Journal of Molecular Biology

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  • Editorial Board
    [Aug 2014]

    Publication date: 9 September 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 18









    Categories: Journal Articles
  • Contents List
    [Aug 2014]

    Publication date: 9 September 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 18









    Categories: Journal Articles
  • The Presence of an RNA:DNA Hybrid That Is Prone to Slippage Promotes Termination by T7 RNA Polymerase
    [Aug 2014]

    Publication date: 9 September 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 18

    Author(s): Vadim Molodtsov , Michael Anikin , William T. McAllister

    Intrinsic termination signals for multisubunit bacterial RNA polymerases (RNAPs) encode a GC-rich stem–loop structure followed by a polyuridine [poly(U)] tract, and it has been proposed that steric clash of the stem–loop with the exit pore of the RNAP imposes a shearing force on the RNA in the downstream RNA:DNA hybrid, resulting in misalignment of the active site. The structurally unrelated T7 RNAP terminates at a similar type of signal (TΦ), suggesting a common mechanism for termination. In the absence of a hairpin (passive conditions), T7 RNAP slips efficiently in both homopolymeric A and U tracts, and we have found that replacement of the U tract in TΦ with a slippage-prone A tract still allows efficient termination. Under passive conditions, incorporation of a single G residue following a poly(U) tract (which is the situation during termination at TΦ) results in a “locked” complex that is unable to extend the transcript. Our results support a model in which transmission of the shearing force generated by steric clash of the hairpin with the exit pore is promoted by the presence of a slippery tracts downstream, resulting in alterations in the active site and the formation of a locked complex that represents an early step in the termination pathway.
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    Categories: Journal Articles
  • Interactions of the natural product kendomycin and the 20S proteasome
    [Aug 2014]

    Publication date: 9 September 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 18

    Author(s): Philipp Beck , Wolfgang Heinemeyer , Anna-Lena Späth , Yasser Elnakady , Rolf Müller , Michael Groll

    Natural products are a valuable source for novel lead structures in drug discovery, but for the majority of isolated bioactive compounds, the cellular targets are unknown. The structurally unique ansa-polyketide kendomycin (KM) was reported to exert its potent cytotoxic effects via impairment of the ubiquitin proteasome system, but the exact mode of action remained unclear. Here, we present a systematic biochemical characterization of KM–proteasome interactions in vitro and in vivo, including complex structures of wild type and mutant yeast 20S proteasome with KM. Our results provide evidence for a polypharmacological mode of action for KM's cytotoxic effect on cancer cells.
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    Categories: Journal Articles
  • Direct Interaction of 14-3-3ζ with Ezrin Promotes Cell Migration by Regulating the Formation of Membrane Ruffle
    [Aug 2014]

    Publication date: 9 September 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 18

    Author(s): Miaojuan Chen , Tengfei Liu , Lina Xu , Xuejuan Gao , Xiaohui Liu , Cuihua Wang , Qingyu He , Gong Zhang , Langxia Liu

    14-3-3 proteins have been shown to regulate the actin cytoskeleton remodeling, cell adhesion and migration. In this study, we identified ezrin, a cross-linker between plasma membrane and actin cytoskeleton, as a novel 14-3-3ζ interacting partner. The direct interaction between 14-3-3ζ and ezrin was validated in the cells and by in vitro assays. We showed that the 14-3-3ζ binding region in ezrin was located within the N-terminal and central α-helical domains and that the αG-to-αI helices of 14-3-3ζ are responsible for the binding to ezrin. Functional analyses revealed that the regulation of cell migration and membrane ruffling by 14-3-3ζ is ezrin dependent, for which the integrity of ezrin protein was required. Conversely, the knockdown of 14-3-3ζ abrogates also the stimulatory effect of ezrin on cell migration and membrane ruffling. Moreover, we found that the phosphorylation of Thr567 in ezrin facilitates the 14-3-3ζ–ezrin interaction and the formation of membrane ruffles. Taken together, these results suggest strongly that the functions of these two proteins in cell migration are linked and might be mediated by their direct physical interaction, which is important for the formation of membrane ruffles.
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    Categories: Journal Articles
  • Structure of a C. perfringens Enterotoxin Mutant in Complex with a Modified Claudin-2 Extracellular Loop 2
    [Aug 2014]

    Publication date: 9 September 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 18

    Author(s): Tamas S. Yelland , Claire E. Naylor , Tannya Bagoban , Christos G. Savva , David S. Moss , Bruce A. McClane , Ingolf E. Blasig , M. Popoff , Ajit K. Basak

    CPE (Clostridium perfringens enterotoxin) is the major virulence determinant for C. perfringens type-A food poisoning, the second most common bacterial food-borne illness in the UK and USA. After binding to its receptors, which include particular human claudins, the toxin forms pores in the cell membrane. The mature pore apparently contains a hexamer of CPE, claudin and, possibly, occludin. The combination of high binding specificity with cytotoxicity has resulted in CPE being investigated, with some success, as a targeted cytotoxic agent for oncotherapy. In this paper, we present the X-ray crystallographic structure of CPE in complex with a peptide derived from extracellular loop 2 of a modified, CPE-binding Claudin-2, together with high-resolution native and pore-formation mutant structures. Our structure provides the first atomic-resolution data on any part of a claudin molecule and reveals that claudin's CPE-binding fingerprint (NPLVP) is in a tight turn conformation and binds, as expected, in CPE's C-terminal claudin-binding groove. The leucine and valine residues insert into the binding groove while the first residue, asparagine, tethers the peptide via an interaction with CPE's aspartate 225 and the two prolines are required to maintain the tight turn conformation. Understanding the structural basis of the contribution these residues make to binding will aid in engineering CPE to target tumor cells.
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    Categories: Journal Articles
  • Pathways for Virus Assembly around Nucleic Acids
    [Aug 2014]

    Publication date: 9 September 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 18

    Author(s): Jason D. Perlmutter , Matthew R. Perkett , Michael F. Hagan

    Understanding the pathways by which viral capsid proteins assemble around their genomes could identify key intermediates as potential drug targets. In this work, we use computer simulations to characterize assembly over a wide range of capsid protein–protein interaction strengths and solution ionic strengths. We find that assembly pathways can be categorized into two classes, in which intermediates are either predominantly ordered or disordered. Our results suggest that estimating the protein–protein and the protein–genome binding affinities may be sufficient to predict which pathway occurs. Furthermore, the calculated phase diagrams suggest that knowledge of the dominant assembly pathway and its relationship to control parameters could identify optimal strategies to thwart or redirect assembly to block infection. Finally, analysis of simulation trajectories suggests that the two classes of assembly pathways can be distinguished in single-molecule fluorescence correlation spectroscopy or bulk time-resolved small-angle X-ray scattering experiments.





    Categories: Journal Articles
  • Structural Characterization of Anti-Inflammatory Immunoglobulin G Fc Proteins
    [Aug 2014]

    Publication date: 9 September 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 18

    Author(s): Alysia A. Ahmed , John Giddens , Andrew Pincetic , Joseph V. Lomino , Jeffrey V. Ravetch , Lai-Xi Wang , Pamela J. Bjorkman

    Immunoglobulin G (IgG) is a central mediator of host defense due to its ability to recognize and eliminate pathogens. The recognition and effector responses are encoded on distinct regions of IgGs. The diversity of the antigen recognition Fab domains accounts for IgG's ability to bind with high specificity to essentially any antigen. Recent studies have indicated that the Fc effector domain also displays considerable heterogeneity, accounting for its complex effector functions of inflammation, modulation, and immune suppression. Therapeutic anti-tumor antibodies, for example, require the pro-inflammatory properties of the IgG Fc to eliminate tumor cells, while the anti-inflammatory activity of intravenous IgG requires specific Fc glycans for activity. In particular, the anti-inflammatory activity of intravenous IgG is ascribed to a small population of IgGs in which the Asn297-linked complex N-glycans attached to each Fc CH2 domain include terminal α2,6-linked sialic acids. We used chemoenzymatic glycoengineering to prepare fully disialylated IgG Fc and solved its crystal structure. Comparison of the structures of asialylated Fc, sialylated Fc, and F241A Fc, a mutant that displays increased glycan sialylation, suggests that increased conformational flexibility of the CH2 domain is associated with the switch from pro-inflammatory to anti-inflammatory activity of the Fc.
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    Categories: Journal Articles
  • Sorting Nexin 31 Binds Multiple β Integrin Cytoplasmic Domains and Regulates β1 Integrin Surface Levels and Stability
    [Aug 2014]

    Publication date: 9 September 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 18

    Author(s): Hui-Yuan Tseng , Niko Thorausch , Tilman Ziegler , Alexander Meves , Reinhard Fässler , Ralph T. Böttcher

    Trafficking of α5β1 integrin to lysosomes and its subsequent degradation is influenced by ligand occupancy and the binding of SNX17 via its protein 4.1, ezrin, radixin, moesin (FERM) domain to the membrane-distal NPxY motif in the cytoplasmic domain of β1 integrin in early endosomes. Two other sorting nexin (SNX) family members, namely SNX27 and SNX31, share with SNX17 next to their obligate phox domain a FERM domain, which may enable them to bind β integrin tails. Here we report that, in addition to SNX17, SNX31 but not SNX27 binds several β integrin tails in early endosomes in a PI3 (phosphatidylinositide 3)-kinase-dependent manner. Similarly like SNX17, binding of SNX31 with β1 integrin tails in early endosomes occurs between the FERM domain and the membrane-distal NPxY motif in the β1 integrin cytoplasmic domain. Furthermore, expression of SNX31 rescues β1 integrin surface levels and stability in SNX17-depleted cells. In contrast to SNX17, expression of SNX31 is restricted and found highly expressed in bladder and melanoma tissue. Altogether, these results demonstrate that SNX31 is an endosomal regulator of β integrins with a restricted expression pattern.
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    Categories: Journal Articles
  • Corrigendum to “Context-Dependent Remodeling of Rad51–DNA Complexes by Srs2 Is Mediated by a Specific Protein–Protein Interaction” [J. Mol. Biol. 426 (2014) 1883–1897]
    [Aug 2014]

    Publication date: 9 September 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 18

    Author(s): Anna K. Lytle , Sofia S. Origanti , Yupeng Qiu , Jeffrey VonGermeten , Sua Myong , Edwin Antony







    Categories: Journal Articles
  • Editorial Board
    [Aug 2014]

    Publication date: 26 August 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 17









    Categories: Journal Articles
  • Contents List
    [Aug 2014]

    Publication date: 26 August 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 17









    Categories: Journal Articles
  • Structure of Kif14: An Engaging Molecular Motor
    [Aug 2014]

    Publication date: 26 August 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 17

    Author(s): Sarah Rice







    Categories: Journal Articles
  • KIF14 Binds Tightly to Microtubules and Adopts a Rigor-Like Conformation
    [Aug 2014]

    Publication date: 26 August 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 17

    Author(s): Kritica Arora , Lama Talje , Ana B. Asenjo , Parker Andersen , Kaleem Atchia , Monika Joshi , Hernando Sosa , John S. Allingham , Benjamin H. Kwok

    The mitotic kinesin motor protein KIF14 is essential for cytokinesis during cell division and has been implicated in cerebral development and a variety of human cancers. Here we show that the mouse KIF14 motor domain binds tightly to microtubules and does not display typical nucleotide-dependent changes in this affinity. It also has robust ATPase activity but very slow motility. A crystal structure of the ADP-bound form of the KIF14 motor domain reveals a dramatically opened ATP-binding pocket, as if ready to exchange its bound ADP for Mg·ATP. In this state, the central β-sheet is twisted ~10° beyond the maximal amount observed in other kinesins. This configuration has only been seen in the nucleotide-free states of myosins—known as the “rigor-like” state. Fitting of this atomic model to electron density maps from cryo-electron microscopy indicates a distinct binding configuration of the motor domain to microtubules. We postulate that these properties of KIF14 are well suited for stabilizing midbody microtubules during cytokinesis.
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    Categories: Journal Articles
  • Structural Basis for the Specific Recognition of the Major Antigenic Peptide from the Japanese Cedar Pollen Allergen Cry j 1 by HLA-DP5
    [Aug 2014]

    Publication date: 26 August 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 17

    Author(s): Seisuke Kusano , Mutsuko Kukimoto-Niino , Yoko Satta , Noboru Ohsawa , Tomomi Uchikubo-Kamo , Motoaki Wakiyama , Mariko Ikeda , Takaho Terada , Ken Yamamoto , Yasuharu Nishimura , Mikako Shirouzu , Takehiko Sasazuki , Shigeyuki Yokoyama

    The major allergen, Cry j 1, was isolated from Japanese cedar Cryptomeria japonica (Cry j) pollen and was shown to react with immunoglobulin E antibodies in the sera from pollinosis patients. We previously reported that the frequency of HLA-DP5 was significantly higher in pollinosis patients and the immunodominant peptides from Cry j 1 bound to HLA-DP5 to activate Th2 cells. In the present study, we determined the crystal structure of the HLA-DP5 heterodimer in complex with a Cry j 1-derived nine-residue peptide, at 2.4Å resolution. The peptide-binding groove recognizes the minimal peptide with 10 hydrogen bonds, including those between the negatively charged P1 pocket and the Lys side chain at the first position in the peptide sequence. We confirmed that HLA-DP5 exhibits the same Cry j 1-binding mode in solution, through pull-down experiments using structure-based mutations of Cry j 1. We also identified the characteristic residues of HLA-DP5 that are responsible for the distinct properties of the groove, by comparing the structure of HLA-DP5 and the previously reported structures of HLA-DP2 in complexes with pDRA of the self-antigen. The comparison revealed that the HLA-DP5·pCry j 1 complex forms several hydrogen bond/salt bridge networks between the receptor and the antigen that were not observed in the HLA-DP2·pDRA complex. Evolutionary considerations have led us to conclude that HLA-DP5 and HLA-DP2 represent two major groups of the HLA-DP family, in which the properties of the P1 and P4 pockets have evolved and acquired the present ranges of epitope peptide-binding specificities.
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    Categories: Journal Articles
  • Structural Insights into the Substrate Specificity of (S)-Ureidoglycolate Amidohydrolase and Its Comparison with Allantoate Amidohydrolase
    [Aug 2014]

    Publication date: 26 August 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 17

    Author(s): Inchul Shin , Kitae Han , Sangkee Rhee

    In plants, the ureide pathway is a metabolic route that converts the ring nitrogen atoms of purine into ammonia via sequential enzymatic reactions, playing an important role in nitrogen recovery. In the final step of the pathway, (S)-ureidoglycolate amidohydrolase (UAH) catalyzes the conversion of (S)-ureidoglycolate into glyoxylate and releases two molecules of ammonia as by-products. UAH is homologous in structure and sequence with allantoate amidohydrolase (AAH), an upstream enzyme in the pathway with a similar function as that of an amidase but with a different substrate. Both enzymes exhibit strict substrate specificity and catalyze reactions in a concerted manner, resulting in purine degradation. Here, we report three crystal structures of Arabidopsis thaliana UAH (bound with substrate, reaction intermediate, and product) and a structure of Escherichia coli AAH complexed with allantoate. Structural analyses of UAH revealed a distinct binding mode for each ligand in a bimetal reaction center with the active site in a closed conformation. The ligand directly participates in the coordination shell of two metal ions and is stabilized by the surrounding residues. In contrast, AAH, which exhibits a substrate-binding site similar to that of UAH, requires a larger active site due to the additional ureido group in allantoate. Structural analyses and mutagenesis revealed that both enzymes undergo an open-to-closed conformational transition in response to ligand binding and that the active-site size and the interaction environment in UAH and AAH are determinants of the substrate specificities of these two structurally homologous enzymes.
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    Categories: Journal Articles
  • Functional Evolution of Ribonuclease Inhibitor: Insights from Birds and Reptiles
    [Aug 2014]

    Publication date: 26 August 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 17

    Author(s): Jo E. Lomax , Christopher M. Bianchetti , Aram Chang , George N. Phillips Jr. , Brian G. Fox , Ronald T. Raines

    Ribonuclease inhibitor (RI) is a conserved protein of the mammalian cytosol. RI binds with high affinity to diverse secretory ribonucleases (RNases) and inhibits their enzymatic activity. Although secretory RNases are found in all vertebrates, the existence of a non-mammalian RI has been uncertain. Here, we report on the identification and characterization of RI homologs from chicken and anole lizard. These proteins bind to RNases from multiple species but exhibit much greater affinity for their cognate RNases than for mammalian RNases. To reveal the basis for this differential affinity, we determined the crystal structure of mouse, bovine, and chicken RI·RNase complexes to a resolution of 2.20, 2.21, and 1.92Å, respectively. A combination of structural, computational, and bioinformatic analyses enabled the identification of two residues that appear to contribute to the differential affinity for RNases. We also found marked differences in oxidative instability between mammalian and non-mammalian RIs, indicating evolution toward greater oxygen sensitivity in RIs from mammalian species. Taken together, our results illuminate the structural and functional evolution of RI, along with its dynamic role in vertebrate biology.
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    Categories: Journal Articles
  • Crystal Structures of Ricin Toxin's Enzymatic Subunit (RTA) in Complex with Neutralizing and Non-Neutralizing Single-Chain Antibodies
    [Aug 2014]

    Publication date: 26 August 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 17

    Author(s): Michael J. Rudolph , David J. Vance , Jonah Cheung , Matthew C. Franklin , Fiana Burshteyn , Michael S. Cassidy , Ebony N. Gary , Cristina Herrera , Charles B. Shoemaker , Nicholas J. Mantis

    Ricin is a select agent toxin and a member of the RNA N-glycosidase family of medically important plant and bacterial ribosome-inactivating proteins. In this study, we determined X-ray crystal structures of the enzymatic subunit of ricin (RTA) in complex with the antigen binding domains (VHH) of five unique single-chain monoclonal antibodies that differ in their respective toxin-neutralizing activities. None of the VHHs made direct contact with residues involved in RTA's RNA N-glycosidase activity or induced notable allosteric changes in the toxin's subunit. Rather, the five VHHs had overlapping structural epitopes on the surface of the toxin and differed in the degree to which they made contact with prominent structural elements in two folding domains of the RTA. In general, RTA interactions were influenced most by the VHH CDR3 (CDR, complementarity-determining region) elements, with the most potent neutralizing antibody having the shortest and most conformationally constrained CDR3. These structures provide unique insights into the mechanisms underlying toxin neutralization and provide critically important information required for the rational design of ricin toxin subunit vaccines.
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    Categories: Journal Articles
  • A Conserved Noncoding Sequence Can Function as a Spermatocyte-Specific Enhancer and a Bidirectional Promoter for a Ubiquitously Expressed Gene and a Testis-Specific Long Noncoding RNA
    [Aug 2014]

    Publication date: 26 August 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 17

    Author(s): Misuzu Kurihara , Akira Shiraishi , Honoo Satake , Atsushi P. Kimura

    Tissue-specific gene expression is tightly regulated by various elements such as promoters, enhancers, and long noncoding RNAs (lncRNAs). In the present study, we identified a conserved noncoding sequence (CNS1) as a novel enhancer for the spermatocyte-specific mouse testicular cell adhesion molecule 1 (Tcam1) gene. CNS1 was located 3.4kb upstream of the Tcam1 gene and associated with histone H3K4 mono-methylation in testicular germ cells. By the in vitro reporter gene assay, CNS1 could enhance Tcam1 promoter activity only in GC-2spd(ts) cells, which were derived from mouse spermatocytes. When we integrated the 6.9-kb 5′-flanking sequence of Tcam1 with or without a deletion of CNS1 linked to the enhanced green fluorescent protein gene into the chromatin of GC-2spd(ts) cells, CNS1 significantly enhanced Tcam1 promoter activity. These results indicate that CNS1 could function as a spermatocyte-specific enhancer. Interestingly, CNS1 also showed high bidirectional promoter activity in the reporter assay, and consistent with this, the Smarcd2 gene and lncRNA, designated lncRNA-Tcam1, were transcribed from adjacent regions of CNS1. While Smarcd2 was ubiquitously expressed, lncRNA-Tcam1 expression was restricted to testicular germ cells, although this lncRNA did not participate in Tcam1 activation. Ubiquitous Smarcd2 expression was correlated to CpG hypo-methylation of CNS1 and partially controlled by Sp1. However, for lncRNA-Tcam1 transcription, the strong association with histone acetylation and histone H3K4 tri-methylation also appeared to be required. The present data suggest that CNS1 is a spermatocyte-specific enhancer for the Tcam1 gene and a bidirectional promoter of Smarcd2 and lncRNA-Tcam1.
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    Categories: Journal Articles
  • DNA-recognition by a σ54 transcriptional activator from Aquifex aeolicus
    [Aug 2014]

    Publication date: Available online 23 August 2014
    Source:Journal of Molecular Biology

    Author(s): Natasha K. Vidangos , Johanna Heideker , Artem Lyubimov , Meindert Lamers , Yixin Huo , Jeffrey G. Pelton , Jimmy Ton , Jay Gralla , James Berger , David E. Wemmer

    Transcription initiation by bacterial σ54-polymerase requires the action of a transcriptional activator protein. Activators bind sequence-specifically upstream of the transcription initiation site via a DNA-binding domain. The structurally characterized DNA-binding domains from activators all belong to the Factor for Inversion Stimulation (Fis) family of helix-turn-helix DNA-binding proteins. We report here structures of the free and DNA-bound forms of the DNA-binding domain of NtrC4 (4DBD) from Aquifex aeolicus, a member of the NtrC family of σ54 activators. Two NtrC4 binding sites were identified upstream (–145 and –85 base pairs) from the start of the lpxC gene, which is responsible for the first committed step in Lipid A biosynthesis. This is the first experimental evidence for σ54 regulation in lpxC expression. 4DBD was crystallized both without DNA and in complex with the –145 binding site. The structures, together with biochemical data, indicate that NtrC4 binds to DNA in a manner that is similar to that of its close homologue, Fis. The greater sequence specificity for the binding of 4DBD relative to Fis seems to arise from a larger number of base specific contacts contributing to affinity than for Fis.
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    Categories: Journal Articles