Journal of Molecular Biology

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  • Structure and interactions of the CS domain of human H/ACA RNP assembly protein Shq1
    [Jan 2015]

    Publication date: Available online 29 December 2014
    Source:Journal of Molecular Biology

    Author(s): Mahavir Singh , Zhonghua Wang , Duilio Cascio , Juli Feigon

    Shq1 is an essential protein involved in the early steps of biogenesis and assembly of H/ACA RNPs. Shq1 binds to dyskerin (Cbf5 in yeast) at an early step of H/ACA RNP assembly and is subsequently displaced by the H/ACA RNA. Shq1 contains an N-terminal CS and a C-terminal Shq1-specific domain (SSD). Dyskerin harbors many dyskeratosis congenita (DC) associated mutations. Structures of yeast Shq1 SSD bound to Cbf5 revealed that only a subset of these mutations is in the SSD binding site, implicating another subset in the putative CS binding site. Here we present the crystal structure of human Shq1 CS (hCS) and the NMR and crystal structure of hCS containing a serine substitution for proline 22 that is associated with some prostate cancers. The structure of hCS is similar to yeast Shq1 CS domain (yCS) and consists of two β-sheets that form an immunoglobulin-like β-sandwich fold. The N-terminal affinity tag sequence AHHHHHH associates with a neighboring protein in the crystal lattice to form an extra β-strand. Deletion of this tag was required to get spectra suitable for NMR structure determination, while the tag was required for crystallization. NMR chemical shift perturbation (CSP) experiments with dyskerin and Cbf5 derived peptides from putative CS binding sites revealed a conserved surface on CS important for Cbf5/dyskerin binding. A HADDOCK docking model of a Shq1-Cbf5 complex that defines the position of CS domain in the pre-H/ACA RNP was calculated using the CSP data.
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  • Conformational Preferences Underlying Reduced Activity of a Thermophilic Ribonuclease H
    [Jan 2015]

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

    Author(s): Kate A. Stafford , Nikola Trbovic , Joel A. Butterwick , Robert Abel , Richard A. Friesner , Palmer Arthur G. III

    The conformational basis for reduced activity of the thermophilic ribonuclease HI enzyme from Thermus thermophilus, compared to its mesophilic homolog from Escherichia coli, is elucidated using a combination of NMR spectroscopy and molecular dynamics (MD) simulations. Explicit-solvent all-atom MD simulations of the two wild-type proteins as well as an E. coli mutant in which a glycine residue is inserted after position 80 to mimic the T. thermophilus protein reproduce the differences in conformational dynamics determined from 15 N spin relaxation NMR spectroscopy of three loop regions that surround the active site and contain functionally important residues: the glycine-rich region, the handle region, and the β5/αE loop. Examination of the MD trajectories indicates that the thermophilic protein samples conformations productive for substrate binding and activity less frequently than the mesophilic enzyme, although these differences may manifest as either increased or decreased relative flexibility of the different regions. Additional MD simulations indicate that mutations increasing activity of the T. thermophilus enzyme at mesophilic temperatures do so by reconfiguring the local environments of the mutated sites to more closely resemble active conformations. Taken together, the results show that both locally increased and decreased flexibility contribute to an overall reduction in activity of T. thermophilus ribonuclease H compared to its mesophilic E. coli homolog.
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  • Novel essential gene involved in 16S rRNA processing in Escherichia coli
    [Jan 2015]

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

    Author(s): Tatsuaki Kurata , Shinobu Nakanishi , Masayuki Hashimoto , Masato Taoka , Yukiko Yamazaki , Toshiaki Isobe , Jun-ichi Kato

    Biogenesis of ribosomes is a complex process mediated by many factors. While its transcription proceeds, ribosomal RNA (rRNA) folds itself into a characteristic three dimensional structure through interaction with ribosomal proteins, during which its ends are processed. Here, we show that the essential protein YqgF, a RuvC family protein with an RNase H-like motif, is involved in the processing of pre-16S rRNA during ribosome maturation. Indeed, pre-16S rRNA accumulated in cells of a temperature-sensitive yqgF mutant (yqgFts ) cultured at a non-permissive temperature. In addition, purified YqgF was shown to process the 5’-end of pre-16S rRNA within 70S ribosomes in vitro. Mass spectrometry analysis of the total proteins in the yqgFts mutant cells showed that the expression of genes containing multiple Shine-Dalgarno-like sequences was observed to be lower than in wild-type. These results are interpreted to indicate that YqgF is involved in a novel enzymic activity necessary for the processing of pre-16S rRNA, thereby affecting elongation of translation.
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  • The Cobalamin-Independent Methionine Synthase Enzyme Captured in a Substrate-Induced Closed Conformation
    [Jan 2015]

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

    Author(s): Devinder K. Ubhi , Jon D. Robertus

    The cobalamin-independent methionine synthase enzyme catalyzes a challenging reaction: the direct transfer of a methyl from 5-methyl-tetrahydrofolate-glutamate3 to the l-homocysteine thiol. The enzyme has a dual (βα)8 TIM barrel structure that binds, activates and brings the reactants into reaction proximity by conformational movements. In the previously observed open structures, the substrates bind too far apart to react, but we have captured a ternary complex with both substrates bound in a closed form of the enzyme. The closing is described in terms of a hinge between the N- and C-terminal TIM barrels and a rearrangement of key loops within the C domain. The substrate specificity can now be rationalized and the structure reveals His707 as the acid that protonates the THF leaving group through a water molecule trapped in the closed active site. The substrates are correctly oriented for an in-line attack by l-homocysteine on the N 5-methyl.
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  • Structure of Salmonella FlhE, conserved member of a flagellar Type III secretion operon
    [Jan 2015]

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

    Author(s): Jaemin Lee , Arthur F. Monzingo , Adrian T. Keatinge-Clay , Rasika M. Harshey

    The bacterial flagellum is assembled by a multicomponent transport apparatus categorized as a Type III secretion (T3S) system. The secretion of proteins that assemble into the flagellum is driven by the proton motive force. The periplasmic protein FlhE is a member of the flhBAE operon in the majority of bacteria where FlhE is found. FlhA and FlhB are established components of the flagellar T3S system. The absence of FlhE results in a proton leak through the flagellar system, inappropriate secretion patterns, and cell death, indicating that FlhE regulates an important aspect of proper flagellar biosynthesis. We isolated FlhE from the periplasm of Salmonella and solved its structure to 1.5Å resolution. Possible roles of FlhE, including that of a chaperone, are discussed.
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  • Differential Role of Base Pairs on gal Promoters Strength
    [Jan 2015]

    Publication date: Available online 24 December 2014
    Source:Journal of Molecular Biology

    Author(s): Dale E.A. Lewis , Phuoc Le , Sankar Adhya

    Sequence alignments of promoters in prokaryotes postulated that the frequency of occurrence of a base pair at a given position of promoter elements reflects its contribution to intrinsic promoter strength. We directly assessed the contribution of the four base pairs in each position in the intrinsic promoter strength by keeping the context constant in Escherichia coli cAMP-CRP (cAMP receptor protein) regulated gal promoters by in vitro transcription assays. First, we show that base pair frequency within known consensus elements correlates well with promoter strength. Second, we observe some substitutions upstream of the ex-10 TG motif that are important for promoter function. Although the galP1 and P2 promoters overlap, only three positions where substitutions inactivated both promoters were found. We propose that RNA polymerase binds to the −12T base pair as part of double-stranded DNA while opening base pairs from −11A to +3 to form the single-stranded transcription bubble DNA during isomerization. The cAMP-CRP complex rescued some deleterious substitutions in the promoter region. The base pair roles and their flexibilities reported here for E. coli gal promoters may help construction of synthetic promoters in gene circuitry experiments in which overlapping promoters with differential controls may be warranted.
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  • Phosphomimetic mutation of the N-terminal lid of MDM2 enhances the polyubiquitination of p53 through stimulation of E2-ubiquitin thioester hydrolysis
    [Jan 2015]

    Publication date: Available online 24 December 2014
    Source:Journal of Molecular Biology

    Author(s): Jennifer A. Fraser , Erin G. Worrall , Yao Lin , Vivien Landre , Susanne Pettersson , Elizabeth Blackburn , Malcolm Walkinshaw , Petr Muller , Borek Vojtesek , Kathryn Ball , Ted R. Hupp

    MDM2 has a phosphorylation site within a lid motif at Ser17 whose phospho-mimetic mutation to Asp17 stimulates MDM2-mediated polyubiquitination of p53. MDM2 lid deletion, but not Asp17 mutation, induced a blue-shift in the λmax of intrinsic fluorescence derived from residues in the central domain including Trp235, Trp303, Trp323, and Trp329. This indicates that the Asp17 mutation does not alter the conformation of MDM2 surrounding the tryptophan residues. In addition, Phe235 mutation enhanced MDM2 binding to p53 but did not stimulate its’ ubiquitination function, thus uncoupling increases in p53 binding from its’ E3 ubiquitin ligase function. However, the Asp17 mutation in MDM2 stimulated its discharge of the UBCH5a-Ubiquitin thioester adduct. This stimulation of ubiquitin discharge from E2 was independent of the p53 substrate. There are now four known effects of the Asp17 mutation on MDM2; (i) it alters the conformation of the isolated N-terminus as defined by NMR; (ii) it induces increased thermostability of the isolated N-terminal domain; (iii) it stimulates the allosteric interaction of MDM2 with the DNA-binding domain of p53; and (iv) it stimulates a novel protein-protein interaction with the E2- ubiquitin complex in the absence of substrate p53 that in turns increases hydrolysis of the E2- ubiquitin thioester bond. These data also suggest a new strategy to disrupt MDM2 function by targetting the E2- ubiquitin discharge reaction.
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  • Assembly States of FliM and FliG within the Flagellar Switch Complex
    [Jan 2015]

    Publication date: Available online 20 December 2014
    Source:Journal of Molecular Biology

    Author(s): Ria Sircar , Peter P. Borbat , Michael J. Lynch , Jaya Bhatnagar , Matthew S. Beyersdorf , Christopher J. Halkides , Jack H. Freed , Brian R. Crane

    At the base of the bacterial flagella, a cytoplasmic rotor (the C-ring) generates torque and reverses rotation sense in response to stimuli. The bulk of the C-ring forms from many copies of the proteins FliG, FliM, and FliN, which together constitute the switch complex. To help resolve outstanding issues regarding C-ring architecture, we have investigated interactions between FliM and FliG from Thermotoga maritima with X-ray crystallography and pulsed dipolar ESR spectroscopy (PDS). A new crystal structure of an 11-unit FliG:FliM complex produces a large arc with a curvature consistent with the dimensions of the C-ring. Previously determined structures along with this new structure provided a basis to test switch complex assembly models. PDS combined with mutational studies and targeted cross-linking reveal that FliM and FliG interact through their middle domains to form both parallel and antiparallel arrangements in solution. Residue substitutions at predicted interfaces disrupt higher-order complexes that are primarily mediated by contacts between the C-terminal domain of FliG and the middle domain of a neighboring FliG molecule. Spin separations among multi-labeled components fit a self-consistent model that agree well with electron microscopy images of the C-ring. An activated form of the response regulator CheY destabilizes the parallel arrangement of FliM molecules to perturb FliG alignment in a process that may reflect the onset of rotation switching. These data suggest a model of C-ring assembly in which intermolecular contacts among FliG domains provide a template for FliM assembly and cooperative transitions.
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  • The Basis of Asymmetry in the SecA:SecB Complex
    [Jan 2015]

    Publication date: Available online 19 December 2014
    Source:Journal of Molecular Biology

    Author(s): Yuying Suo , Simon J.S. Hardy , Linda L. Randall

    During export in Escherichia coli, SecB, a homotetramer structurally organized as a dimer of dimers, forms a complex with two protomers of SecA, which is the ATPase that provides energy to transfer a precursor polypeptide through the membrane via the SecYEG translocon. There are two areas of contact on SecB that stabilize the SecA:SecB complex: the flat sides of the SecB tetramer and the C-terminal 13 residues of SecB. These contacts within the complex are distributed asymmetrically. Breaking contact between SecA and the sides of SecB results in release of only one protomer of SecA yielding a complex of stoichiometry SecA1:SecB4. This complex mediates export; however, the coupling of ATP hydrolysis to movements of the precursor through the translocon is much less efficient than the coupling by the SecA2:SecB4 complex. Here we used heterotetrameric species of SecB to understand the source of the asymmetry in the contacts and its role in the functioning of the complex. The model of interactions presented suggests a way that binding between SecA and SecB might decrease the affinity of precursor polypeptides for SecB and facilitate the transfer to SecA.
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  • Simultaneous Sequence-Based Prediction of the Statistical Populations of Ordered and Disordered Regions in Proteins
    [Jan 2015]

    Publication date: Available online 19 December 2014
    Source:Journal of Molecular Biology

    Author(s): Pietro Sormanni , Carlo Camilloni , Piero Fariselli , Michele Vendruscolo

    Extensive amounts of information about protein sequences are becoming available, as demonstrated by the over 79 million entries in the UniProt database. Yet, it is still challenging to obtain proteome-wide experimental information on the structural properties associated with these sequences. To bridge this gap, fast computational predictors of secondary structure and of intrinsic disorder of proteins have been developed. These two types of predictions, however, have remained largely separated, often preventing a clear characterisation of the structure and dynamics of proteins. Here, we introduce a computational method to predict secondary structure populations from amino acid sequences, which characterises simultaneously structure and disorder in a unified statistical mechanics framework. To develop this method, called s2D, we exploited recent advances made in the analysis of NMR chemical shifts that provide quantitative information about the probability distributions of secondary structure elements in disordered states. The results that we discuss show that the s2D method predicts secondary structure populations with an average error of about 14%. A validation on three datasets of, respectively, mostly disordered, mostly structured, and partly structured proteins show that its performance is comparable or better than that of existing predictors of intrinsic disorder and of secondary structure. These results indicate that it is possible to perform rapid and quantitative sequence-based characterisations of the structure and dynamics of proteins through the predictions of the statistical distributions of their ordered and disordered regions.
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  • Flexible Stoichiometry and Asymmetry of the PIDDosome Core Complex by Heteronuclear NMR Spectroscopy and Mass Spectrometry
    [Jan 2015]

    Publication date: Available online 18 December 2014
    Source:Journal of Molecular Biology

    Author(s): Lily A. Nematollahi , Acely Garza-Garcia , Chérine Bechara , Diego Esposito , Nina Morgner , Carol V. Robinson , Paul C. Driscoll

    Homotypic death domain (DD)–DD interactions are important in the assembly of oligomeric signaling complexes such as the PIDDosome that acts as a platform for activation of caspase-2-dependent apoptotic signaling. The structure of the PIDDosome core complex exhibits an asymmetric three-layered arrangement containing five PIDD-DDs in one layer, five RAIDD-DDs in a second layer and an additional two RAIDD-DDs. We addressed complex formation between PIDD-DD and RAIDD-DD in solution using heteronuclear nuclear magnetic resonance (NMR) spectroscopy, nanoflow electrospray ionization mass spectrometry and size-exclusion chromatography with multi-angle light scattering. The DDs assemble into complexes displaying molecular masses in the range 130–158kDa and RAIDD-DD:PIDD-DD stoichiometries of 5:5, 6:5 and 7:5. These data suggest that the crystal structure is representative of only the heaviest species in solution and that two RAIDD-DDs are loosely attached to the 5:5 core. Two-dimensional 1H,15N-NMR experiments exhibited signal loss upon complexation consistent with the formation of high-molecular-weight species. 13C-Methyl-transverse relaxation optimized spectroscopy measurements of the PIDDosome core exhibit signs of differential line broadening, cross-peak splitting and chemical shift heterogeneity that reflect the presence of non-equivalent sites at interfaces within an asymmetric complex. Experiments using a mutant RAIDD-DD that forms a monodisperse 5:5 complex with PIDD-DD show that the spectroscopic signature derives from the quasi- but non-exact equivalent environments of each DD. Since this characteristic was previously demonstrated for the complex between the DDs of CD95 and FADD, the NMR data for this system are consistent with the formation of a structure homologous to the PIDDosome core.
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  • Uncovering the role of Sgf73 in maintaining SAGA Deubiquitinating Module Structure and Activity
    [Jan 2015]

    Publication date: Available online 17 December 2014
    Source:Journal of Molecular Biology

    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 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 ubiquitin-binding conformation of Ubp8, thereby contributing to overall DUBm activity.
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  • The Amyloid Precursor Protein Shows a pH-Dependent Conformational Switch in Its E1 Domain
    [Jan 2015]

    Publication date: Available online 17 December 2014
    Source:Journal of Molecular Biology

    Author(s): Sandra Hoefgen , Sven O. Dahms , Kathrin Oertwig , Manuel E. Than

    The amyloid precursor protein (APP) and its proteolytic cleavage product Aβ are widely believed to be central to the etiology of Alzheimer's disease (AD). APP and its family members are also essential for proper neuronal development and homeostasis. APP is located at the cell surface and within intracellular compartments, cellular regions that exhibit different pH values. The AD-associated amyloidogenic processing of APP is initiated predominantly in intracellular acidic compartments, whereas its non-amyloidogenic cleavage is initiated at the cell surface at slightly basic pH. We analyzed the influence of pH on the APP-E1 domain and found that its two constituting subdomains, GFLD and CuBD, interact with each other in a pH-dependent manner. Dynamic light scattering showed that APP-E1 represents a more open conformation at neutral pH and a more closed conformation at acidic pH. Analyzing a 1.4 Å, high-resolution X-ray structure of E1 derived from merohedrally twinned crystals resulted in the identification of individual residues that are responsible for these pH-dependent interactions. Mutational studies and dynamic light scattering measurements further proved that specific hydrogen bonds between the two carboxylates of D177 and E87, as well as between N89 and H147, are major determinants of this pH-driven conformational switch in APP-E1. These findings show how APP can adopt different conformations depending on pH and suggest that the protein fulfils different functions at distinct localizations within the cell. Additionally, our data suggest a novel strategy for treating AD based on regulating the amyloidogenic processing of APP by the specific interruption of the interaction between the APP-E1 subdomains.
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    Categories: Journal Articles
  • Editorial Board
    [Jan 2015]

    Publication date: 12 December 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 24









    Categories: Journal Articles
  • Contents List
    [Jan 2015]

    Publication date: 12 December 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 24









    Categories: Journal Articles
  • Accessory Replicative Helicases and the Replication of Protein-Bound DNA
    [Jan 2015]

    Publication date: 12 December 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 24

    Author(s): Jan-Gert Brüning , Jamieson L. Howard , Peter McGlynn

    Complete, accurate duplication of the genetic material is a prerequisite for successful cell division. Achieving this accuracy is challenging since there are many barriers to replication forks that may cause failure to complete genome duplication or result in possibly catastrophic corruption of the genetic code. One of the most important types of replicative barriers are proteins bound to the template DNA, especially transcription complexes. Removal of these barriers demands energy input not only to separate the DNA strands but also to disrupt multiple bonds between the protein and DNA. Replicative helicases that unwind the template DNA for polymerases at the fork can displace proteins bound to the template. However, even occasional failures in protein displacement by the replicative helicase could spell disaster. In such circumstances, failure to restart replication could result in incomplete genome duplication. Avoiding incomplete genome duplication via the repair and restart of blocked replication forks also challenges viability since the involvement of recombination enzymes is associated with the risk of genome rearrangements. Organisms have therefore evolved accessory replicative helicases that aid replication fork movement along protein-bound DNA. These helicases reduce the dangers associated with replication blockage by protein–DNA complexes, aiding clearance of blocks and resumption of replication by the same replisome thus circumventing the need for replication repair and restart. This review summarises recent work in bacteria and eukaryotes that has begun to delineate features of accessory replicative helicases and their importance in genome stability.
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  • Phosphorylation and Prolyl Isomerization Independently Regulate the Signal Adapter Function of CrkII
    [Jan 2015]

    Publication date: 12 December 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 24

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

    The signaling protein CrkII switches between forms with high or low binding affinity. Both phosphorylation and native-state prolyl isomerization were suggested to regulate the transition between these forms. Here we analyzed how phosphorylation at Tyr222 and Tyr252 and the Pro238Ala substitution affect signal transfer of human and chicken CrkII to a downstream target. Human CrkII is regulated by phosphorylation only, but chicken CrkII is regulated by Pro238 trans → cis isomerization and by Tyr222 phosphorylation. Surprisingly, they act in an independent fashion. Apparently, the allosteric transition to a low-activity form can be induced by phosphorylation or prolyl isomerization located at distant sites in CrkII.
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  • An Ancient Autoproteolytic Domain Found in GAIN, ZU5 and Nucleoporin98
    [Jan 2015]

    Publication date: 12 December 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 24

    Author(s): Yuxing Liao , Jimin Pei , Hua Cheng , Nick V. Grishin

    A large family of G protein-coupled receptors (GPCRs) involved in cell adhesion has a characteristic autoproteolysis motif of HLT/S known as the GPCR proteolysis site (GPS). GPS is also shared by polycystic kidney disease proteins and it precedes the first transmembrane segment in both families. Recent structural studies have elucidated the GPS to be part of a larger domain named GPCR autoproteolysis inducing (GAIN) domain. Here we demonstrate the remote homology relationships of GAIN domain to ZU5 domain and Nucleoporin98 (Nup98) C-terminal domain by structural and sequence analysis. Sequence homology searches were performed to extend ZU5-like domains to bacteria and archaea, as well as new eukaryotic families. We found that the consecutive ZU5-UPA-death domain domain organization is commonly used in human cytoplasmic proteins with ZU5 domains, including CARD8 (caspase recruitment domain-containing protein 8) and NLRP1 (NACHT, LRR and PYD domain-containing protein 1) from the FIIND (Function to Find) family. Another divergent family of extracellular ZU5-like domains was identified in cartilage intermediate layer proteins and FAM171 proteins. Current diverse families of GAIN domain subdomain B, ZU5 and Nup98 C-terminal domain likely evolved from an ancient autoproteolytic domain with an HFS motif. The autoproteolytic site was kept intact in Nup98, p53-induced protein with a death domain and UNC5C-like, deteriorated in many ZU5 domains and changed in GAIN and FIIND. Deletion of the strand after the cleavage site was observed in zonula occluden-1 and some Nup98 homologs. These findings link several autoproteolytic domains, extend our understanding of GAIN domain origination in adhesion GPCRs and provide insights into the evolution of an ancient autoproteolytic domain.
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    Categories: Journal Articles
  • GEN1 from a Thermophilic Fungus Is Functionally Closely Similar to Non-Eukaryotic Junction-Resolving Enzymes
    [Jan 2015]

    Publication date: 12 December 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 24

    Author(s): Alasdair D.J. Freeman , Yijin Liu , Anne-Cécile Déclais , Anton Gartner , David M.J. Lilley

    Processing of Holliday junctions is essential in recombination. We have identified the gene for the junction-resolving enzyme GEN1 from the thermophilic fungus Chaetomium thermophilum and expressed the N-terminal 487-amino-acid section. The protein is a nuclease that is highly selective for four-way DNA junctions, cleaving 1nt 3′ to the point of strand exchange on two strands symmetrically disposed about a diagonal axis. CtGEN1 binds to DNA junctions as a discrete homodimer with nanomolar affinity. Analysis of the kinetics of cruciform cleavage shows that cleavage of the second strand occurs an order of magnitude faster than the first cleavage so as to generate a productive resolution event. All these properties are closely similar to those described for bacterial, phage and mitochondrial junction-resolving enzymes. CtGEN1 is also similar in properties to the human enzyme but lacks the problems with aggregation that currently prevent detailed analysis of the latter protein. CtGEN1 is thus an excellent enzyme with which to engage in biophysical and structural analysis of eukaryotic GEN1.
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  • The PHD finger of p300 Influences Its Ability to Acetylate Histone and Non-Histone Targets
    [Jan 2015]

    Publication date: 12 December 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 24

    Author(s): Johannes G.M. Rack , Timo Lutter , Gro Elin Kjæreng Bjerga , Corina Guder , Christine Ehrhardt , Signe Värv , Mathias Ziegler , Rein Aasland

    In enzymes that regulate chromatin structure, the combinatorial occurrence of modules that alter and recognise histone modifications is a recurrent feature. In this study, we explored the functional relationship between the acetyltransferase domain and the adjacent bromodomain/PHD finger (bromo/PHD) region of the transcriptional coactivator p300. We found that the bromo/PHD region of p300 can bind to the acetylated catalytic domain in vitro and augment the catalytic activity of the enzyme. Deletion of the PHD finger, but not the bromodomain, impaired the ability of the enzyme to acetylate histones in vivo, whilst it enhanced p300 self-acetylation. A point mutation in the p300 PHD finger that is related to the Rubinstein-Taybi syndrome resulted in increased self-acetylation but retained the ability to acetylate histones. Hence, the PHD finger appears to negatively regulate self-acetylation. Furthermore, our data suggest that the PHD finger has a role in the recruitment of p300 to chromatin.
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    Categories: Journal Articles