent Wild Isolates in Caenorhabditis elegans
    [May 2014]

    Publication date: 29 May 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 11

    Author(s): Stephen D. Dingley , Erzsebet Polyak , Julian Ostrovsky , Satish Srinivasan , Icksoo Lee , Amy B. Rosenfeld , Mai Tsukikawa , Rui Xiao , Mary A. Selak , Joshua J. Coon , Alexander S. Hebert , Paul A. Grimsrud , Young Joon Kwon , David J. Pagliarini , Xiaowu Gai , Theodore G. Schurr , Maik Hüttemann , Eiko Nakamaru-Ogiso , Marni J. Falk

    Mitochondrial DNA (mtDNA) sequence variation can influence the penetrance of complex diseases and climatic adaptation. While studies in geographically defined human populations suggest that mtDNA mutations become fixed when they have conferred metabolic capabilities optimally suited for a specific environment, it has been challenging to definitively assign adaptive functions to specific mtDNA sequence variants in mammals. We investigated whether mtDNA genome variation functionally influences Caenorhabditis elegans wild isolates of distinct mtDNA lineages and geographic origins. We found that, relative to N2 (England) wild-type nematodes, CB4856 wild isolates from a warmer native climate (Hawaii) had a unique p.A12S amino acid substitution in the mtDNA-encoded COX1 core catalytic subunit of mitochondrial complex IV (CIV). Relative to N2, CB4856 worms grown at 20°C had significantly increased CIV enzyme activity, mitochondrial matrix oxidant burden, and sensitivity to oxidative stress but had significantly reduced lifespan and mitochondrial membrane potential. Interestingly, mitochondrial membrane potential was significantly increased in CB4856 grown at its native temperature of 25°C. A transmitochondrial cybrid worm strain, chpIR (M, CB4856>N2), was bred as homoplasmic for the CB4856 mtDNA genome in the N2 nuclear background. The cybrid strain also displayed significantly increased CIV activity, demonstrating that this difference results from the mtDNA-encoded p.A12S variant. However, chpIR (M, CB4856>N2) worms had significantly reduced median and maximal lifespan relative to CB4856, which may relate to their nuclear–mtDNA genome mismatch. Overall, these data suggest that C. elegans wild isolates of varying geographic origins may adapt to environmental challenges through mtDNA variation to modulate critical aspects of mitochondrial energy metabolism.
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    Categories: Journal Articles
  • Assembly of Robust Bacterial Microcompartment Shells Using Building Blocks from an Organelle of Unknown Function
    [May 2014]

    Publication date: 29 May 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 11

    Author(s): Jonathan K. Lassila , Susan L. Bernstein , James N. Kinney , Seth D. Axen , Cheryl A. Kerfeld

    Bacterial microcompartments (BMCs) sequester enzymes from the cytoplasmic environment by encapsulation inside a selectively permeable protein shell. Bioinformatic analyses indicate that many bacteria encode BMC clusters of unknown function and with diverse combinations of shell proteins. The genome of the halophilic myxobacterium Haliangium ochraceum encodes one of the most atypical sets of shell proteins in terms of composition and primary structure. We found that microcompartment shells could be purified in high yield when all seven H. ochraceum BMC shell genes were expressed from a synthetic operon in Escherichia coli. These shells differ substantially from previously isolated shell systems in that they are considerably smaller and more homogeneous, with measured diameters of 39±2nm. The size and nearly uniform geometry allowed the development of a structural model for the shells composed of 260 hexagonal units and 13 hexagons per icosahedral face. We found that new proteins could be recruited to the shells by fusion to a predicted targeting peptide sequence, setting the stage for the use of these remarkably homogeneous shells for applications such as three-dimensional scaffolding and the construction of synthetic BMCs. Our results demonstrate the value of selecting from the diversity of BMC shell building blocks found in genomic sequence data for the construction of novel compartments.
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    Categories: Journal Articles
  • Architectures of Whole-Module and Bimodular Proteins from the 6-Deoxyerythronolide B Synthase
    [May 2014]

    Publication date: 29 May 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 11

    Author(s): Andrea L. Edwards , Tsutomu Matsui , Thomas M. Weiss , Chaitan Khosla

    The 6-deoxyerythronolide B synthase (DEBS) is a prototypical assembly line polyketide synthase produced by the actinomycete Saccharopolyspora erythraea that synthesizes the macrocyclic core of the antibiotic erythromycin 6-deoxyerythronolide B. The megasynthase is a 2-MDa trimeric complex composed of three unique homodimers assembled from the gene products DEBS1, DEBS2, and DEBS3, which are housed within the erythromycin biosynthetic gene cluster. Each homodimer contains two clusters of catalytically independent enzymatic domains, each referred to as a module, which catalyzes one round of polyketide chain extension and modification. Modules are named sequentially to indicate the order in which they are utilized during synthesis of 6-deoxyerythronolide B. We report small-angle X-ray scattering (SAXS) analyses of a whole module and a bimodule from DEBS, as well as a set of domains for which high-resolution structures are available. In all cases, the solution state was probed under previously established conditions ensuring that each protein is catalytically active. SAXS data are consistent with atomic-resolution structures of DEBS fragments. Therefore, we used the available high-resolution structures of DEBS domains to model the architectures of the larger protein assemblies using rigid-body refinement. Our data support a model in which the third module of DEBS forms a disc-shaped structure capable of caging the acyl carrier protein domain proximal to each active site. The molecular envelope of DEBS3 is a thin elongated ellipsoid, and the results of rigid-body modeling suggest that modules 5 and 6 stack collinearly along the 2-fold axis of symmetry.
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    Categories: Journal Articles
  • Why Have Small Multidrug Resistance Proteins Not Evolved into Fused, Internally Duplicated Structures?
    [May 2014]

    Publication date: 29 May 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 11

    Author(s): Pilar Lloris-Garcerá , Susanna Seppälä , Joanna S.G. Slusky , Mikaela Rapp , Gunnar von Heijne

    The increasing number of solved membrane protein structures has led to the recognition of a common feature in a large fraction of the small-molecule transporters: inverted repeat structures, formed by two fused homologous membrane domains with opposite orientation in the membrane. An evolutionary pathway in which the ancestral state is a single gene encoding a dual-topology membrane protein capable of forming antiparallel homodimers has been posited. A gene duplication event enables the evolution of two oppositely orientated proteins that form antiparallel heterodimers. Finally, fusion of the two genes generates an internally duplicated transporter with two oppositely orientated membrane domains. Strikingly, however, in the small multidrug resistance (SMR) family of transporters, no fused, internally duplicated proteins have been found to date. Here, we have analyzed fused versions of the dual-topology transporter EmrE, a member of the SMR family, by blue-native PAGE and in vivo activity measurements. We find that fused constructs give rise to both intramolecular inverted repeat structures and competing intermolecular dimers of varying activity. The formation of several intramolecularly and intermolecularly paired species indicates that a gene fusion event may lower the overall amount of active protein, possibly explaining the apparent absence of fused SMR proteins in nature.
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    Categories: Journal Articles
  • Systematic Detection of Internal Symmetry in Proteins Using CE-Symm
    [May 2014]

    Publication date: 29 May 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 11

    Author(s): Douglas Myers-Turnbull , Spencer E. Bliven , Peter W. Rose , Zaid K. Aziz , Philippe Youkharibache , Philip E. Bourne , Andreas Prlić

    Symmetry is an important feature of protein tertiary and quaternary structures that has been associated with protein folding, function, evolution, and stability. Its emergence and ensuing prevalence has been attributed to gene duplications, fusion events, and subsequent evolutionary drift in sequence. This process maintains structural similarity and is further supported by this study. To further investigate the question of how internal symmetry evolved, how symmetry and function are related, and the overall frequency of internal symmetry, we developed an algorithm, CE-Symm, to detect pseudo-symmetry within the tertiary structure of protein chains. Using a large manually curated benchmark of 1007 protein domains, we show that CE-Symm performs significantly better than previous approaches. We use CE-Symm to build a census of symmetry among domain superfamilies in SCOP and note that 18% of all superfamilies are pseudo-symmetric. Our results indicate that more domains are pseudo-symmetric than previously estimated. We establish a number of recurring types of symmetry–function relationships and describe several characteristic cases in detail. With the use of the Enzyme Commission classification, symmetry was found to be enriched in some enzyme classes but depleted in others. CE-Symm thus provides a methodology for a more complete and detailed study of the role of symmetry in tertiary protein structure [availability: CE-Symm can be run from the Web at http://source.rcsb.org/jfatcatserver/symmetry.jsp. Source code and software binaries are also available under the GNU Lesser General Public License (version 2.1) at https://github.com/rcsb/symmetry. An interactive census of domains identified as symmetric by CE-Symm is available from http://source.rcsb.org/jfatcatserver/scopResults.jsp].
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    Categories: Journal Articles
  • Editorial Board
    [May 2014]

    Publication date: 15 May 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 10









    Categories: Journal Articles
  • Contents List
    [May 2014]

    Publication date: 15 May 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 10









    Categories: Journal Articles
  • A New Crystal Structure of the Bifunctional Antibiotic Simocyclinone D8 Bound to DNA Gyrase Gives Fresh Insight into the Mechanism of Inhibition
    [May 2014]

    Publication date: 15 May 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 10

    Author(s): Stephen J. Hearnshaw , Marcus J. Edwards , Clare E. Stevenson , David M. Lawson , Anthony Maxwell

    Simocyclinone D8 (SD8) is an antibiotic produced by Streptomyces antibioticus that targets DNA gyrase. A previous structure of SD8 complexed with the N-terminal domain of the DNA gyrase A protein (GyrA) suggested that four SD8 molecules stabilized a tetramer of the protein; subsequent mass spectrometry experiments suggested that a protein dimer with two symmetry-related SD8s was more likely. This work describes the structures of a further truncated form of the GyrA N-terminal domain fragment with and without SD8 bound. The structure with SD8 has the two SD8 molecules bound within the same GyrA dimer. This new structure is entirely consistent with the mutations in GyrA that confer SD8 resistance and, by comparison with a new apo structure of the GyrA N-terminal domain, reveals the likely conformation changes that occur upon SD8 binding and the detailed mechanism of SD8 inhibition of gyrase. Isothermal titration calorimetry experiments are consistent with the crystallography results and further suggest that a previously observed complex between SD8 and GyrB is ~1000-fold weaker than the interaction with GyrA.
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    Categories: Journal Articles
  • The Histone H4 Tail Regulates the Conformation of the ATP-Binding Pocket in the SNF2h Chromatin Remodeling Enzyme
    [May 2014]

    Publication date: 15 May 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 10

    Author(s): Lisa R. Racki , Nariman Naber , Ed Pate , John D. Leonard , Roger Cooke , Geeta J. Narlikar

    The chromatin remodeling complex ACF helps establish the appropriate nucleosome spacing for generating repressed chromatin states. ACF activity is stimulated by two defining features of the nucleosomal substrate: a basic patch on the histone H4 N-terminal tail and the specific length of flanking DNA. However, the mechanisms by which these two substrate cues function in the ACF remodeling reaction is not well understood. Using electron paramagnetic resonance spectroscopy with spin-labeled ATP analogs to probe the structure of the ATP active site under physiological solution conditions, we identify a closed state of the ATP-binding pocket that correlates with ATPase activity. We find that the H4 tail promotes pocket closure. We further show that ATPase stimulation by the H4 tail does not require a specific structure connecting the H4 tail and the globular domain. In the case of many DNA helicases, closure of the ATP-binding pocket is regulated by specific DNA substrates. Pocket closure by the H4 tail may analogously provide a mechanism to directly couple substrate recognition to activity. Surprisingly, the flanking DNA, which also stimulates ATP hydrolysis, does not promote pocket closure, suggesting that the H4 tail and flanking DNA may be recognized in different reaction steps.
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    Categories: Journal Articles
  • In Silico Derived Small Molecules Bind the Filovirus VP35 Protein and Inhibit Its Polymerase Cofactor Activity
    [May 2014]

    Publication date: 15 May 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 10

    Author(s): Craig S. Brown , Michael S. Lee , Daisy W. Leung , Tianjiao Wang , Wei Xu , Priya Luthra , Manu Anantpadma , Reed S. Shabman , Lisa M. Melito , Karen S. MacMillan , Dominika M. Borek , Zbyszek Otwinowski , Parameshwaran Ramanan , Alisha J. Stubbs , Dayna S. Peterson , Jennifer M. Binning , Marco Tonelli , Mark A. Olson , Robert A. Davey , Joseph M. Ready , Christopher F. Basler , Gaya K. Amarasinghe

    The Ebola virus (EBOV) genome only encodes a single viral polypeptide with enzymatic activity, the viral large (L) RNA-dependent RNA polymerase protein. However, currently, there is limited information about the L protein, which has hampered the development of antivirals. Therefore, antifiloviral therapeutic efforts must include additional targets such as protein–protein interfaces. Viral protein 35 (VP35) is multifunctional and plays important roles in viral pathogenesis, including viral mRNA synthesis and replication of the negative-sense RNA viral genome. Previous studies revealed that mutation of key basic residues within the VP35 interferon inhibitory domain (IID) results in significant EBOV attenuation, both in vitro and in vivo. In the current study, we use an experimental pipeline that includes structure-based in silico screening and biochemical and structural characterization, along with medicinal chemistry, to identify and characterize small molecules that target a binding pocket within VP35. NMR mapping experiments and high-resolution x-ray crystal structures show that select small molecules bind to a region of VP35 IID that is important for replication complex formation through interactions with the viral nucleoprotein (NP). We also tested select compounds for their ability to inhibit VP35 IID–NP interactions in vitro as well as VP35 function in a minigenome assay and EBOV replication. These results confirm the ability of compounds identified in this study to inhibit VP35–NP interactions in vitro and to impair viral replication in cell-based assays. These studies provide an initial framework to guide development of antifiloviral compounds against filoviral VP35 proteins.
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    Categories: Journal Articles
  • 3D Cryo-Electron Reconstruction of BmrA, a Bacterial Multidrug ABC Transporter in an Inward-Facing Conformation and in a Lipidic Environment
    [May 2014]

    Publication date: 15 May 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 10

    Author(s): Pierre Frederic Fribourg , Mohamed Chami , Carlos Oscar S. Sorzano , Francesca Gubellini , Roberto Marabini , Sergio Marco , Jean-Michel Jault , Daniel Lévy

    ABC (ATP-binding cassette) membrane exporters are efflux transporters of a wide diversity of molecule across the membrane at the expense of ATP. A key issue regarding their catalytic cycle is whether or not their nucleotide-binding domains (NBDs) are physically disengaged in the resting state. To settle this controversy, we obtained structural data on BmrA, a bacterial multidrug homodimeric ABC transporter, in a membrane-embedded state. BmrA in the apostate was reconstituted in lipid bilayers forming a mixture of ring-shaped structures of 24 or 39 homodimers. Three-dimensional models of the ring-shaped structures of 24 or 39 homodimers were calculated at 2.3nm and 2.5nm resolution from cryo-electron microscopy, respectively. In these structures, BmrA adopts an inward-facing open conformation similar to that found in mouse P-glycoprotein structure with the NBDs separated by 3nm. Both lipidic leaflets delimiting the transmembrane domains of BmrA were clearly resolved. In planar membrane sheets, the NBDs were even more separated. BmrA in an ATP-bound conformation was determined from two-dimensional crystals grown in the presence of ATP and vanadate. A projection map calculated at 1.6nm resolution shows an open outward-facing conformation. Overall, the data are consistent with a mechanism of drug transport involving large conformational changes of BmrA and show that a bacterial ABC exporter can adopt at least two open inward conformations in lipid membrane.
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    Categories: Journal Articles
  • Crystal Structure of Human Myosin 1c—The Motor in GLUT4 Exocytosis: Implications for Ca2+ Regulation and 14-3-3 Binding
    [May 2014]

    Publication date: 15 May 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 10

    Author(s): Stefan Münnich , Manuel H. Taft , Dietmar J. Manstein

    Myosin 1c (Myo1c) plays a key role in supporting motile events that underlie cell migration, vesicle trafficking, insulin-stimulated glucose uptake and hearing. Here, we present the crystal structure of the human Myo1c motor in complex with its light chain calmodulin. Our structure reveals tight interactions of the motor domain with calmodulin bound to the first IQ motif in the neck region. Several of the calmodulin residues contributing to this interaction are also involved in Ca2+ binding. Contact residues in the motor domain are linked to the central β-sheet and the HO helix, suggesting a mechanism for communicating changes in Ca2+ binding in the neck region to the actin and nucleotide binding regions of the motor domain. The structural context and the chemical environment of Myo1c mutations that are involved in sensorineural hearing loss in humans are described and their impact on motor function is discussed. We show that a construct consisting of the motor domain of Myo1c and the first IQ motif is sufficient to establish a tight interaction with 14-3-3β (K D =0.9μM) and present the model of a double-headed Myo1c–14-3-3 complex. This complex has been implicated in the exocytosis of glucose transporter 4 storage vesicles during insulin-stimulated glucose uptake.
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    Categories: Journal Articles
  • Intrinsic Disorder Mediates Cooperative Signal Transduction in STIM1
    [May 2014]

    Publication date: 15 May 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 10

    Author(s): Yukio Furukawa , Shunsuke Teraguchi , Takahisa Ikegami , Onur Dagliyan , Lin Jin , Damien Hall , Nikolay V. Dokholyan , Keiichi Namba , Shizuo Akira , Tomohiro Kurosaki , Yoshihiro Baba , Daron M. Standley

    Intrinsically disordered domains have been reported to play important roles in signal transduction networks by introducing cooperativity into protein–protein interactions. Unlike intrinsically disordered domains that become ordered upon binding, the EF-SAM domain in the stromal interaction molecule (STIM) 1 is distinct in that it is ordered in the monomeric state and partially unfolded in its oligomeric state, with the population of the two states depending on the local Ca2+ concentration. The oligomerization of STIM1, which triggers extracellular Ca2+ influx, exhibits cooperativity with respect to the local endoplasmic reticulum Ca2+ concentration. Although the physiological importance of the oligomerization reaction is well established, the mechanism of the observed cooperativity is not known. Here, we examine the response of the STIM1 EF-SAM domain to changes in Ca2+ concentration using mathematical modeling based on in vitro experiments. We find that the EF-SAM domain partially unfolds and dimerizes cooperatively with respect to Ca2+ concentration, with Hill coefficients and half-maximal activation concentrations very close to the values observed in vivo for STIM1 redistribution and extracellular Ca2+ influx. Our mathematical model of the dimerization reaction agrees quantitatively with our analytical ultracentrifugation-based measurements and previously published free energies of unfolding. A simple interpretation of these results is that Ca2+ loss effectively acts as a denaturant, enabling cooperative dimerization and robust signal transduction. We present a structural model of the Ca2+-unbound EF-SAM domain that is consistent with a wide range of evidence, including resistance to proteolytic cleavage of the putative dimerization portion.
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    Categories: Journal Articles
  • Exploring the Biological and Chemical Complexity of the Ligases
    [May 2014]

    Publication date: 15 May 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 10

    Author(s): Gemma L. Holliday , Syed Asad Rahman , Nicholas Furnham , Janet M. Thornton

    Using a novel method to map and cluster chemical reactions, we have re-examined the chemistry of the ligases [Enzyme Commission (EC) Class 6] and their associated protein families in detail. The type of bond formed by the ligase can be automatically extracted from the equation of the reaction, replicating the EC subclass division. However, this subclass division hides considerable complexities, especially for the C–N forming ligases, which fall into at least three distinct types. The lower levels of the EC classification for ligases are somewhat arbitrary in their definition and add little to understanding their chemistry or evolution. By comparing the multi-domain architecture of the enzymes and using sequence similarity networks, we examined the links between overall reaction and evolution of the ligases. These show that, whilst many enzymes that perform the same overall chemistry group together, both convergent (similar function, different ancestral lineage) and divergent (different function, common ancestor) evolution of function are observed. However, a common theme is that a single conserved domain (often the nucleoside triphosphate binding domain) is combined with ancillary domains that provide the variation in substrate binding and function.
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    Categories: Journal Articles