Journal of Molecular Biology

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  • Galectin-3 Interactions with Glycosphingolipids
    [Apr 2014]

    Publication date: 3 April 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 7

    Author(s): Patrick M. Collins , Khuchtumur Bum-Erdene , Xing Yu , Helen Blanchard

    Galectins have essential roles in pathological states including cancer, inflammation, angiogenesis and microbial infections. Endogenous receptors include members of the lacto- and neolacto-series glycosphingolipids present on mammalian cells and contain the tetrasaccharides lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT) that form their core structural components and also ganglio-series glycosphingolipids. We present crystallographic structures of the carbohydrate recognition domain of human galectin-3, both wild type and a mutant (K176L) that influenced ligand affinity, in complex with LNT, LNnT and acetamido ganglioside a-GM3 (α2,3-sialyllactose). Key structural features revealed include galectin-3's demonstration of a binding mode towards gangliosides distinct from that to the lacto/neolacto-glycosphingolipids, with its capacity for recognising the core β-galactoside region being challenged when the core oligosaccharide epitope of ganglio-series glycosphingolipids (GM3) is embedded within particular higher-molecular-weight glycans. The lacto- and neolacto- glycosphingolipids revealed different orientations of their terminal galactose in the galectin-3-bound LNT and LNnT structures that has significant ramifications for the capacity of galectin-3 to interact with higher-order lacto/neolacto-series glycosphingolipids such as ABH blood group antigens and the HNK-1 antigen that is common on leukocytes. LNnT also presents an important model for poly-N-acetyllactosamine-containing glycans and provides insight into galectin-3's accommodation of extended oligosaccharides such as the poly-N-acetyllactosamine-modified N- and O-glycans that, via galectin-3 interaction, facilitate progression of lung and bladder cancers, respectively. These findings provide the first atomic detail of galectin-3's interactions with the core structures of mammalian glycosphingolipids, providing information important in understanding the capacity of galectin-3 to engage with receptors identified as facilitators of major disease.
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  • Structural Characterization of the Glycoprotein GP2 Core Domain from the CAS Virus, a Novel Arenavirus-Like Species
    [Apr 2014]

    Publication date: 3 April 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 7

    Author(s): Jayne F. Koellhoffer , Zhou Dai , Vladimir N. Malashkevich , Mark D. Stenglein , Yanyun Liu , Rafael Toro , Joseph S. Harrison , Kartik Chandran , Joseph L. DeRisi , Steven C. Almo , Jonathan R. Lai

    Fusion of the viral and host cell membranes is a necessary first step for infection by enveloped viruses and is mediated by the envelope glycoprotein. The transmembrane subunits from the structurally defined “class I” glycoproteins adopt an α-helical “trimer-of-hairpins” conformation during the fusion pathway. Here, we present our studies on the envelope glycoprotein transmembrane subunit, GP2, of the CAS virus (CASV). CASV was recently identified from annulated tree boas (Corallus annulatus) with inclusion body disease and is implicated in the disease etiology. We have generated and characterized two protein constructs consisting of the predicted CASV GP2 core domain. The crystal structure of the CASV GP2 post-fusion conformation indicates a trimeric α-helical bundle that is highly similar to those of Ebola virus and Marburg virus GP2 despite CASV genome homology to arenaviruses. Denaturation studies demonstrate that the stability of CASV GP2 is pH dependent with higher stability at lower pH; we propose that this behavior is due to a network of interactions among acidic residues that would destabilize the α-helical bundle under conditions where the side chains are deprotonated. The pH-dependent stability of the post-fusion structure has been observed in Ebola virus and Marburg virus GP2, as well as other viruses that enter via the endosome. Infection experiments with CASV and the related Golden Gate virus support a mechanism of entry that requires endosomal acidification. Our results suggest that, despite being primarily arenavirus like, the transmembrane subunit of CASV is extremely similar to the filoviruses.
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  • Structural and Functional Analysis of Fucose-Processing Enzymes from Streptococcus pneumoniae
    [Apr 2014]

    Publication date: 3 April 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 7

    Author(s): Melanie A. Higgins , Michael D. Suits , Candace Marsters , Alisdair B. Boraston

    Fucose metabolism pathways are present in many bacterial species and typically contain the central fucose-processing enzymes fucose isomerase (FcsI), fuculose kinase (FcsK), and fuculose-1-phosphate aldolase (FcsA). Fucose initially undergoes isomerization by FcsI producing fuculose, which is then phosphorylated by FcsK. FcsA cleaves the fuculose-1-phosphate product into lactaldehyde and dihydroxyacetone phosphate, which can be incorporated into central metabolism allowing the bacterium to use fucose as an energy source. Streptococcus pneumoniae has fucose-processing operons containing homologs of FcsI, FcsK, and FcsA; however, this bacterium appears unable to utilize fucose as an energy source. To investigate this contradiction, we performed biochemical and structural studies of the S. pneumoniae fucose-processing enzymes SpFcsI, SpFcsK, and SpFcsA. These enzymes are demonstrated to act in a sequential manner to ultimately produce dihydroxyacetone phosphate and have structural features entirely consistent with their observed biochemical activities. Analogous to the regulation of the Escherichia coli fucose utilization operon, fuculose-1-phosphate appears to act as an inducing molecule for activation of the S. pneumoniae fucose operon. Despite our evidence that S. pneumoniae appears to have the appropriate regulatory and biochemical machinery for fucose metabolism, we confirmed the inability of the S. pneumoniae TIGR4 strain to grow on fucose or on the H-disaccharide, which is the probable substrate of the transporter for the pathway. On the basis of these observations, we postulate that the S. pneumoniae fucose-processing pathway has a non-metabolic role in the interaction of this bacterium with its human host.
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  • The Solution Structure of the Regulatory Domain of Tyrosine Hydroxylase
    [Apr 2014]

    Publication date: 3 April 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 7

    Author(s): Shengnan Zhang , Tao Huang , Udayar Ilangovan , Andrew P. Hinck , Paul F. Fitzpatrick

    Tyrosine hydroxylase (TyrH) catalyzes the hydroxylation of tyrosine to form 3,4-dihydroxyphenylalanine in the biosynthesis of the catecholamine neurotransmitters. The activity of the enzyme is regulated by phosphorylation of serine residues in a regulatory domain and by binding of catecholamines to the active site. Available structures of TyrH lack the regulatory domain, limiting the understanding of the effect of regulation on structure. We report the use of NMR spectroscopy to analyze the solution structure of the isolated regulatory domain of rat TyrH. The protein is composed of a largely unstructured N-terminal region (residues 1–71) and a well-folded C-terminal portion (residues 72–159). The structure of a truncated version of the regulatory domain containing residues 65–159 has been determined and establishes that it is an ACT domain. The isolated domain is a homodimer in solution, with the structure of each monomer very similar to that of the core of the regulatory domain of phenylalanine hydroxylase. Two TyrH regulatory domain monomers form an ACT domain dimer composed of a sheet of eight strands with four α-helices on one side of the sheet. Backbone dynamic analyses were carried out to characterize the conformational flexibility of TyrH65–159. The results provide molecular details critical for understanding the regulatory mechanism of TyrH.
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  • The N-Terminal Amphipathic Helices Determine Regulatory and Effector Functions of Phage Shock Protein A (PspA) in Escherichia coli
    [Apr 2014]

    Publication date: 3 April 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 7

    Author(s): Goran Jovanovic , Parul Mehta , Christopher McDonald , Anthony C. Davidson , Povilas Uzdavinys , Liming Ying , Martin Buck

    The phage shock protein (Psp) systems found in bacteria, archaea and higher plants respond to extracytoplasmic stresses that damage the cytoplasmic membrane and enable cells to repair their membranes. The conserved membrane-associated effector protein PspA has four α-helical domains (HD1–HD4) and helps to repair the membrane as a high-order oligomer. In enterobacteria, under non-stress conditions, PspA as a low-order assembly directly inhibits its cognate transcription activator PspF. Here we show that N-terminal amphipathic helices ahA and ahB in PspA HD1 are functional determinants involved in negative gene control and stress signal perception and its transduction via interactions with the PspBC membrane stress sensors and the inner membrane (IM). The amphipathic helices enable PspA to switch from a low-order gene regulator into an IM-bound high-order effector complex under membrane stress. Conserved residue proline 25 is involved in sequential use of the amphipathic helices and ahA–IM interaction. Single molecule imaging of eGFP-PspA and its amphipathic helices variants in live Escherichia coli cells show distinct spatial and temporal organisations of PspA corresponding to its negative control and effector functions. These findings inform studies on the role of the Psp system in persister cell formation and cell envelope protection in bacterial pathogens and provide a basis for exploring the specialised roles of PspA homologues such as YjfJ, LiaH and Vipp1.
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    Categories: Journal Articles
  • The 1.8-Å Crystal Structure of the N-Terminal Domain of an Archaeal MCM as a Right-Handed Filament
    [Apr 2014]

    Publication date: 3 April 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 7

    Author(s): Yang Fu , Ian M. Slaymaker , Junfeng Wang , Ganggang Wang , Xiaojiang S. Chen

    Mini-chromosome maintenance (MCM) proteins are the replicative helicase necessary for DNA replication in both eukarya and archaea. Most of archaea only have one MCM gene. Here, we report a 1.8-Å crystal structure of the N-terminal MCM from the archaeon Thermoplasma acidophilum (tapMCM). In the structure, the MCM N-terminus forms a right-handed filament that contains six subunits in each turn, with a diameter of 25Å of the central channel opening. The inner surface is highly positively charged, indicating DNA binding. This filament structure with six subunits per turn may also suggests a potential role for an open-ring structure for hexameric MCM and dynamic conformational changes in initiation and elongation stages of DNA replication.
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  • Structure and Dynamics of DNA Duplexes Containing a Cluster of Mutagenic 8-Oxoguanine and Abasic Site Lesions
    [Apr 2014]

    Publication date: 3 April 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 7

    Author(s): Jan Zálešák , Morgane Lourdin , Lumίr Krejčί , Jean-François Constant , Muriel Jourdan

    Clustered DNA damage sites are caused by ionizing radiation. They are much more difficult to repair than are isolated single lesions, and their biological outcomes in terms of mutagenesis and repair inhibition are strongly dependent on the type, relative position and orientation of the lesions present in the cluster. To determine whether these effects on repair mechanism could be due to local structural properties within DNA, we used 1H NMR spectroscopy and restrained molecular dynamics simulation to elucidate the structures of three DNA duplexes containing bistranded clusters of lesions. Each DNA sequence contained an abasic site in the middle of one strand and differed by the relative position of the 8-oxoguanine, staggered on either the 3′ or the 5′ side of the complementary strand. Their repair by base excision repair protein Fpg was either complete or inhibited. All the studied damaged DNA duplexes adopt an overall B-form conformation and the damaged residues remain intrahelical. No striking deformations of the DNA chain have been observed as a result of close proximity of the lesions. These results rule out the possibility that differential recognition of clustered DNA lesions by the Fpg protein could be due to changes in the DNA's structural features induced by those lesions and provide new insight into the Fpg recognition process.
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  • The NreA Protein Functions as a Nitrate Receptor in the Staphylococcal Nitrate Regulation System
    [Apr 2014]

    Publication date: 3 April 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 7

    Author(s): Volker Niemann , Mareike Koch-Singenstreu , Ancilla Neu , Stephanie Nilkens , Friedrich Götz , Gottfried Unden , Thilo Stehle

    Staphylococci are able to use nitrate as an alternative electron acceptor during anaerobic respiration. The regulation of energy metabolism is dependent on the presence of oxygen and nitrate. Under anaerobic conditions, staphylococci employ the nitrate regulatory element (Nre) for transcriptional activation of genes involved in reduction and transport of nitrate and nitrite. Of the three proteins that constitute the Nre system, NreB has been characterized as an oxygen sensor kinase and NreC has been characterized as its cognate response regulator. Here, we present structural and functional data that establish NreA as a new type of nitrate receptor. The structure of NreA with bound nitrate was solved at 2.35Å resolution, revealing a GAF domain fold. Isothermal titration calorimetry experiments showed that NreA binds nitrate with low micromolar affinity (K D =22μM). Two crystal forms for NreA were obtained, with either bound nitrate or iodide. While the binding site is hydrophobic, two helix dipoles and polar interactions contribute to specific binding of the ions. The expression of nitrate reductase (NarGHI) was examined using a narG-lip (lipase) reporter gene assay in vivo. Expression was regulated by the presence of NreA and nitrate. Structure-guided mutations of NreA reduced its nitrate binding affinity and also affected the gene expression, thus providing support for the function of NreA as a nitrate receptor.
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  • Evidence Against the “Y–T Coupling” Mechanism of Activation in the Response Regulator NtrC
    [Apr 2014]

    Publication date: 3 April 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 7

    Author(s): Janice Villali , Francesco Pontiggia , Michael W. Clarkson , Michael F. Hagan , Dorothee Kern

    The dominant theory on the mechanism of response regulators activation in two-component bacterial signaling systems is the “Y–T coupling” mechanism, wherein the χ1 rotameric state of a highly conserved aromatic residue correlates with the activation of the protein via structural rearrangements coupled to a conserved tyrosine. In this paper, we present evidence that, in the receiver domain of the response regulator nitrogen regulatory protein C (NtrCR), the interconversion of this tyrosine (Y101) between its rotameric states is actually faster than the rate of inactive/active conversion and is not correlated to the activation process. Data gathered from NMR relaxation dispersion experiments show that a subset of residues surrounding the conserved tyrosine sense a process that is occurring at a faster rate than the inactive/active conformational transition. We show that this process is related to χ1 rotamer exchange of Y101 and that mutation of this aromatic residue to a leucine eliminated this second faster process without affecting activation. Computational simulations of NtrCR in its active conformation further demonstrate that the rotameric state of Y101 is uncorrelated with the global conformational transition during activation. Moreover, the tyrosine does not appear to be involved in the stabilization of the active form upon phosphorylation and is not essential in propagating the signal downstream for ATPase activity of the central domain. Our data provide experimental evidence against the generally accepted “Y–T coupling” mechanism of activation in NtrCR.
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  • Prokaryotic Ancestry of Eukaryotic Protein Networks Mediating Innate Immunity and Apoptosis
    [Apr 2014]

    Publication date: 3 April 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 7

    Author(s): Stanislaw Dunin-Horkawicz , Klaus O. Kopec , Andrei N. Lupas

    Protein domains characteristic of eukaryotic innate immunity and apoptosis have many prokaryotic counterparts of unknown function. By reconstructing interactomes computationally, we found that bacterial proteins containing these domains are part of a network that also includes other domains not hitherto associated with immunity. This network is connected to the network of prokaryotic signal transduction proteins, such as histidine kinases and chemoreceptors. The network varies considerably in domain composition and degree of paralogy, even between strains of the same species, and its repetitive domains are often amplified recently, with individual repeats sharing up to 100% sequence identity. Both phenomena are evidence of considerable evolutionary pressure and thus compatible with a role in the “arms race” between host and pathogen. In order to investigate the relationship of this network to its eukaryotic counterparts, we performed a cluster analysis of organisms based on a census of its constituent domains across all fully sequenced genomes. We obtained a large central cluster of mainly unicellular organisms, from which multicellular organisms radiate out in two main directions. One is taken by multicellular bacteria, primarily cyanobacteria and actinomycetes, and plants form an extension of this direction, connected via the basal, unicellular cyanobacteria. The second main direction is taken by animals and fungi, which form separate branches with a common root in the α-proteobacteria of the central cluster. This analysis supports the notion that the innate immunity networks of eukaryotes originated from their endosymbionts and that increases in the complexity of these networks accompanied the emergence of multicellularity.
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  • Backbone Flexibility of CDR3 and Immune Recognition of Antigens
    [Apr 2014]

    Publication date: 3 April 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 7

    Author(s): Jaafar N. Haidar , Wei Zhu , Jacqueline Lypowy , Brian G. Pierce , Amtul Bari , Kris Persaud , Xenia Luna , Marshall Snavely , Dale Ludwig , Zhiping Weng

    Conformational entropy is an important component of protein–protein interactions; however, there is no reliable method for computing this parameter. We have developed a statistical measure of residual backbone entropy in folded proteins by using the ϕ–ψ distributions of the 20 amino acids in common secondary structures. The backbone entropy patterns of amino acids within helix, sheet or coil form clusters that recapitulate the branching and hydrogen bonding properties of the side chains in the secondary structure type. The same types of residues in coil and sheet have identical backbone entropies, while helix residues have much smaller conformational entropies. We estimated the backbone entropy change for immunoglobulin complementarity-determining regions (CDRs) from the crystal structures of 34 low-affinity T-cell receptors and 40 high-affinity Fabs as a result of the formation of protein complexes. Surprisingly, we discovered that the computed backbone entropy loss of only the CDR3, but not all CDRs, correlated significantly with the kinetic and affinity constants of the 74 selected complexes. Consequently, we propose a simple algorithm to introduce proline mutations that restrict the conformational flexibility of CDRs and enhance the kinetics and affinity of immunoglobulin interactions. Combining the proline mutations with rationally designed mutants from a previous study led to 2400-fold increase in the affinity of the A6 T-cell receptor for Tax-HLAA2. However, this mutational scheme failed to induce significant binding changes in the already-high-affinity C225–Fab/huEGFR interface. Our results will serve as a roadmap to formulate more effective target functions to design immune complexes with improved biological functions.
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    Categories: Journal Articles
  • The Folding of a Family of Three-Helix Bundle Proteins: Spectrin R15 Has a Robust Folding Nucleus, Unlike Its Homologous Neighbours
    [Apr 2014]

    Publication date: 3 April 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 7

    Author(s): Lee Gyan Kwa , Beth G. Wensley , Crispin G. Alexander , Stuart J. Browning , Benjamin R. Lichman , Jane Clarke

    Three homologous spectrin domains have remarkably different folding characteristics. We have previously shown that the slow-folding R16 and R17 spectrin domains can be altered to resemble the fast folding R15, in terms of speed of folding (and unfolding), landscape roughness and folding mechanism, simply by substituting five residues in the core. Here we show that, by contrast, R15 cannot be engineered to resemble R16 and R17. It is possible to engineer a slow-folding version of R15, but our analysis shows that this protein neither has a rougher energy landscape nor does change its folding mechanism. Quite remarkably, R15 appears to be a rare example of a protein with a folding nucleus that does not change in position or in size when its folding nucleus is disrupted. Thus, while two members of this protein family are remarkably plastic, the third has apparently a restricted folding landscape.
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    Categories: Journal Articles
  • Interrelationship between Cytoplasmic Retroviral Gag Concentration and Gag–Membrane Association
    [Apr 2014]

    Publication date: 3 April 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 7

    Author(s): Keir H. Fogarty , Serkan Berk , Iwen F. Grigsby , Yan Chen , Louis M. Mansky , Joachim D. Mueller

    The early events in the retrovirus assembly pathway, particularly the timing and nature of Gag translocation from the site of protein translation to the inner leaflet of the plasma membrane, are poorly understood. We have investigated the interrelationship between cytoplasmic Gag concentration and plasma membrane association using complementary live-cell biophysical fluorescence techniques in real time with both human T-cell leukemia virus type 1 (HTLV-1) and human immunodeficiency virus type 1 (HIV-1) Gag proteins. In particular, dual-color, z-scan fluorescence fluctuation spectroscopy in conjunction with total internal reflection fluorescence and conventional, epi-illumination imaging were utilized. Our results demonstrate that HTLV-1 Gag is capable of membrane targeting and particle assembly at low (i.e., nanomolar) cytoplasmic concentrations and that there is a critical threshold concentration (approaching micromolar) prior to the observation of HIV-1 Gag associated with the plasma membrane. These observations imply fundamental differences between HIV-1 and HTLV-1 Gag trafficking and membrane association.
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    Categories: Journal Articles
  • APE1 incision activity at abasic sites in tandem repeat sequences
    [Apr 2014]

    Publication date: Available online 1 April 2014
    Source:Journal of Molecular Biology

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

    Repetitive DNA sequences, such as are present in micro- and mini-satellites, 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 position(s) of the lesion(s), and, when not properly processed, can contribute to genome instability that underlies aging and disease development. Knowledge of 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 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
  • Why have Small Multidrug-Resistance proteins not evolved into fused, internally duplicated structures?
    [Apr 2014]

    Publication date: Available online 30 March 2014
    Source:Journal of Molecular Biology

    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 has been posited in which the ancestral state is a single gene encoding a dual-topology membrane protein capable of forming antiparallel homodimers. 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 BN-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 intra- 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
  • Identification of the active sites in the methyltransferases of a transcribing dsRNA virus
    [Apr 2014]

    Publication date: Available online 30 March 2014
    Source:Journal of Molecular Biology

    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- 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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  • A non-active site SET domain surface crucial for the interaction of MLL1 and the RbBP5-ASH2L heterodimer within MLL family core complexes
    [Apr 2014]

    Publication date: Available online 27 March 2014
    Source:Journal of Molecular Biology

    Author(s): Stephen A. Shinsky , Michael Hu , Valarie E. Vought , Sarah B. Ng , Michael J. Bamshad , Jay Shendure , Michael S. Cosgrove

    The Mixed Lineage Leukemia-1 (MLL1) enzyme is a histone H3 lysine 4 (H3K4) monomethyltransferase and has served as a paradigm for understanding the mechanism of action of the human SET1 family of enzymes that include MLL1-4, and SET1d1a,b. Dimethylation of H3K4 requires a sub-complex including WDR5, RbBP5, Ash2L, and DPY-30 (WRAD), which binds to each SET1 family member forming a minimal core complex that is required for multiple lysine methylation. We recently demonstrated that WRAD is a novel histone methyltransferase that preferentially catalyzes H3K4 dimethylation in a manner that is dependent on an unknown non-active site surface from the MLL1 SET domain. Recent genome sequencing studies have identified a number of human disease-associated missense mutations that localize to the SET domains of several MLL family members. In this investigation, we mapped many of these mutations onto the three-dimensional structure of the SET domain and noticed that a subset of MLL2 (KMT2D, ALR, MLL4)-associated Kabuki syndrome (KS) missense mutations map to a common solvent-exposed surface that is not expected to alter enzymatic activity. We introduced these mutations into the MLL1 SET domain and observed that all are defective for H3K4 dimethylation by the MLL1 core complex, which is associated with a loss of the ability of MLL1 to interact with WRAD or with the RbBP5-Ash2L heterodimer. Our results suggest that amino acids from this surface, which we term the Kabuki interaction surface or (KIS), are required for formation of a second active site within SET1 family core complexes.
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    Categories: Journal Articles
  • Systematic detection of internal symmetry in proteins using CE-Symm
    [Apr 2014]

    Publication date: Available online 26 March 2014
    Source:Journal of Molecular Biology

    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 structure 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. Using 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 (v. 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
  • Exploring the biological and chemical complexity of the ligases
    [Apr 2014]

    Publication date: Available online 21 March 2014
    Source:Journal of Molecular Biology

    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 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 sub-class division. However, this sub-class 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
  • Editorial Board
    [Apr 2014]

    Publication date: 20 March 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 6









    Categories: Journal Articles