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  • Structural basis for specific cleavage of Lys6-linked polyubiquitin chains by USP30
    Nat. Struct. Mol. Biol. (IF 12.595) Pub Date : 
    Yusuke Sato, Kei Okatsu, Yasushi Saeki, Koji Yamano, Noriyuki Matsuda, Ai Kaiho, Atsushi Yamagata, Sakurako Goto-Ito, Minoru Ishikawa, Yuichi Hashimoto, Keiji Tanaka, Shuya Fukai

    Parkin ubiquitin (Ub) ligase (also known as PARK2) ubiquitinates damaged mitochondria for their clearance and quality control. USP30 deubiquitinase opposes parkin-mediated Ub-chain formation on mitochondria by preferentially cleaving Lys6-linked Ub chains. Here, we report the crystal structure of zebrafish USP30 in complex with a Lys6-linked diubiquitin (diUb or Ub2) at 1.87-Å resolution. The distal Ub-recognition mechanism of USP30 is similar to those of other USP family members, whereas Phe4 and Thr12 of the proximal Ub are recognized by a USP30-specific surface. Structure-based mutagenesis showed that the interface with the proximal Ub is critical for the specific cleavage of Lys6-linked Ub chains, together with the noncanonical catalytic triad composed of Cys-His-Ser. The structural findings presented here reveal a mechanism for Lys6-linkage-specific deubiquitination.

    更新日期:2017-09-25
  • Mechanism and regulation of the Lys6-selective deubiquitinase USP30
    Nat. Struct. Mol. Biol. (IF 12.595) Pub Date : 
    Malte Gersch, Christina Gladkova, Alexander F Schubert, Martin A Michel, Sarah Maslen, David Komander

    Damaged mitochondria undergo mitophagy, a specialized form of autophagy that is initiated by the protein kinase PINK1 and the ubiquitin E3 ligase Parkin. Ubiquitin-specific protease USP30 antagonizes Parkin-mediated ubiquitination events on mitochondria and is a key negative regulator of mitophagy. Parkin and USP30 both show a preference for assembly or disassembly, respectively, of Lys6-linked polyubiquitin, a chain type that has not been well studied. Here we report crystal structures of human USP30 bound to monoubiquitin and Lys6-linked diubiquitin, which explain how USP30 achieves Lys6-linkage preference through unique ubiquitin binding interfaces. We assess the interplay between USP30, PINK1 and Parkin and show that distally phosphorylated ubiquitin chains impair USP30 activity. Lys6-linkage-specific affimers identify numerous mitochondrial substrates for this modification, and we show that USP30 regulates Lys6-polyubiquitinated TOM20. Our work provides insights into the architecture, activity and regulation of USP30, which will aid drug design against this and related enzymes.

    更新日期:2017-09-25
  • Atomic view of the energy landscape in the allosteric regulation of Abl kinase
    Nat. Struct. Mol. Biol. (IF 12.595) Pub Date : 
    Tamjeed Saleh, Paolo Rossi, Charalampos G Kalodimos

    The activity of protein kinases is often regulated in an intramolecular fashion by signaling domains, which feature several phosphorylation or protein-docking sites. How kinases integrate such distinct binding and signaling events to regulate their activities is unclear, especially in quantitative terms. We used NMR spectroscopy to show how structural elements within the Abl regulatory module (RM) synergistically generate a multilayered allosteric mechanism that enables Abl kinase to function as a finely tuned switch. We dissected the structure and energetics of the regulatory mechanism to precisely measure the effects of various activating or inhibiting stimuli on Abl kinase activity. The data provide a mechanistic basis explaining genetic observations and reveal a previously unknown activator region within Abl. Our findings show that drug-resistance mutations in the Abl RM exert their allosteric effect by promoting the activated state of Abl and not by decreasing the drug affinity for the kinase.

    更新日期:2017-09-25
  • The complete structure of the small-subunit processome
    Nat. Struct. Mol. Biol. (IF 12.595) Pub Date : 
    Jonas Barandun, Malik Chaker-Margot, Mirjam Hunziker, Kelly R Molloy, Brian T Chait, Sebastian Klinge

    The small-subunit processome represents the earliest stable precursor of the eukaryotic small ribosomal subunit. Here we present the cryo-EM structure of the Saccharomyces cerevisiae small-subunit processome at an overall resolution of 3.8 Å, which provides an essentially complete near-atomic model of this assembly. In this nucleolar superstructure, 51 ribosome-assembly factors and two RNAs encapsulate the 18S rRNA precursor and 15 ribosomal proteins in a state that precedes pre-rRNA cleavage at site A1. Extended flexible proteins are employed to connect distant sites in this particle. Molecular mimicry and steric hindrance, as well as protein- and RNA-mediated RNA remodeling, are used in a concerted fashion to prevent the premature formation of the central pseudoknot and its surrounding elements within the small ribosomal subunit.

    更新日期:2017-09-25
  • Structural basis for specific cleavage of Lys6-linked polyubiquitin chains by USP30
    Nat. Struct. Mol. Biol. (IF 12.595) Pub Date : 
    Yusuke Sato, Kei Okatsu, Yasushi Saeki, Koji Yamano, Noriyuki Matsuda, Ai Kaiho, Atsushi Yamagata, Sakurako Goto-Ito, Minoru Ishikawa, Yuichi Hashimoto, Keiji Tanaka, Shuya Fukai

    Parkin ubiquitin (Ub) ligase (also known as PARK2) ubiquitinates damaged mitochondria for their clearance and quality control. USP30 deubiquitinase opposes parkin-mediated Ub-chain formation on mitochondria by preferentially cleaving Lys6-linked Ub chains. Here, we report the crystal structure of zebrafish USP30 in complex with a Lys6-linked diubiquitin (diUb or Ub2) at 1.87-Å resolution. The distal Ub-recognition mechanism of USP30 is similar to those of other USP family members, whereas Phe4 and Thr12 of the proximal Ub are recognized by a USP30-specific surface. Structure-based mutagenesis showed that the interface with the proximal Ub is critical for the specific cleavage of Lys6-linked Ub chains, together with the noncanonical catalytic triad composed of Cys-His-Ser. The structural findings presented here reveal a mechanism for Lys6-linkage-specific deubiquitination.

    更新日期:2017-09-25
  • Mechanism and regulation of the Lys6-selective deubiquitinase USP30
    Nat. Struct. Mol. Biol. (IF 12.595) Pub Date : 
    Malte Gersch, Christina Gladkova, Alexander F Schubert, Martin A Michel, Sarah Maslen, David Komander

    Damaged mitochondria undergo mitophagy, a specialized form of autophagy that is initiated by the protein kinase PINK1 and the ubiquitin E3 ligase Parkin. Ubiquitin-specific protease USP30 antagonizes Parkin-mediated ubiquitination events on mitochondria and is a key negative regulator of mitophagy. Parkin and USP30 both show a preference for assembly or disassembly, respectively, of Lys6-linked polyubiquitin, a chain type that has not been well studied. Here we report crystal structures of human USP30 bound to monoubiquitin and Lys6-linked diubiquitin, which explain how USP30 achieves Lys6-linkage preference through unique ubiquitin binding interfaces. We assess the interplay between USP30, PINK1 and Parkin and show that distally phosphorylated ubiquitin chains impair USP30 activity. Lys6-linkage-specific affimers identify numerous mitochondrial substrates for this modification, and we show that USP30 regulates Lys6-polyubiquitinated TOM20. Our work provides insights into the architecture, activity and regulation of USP30, which will aid drug design against this and related enzymes.

    更新日期:2017-09-25
  • Atomic view of the energy landscape in the allosteric regulation of Abl kinase
    Nat. Struct. Mol. Biol. (IF 12.595) Pub Date : 
    Tamjeed Saleh, Paolo Rossi, Charalampos G Kalodimos

    The activity of protein kinases is often regulated in an intramolecular fashion by signaling domains, which feature several phosphorylation or protein-docking sites. How kinases integrate such distinct binding and signaling events to regulate their activities is unclear, especially in quantitative terms. We used NMR spectroscopy to show how structural elements within the Abl regulatory module (RM) synergistically generate a multilayered allosteric mechanism that enables Abl kinase to function as a finely tuned switch. We dissected the structure and energetics of the regulatory mechanism to precisely measure the effects of various activating or inhibiting stimuli on Abl kinase activity. The data provide a mechanistic basis explaining genetic observations and reveal a previously unknown activator region within Abl. Our findings show that drug-resistance mutations in the Abl RM exert their allosteric effect by promoting the activated state of Abl and not by decreasing the drug affinity for the kinase.

    更新日期:2017-09-25
  • The complete structure of the small-subunit processome
    Nat. Struct. Mol. Biol. (IF 12.595) Pub Date : 
    Jonas Barandun, Malik Chaker-Margot, Mirjam Hunziker, Kelly R Molloy, Brian T Chait, Sebastian Klinge

    The small-subunit processome represents the earliest stable precursor of the eukaryotic small ribosomal subunit. Here we present the cryo-EM structure of the Saccharomyces cerevisiae small-subunit processome at an overall resolution of 3.8 Å, which provides an essentially complete near-atomic model of this assembly. In this nucleolar superstructure, 51 ribosome-assembly factors and two RNAs encapsulate the 18S rRNA precursor and 15 ribosomal proteins in a state that precedes pre-rRNA cleavage at site A1. Extended flexible proteins are employed to connect distant sites in this particle. Molecular mimicry and steric hindrance, as well as protein- and RNA-mediated RNA remodeling, are used in a concerted fashion to prevent the premature formation of the central pseudoknot and its surrounding elements within the small ribosomal subunit.

    更新日期:2017-09-25
  • Structure of a transcribing RNA polymerase II–DSIF complex reveals a multidentate DNA–RNA clamp
    Nat. Struct. Mol. Biol. (IF 12.595) Pub Date : 2017-09-11
    Carrie Bernecky, Jürgen M Plitzko, Patrick Cramer

    During transcription, RNA polymerase II (Pol II) associates with the conserved elongation factor DSIF. DSIF renders the elongation complex stable and functions during Pol II pausing and RNA processing. We combined cryo-EM and X-ray crystallography to determine the structure of the mammalian Pol II–DSIF elongation complex at a nominal resolution of 3.4 Å. Human DSIF has a modular structure with two domains forming a DNA clamp, two domains forming an RNA clamp, and one domain buttressing the RNA clamp. The clamps maintain the transcription bubble, position upstream DNA, and retain the RNA transcript in the exit tunnel. The mobile C-terminal region of DSIF is located near exiting RNA, where it can recruit factors for RNA processing. The structure provides insight into the roles of DSIF during mRNA synthesis.

    更新日期:2017-09-15
  • Structures of the human mitochondrial ribosome in native states of assembly
    Nat. Struct. Mol. Biol. (IF 12.595) Pub Date : 2017-09-11
    Alan Brown, Sorbhi Rathore, Dari Kimanius, Shintaro Aibara, Xiao-chen Bai, Joanna Rorbach, Alexey Amunts, V Ramakrishnan

    Mammalian mitochondrial ribosomes (mitoribosomes) have less rRNA content and 36 additional proteins compared with the evolutionarily related bacterial ribosome. These differences make the assembly of mitoribosomes more complex than the assembly of bacterial ribosomes, but the molecular details of mitoribosomal biogenesis remain elusive. Here, we report the structures of two late-stage assembly intermediates of the human mitoribosomal large subunit (mt-LSU) isolated from a native pool within a human cell line and solved by cryo-EM to ~3-Å resolution. Comparison of the structures reveals insights into the timing of rRNA folding and protein incorporation during the final steps of ribosomal maturation and the evolutionary adaptations that are required to preserve biogenesis after the structural diversification of mitoribosomes. Furthermore, the structures redefine the ribosome silencing factor (RsfS) family as multifunctional biogenesis factors and identify two new assembly factors (L0R8F8 and mt-ACP) not previously implicated in mitoribosomal biogenesis.

    更新日期:2017-09-15
  • Guide-bound structures of an RNA-targeting A-cleaving CRISPR–Cas13a enzyme
    Nat. Struct. Mol. Biol. (IF 12.595) Pub Date : 2017-09-11
    Gavin J Knott, Alexandra East-Seletsky, Joshua C Cofsky, James M Holton, Emeric Charles, Mitchell R O'Connell, Jennifer A Doudna

    CRISPR adaptive immune systems protect bacteria from infections by deploying CRISPR RNA (crRNA)-guided enzymes to recognize and cut foreign nucleic acids. Type VI-A CRISPR–Cas systems include the Cas13a enzyme, an RNA-activated RNase capable of crRNA processing and single-stranded RNA degradation upon target-transcript binding. Here we present the 2.0-Å resolution crystal structure of a crRNA-bound Lachnospiraceae bacterium Cas13a (LbaCas13a), representing a recently discovered Cas13a enzyme subtype. This structure and accompanying biochemical experiments define the Cas13a catalytic residues that are directly responsible for crRNA maturation. In addition, the orientation of the foreign-derived target-RNA-specifying sequence in the protein interior explains the conformational gating of Cas13a nuclease activation. These results describe how Cas13a enzymes generate functional crRNAs and how catalytic activity is blocked before target-RNA recognition, with implications for both bacterial immunity and diagnostic applications.

    更新日期:2017-09-15
  • Structure of a transcribing RNA polymerase II–DSIF complex reveals a multidentate DNA–RNA clamp
    Nat. Struct. Mol. Biol. (IF 12.595) Pub Date : 
    Carrie Bernecky, Jürgen M Plitzko, Patrick Cramer

    During transcription, RNA polymerase II (Pol II) associates with the conserved elongation factor DSIF. DSIF renders the elongation complex stable and functions during Pol II pausing and RNA processing. We combined cryo-EM and X-ray crystallography to determine the structure of the mammalian Pol II–DSIF elongation complex at a nominal resolution of 3.4 Å. Human DSIF has a modular structure with two domains forming a DNA clamp, two domains forming an RNA clamp, and one domain buttressing the RNA clamp. The clamps maintain the transcription bubble, position upstream DNA, and retain the RNA transcript in the exit tunnel. The mobile C-terminal region of DSIF is located near exiting RNA, where it can recruit factors for RNA processing. The structure provides insight into the roles of DSIF during mRNA synthesis.

    更新日期:2017-09-14
  • Structures of the human mitochondrial ribosome in native states of assembly
    Nat. Struct. Mol. Biol. (IF 12.595) Pub Date : 
    Alan Brown, Sorbhi Rathore, Dari Kimanius, Shintaro Aibara, Xiao-chen Bai, Joanna Rorbach, Alexey Amunts, V Ramakrishnan

    Mammalian mitochondrial ribosomes (mitoribosomes) have less rRNA content and 36 additional proteins compared with the evolutionarily related bacterial ribosome. These differences make the assembly of mitoribosomes more complex than the assembly of bacterial ribosomes, but the molecular details of mitoribosomal biogenesis remain elusive. Here, we report the structures of two late-stage assembly intermediates of the human mitoribosomal large subunit (mt-LSU) isolated from a native pool within a human cell line and solved by cryo-EM to ~3-Å resolution. Comparison of the structures reveals insights into the timing of rRNA folding and protein incorporation during the final steps of ribosomal maturation and the evolutionary adaptations that are required to preserve biogenesis after the structural diversification of mitoribosomes. Furthermore, the structures redefine the ribosome silencing factor (RsfS) family as multifunctional biogenesis factors and identify two new assembly factors (L0R8F8 and mt-ACP) not previously implicated in mitoribosomal biogenesis.

    更新日期:2017-09-14
  • Guide-bound structures of an RNA-targeting A-cleaving CRISPR–Cas13a enzyme
    Nat. Struct. Mol. Biol. (IF 12.595) Pub Date : 
    Gavin J Knott, Alexandra East-Seletsky, Joshua C Cofsky, James M Holton, Emeric Charles, Mitchell R O'Connell, Jennifer A Doudna

    CRISPR adaptive immune systems protect bacteria from infections by deploying CRISPR RNA (crRNA)-guided enzymes to recognize and cut foreign nucleic acids. Type VI-A CRISPR–Cas systems include the Cas13a enzyme, an RNA-activated RNase capable of crRNA processing and single-stranded RNA degradation upon target-transcript binding. Here we present the 2.0-Å resolution crystal structure of a crRNA-bound Lachnospiraceae bacterium Cas13a (LbaCas13a), representing a recently discovered Cas13a enzyme subtype. This structure and accompanying biochemical experiments define the Cas13a catalytic residues that are directly responsible for crRNA maturation. In addition, the orientation of the foreign-derived target-RNA-specifying sequence in the protein interior explains the conformational gating of Cas13a nuclease activation. These results describe how Cas13a enzymes generate functional crRNAs and how catalytic activity is blocked before target-RNA recognition, with implications for both bacterial immunity and diagnostic applications.

    更新日期:2017-09-14
  • Eukaryotic ribosome assembly, transport and quality control
    Nat. Struct. Mol. Biol. (IF 12.595) Pub Date : 2017-09-07
    Cohue Peña, Ed Hurt, Vikram Govind Panse

    Eukaryotic ribosome synthesis is a complex, energy-consuming process that takes place across the nucleolus, nucleoplasm and cytoplasm and requires more than 200 conserved assembly factors. Here, we discuss mechanisms by which the ribosome assembly and nucleocytoplasmic transport machineries collaborate to produce functional ribosomes. We also highlight recent cryo-EM studies that provided unprecedented snapshots of ribosomes during assembly and quality control.

    更新日期:2017-09-09
  • Oocyte-derived histone H3 lysine 27 methylation controls gene expression in the early embryo
    Nat. Struct. Mol. Biol. (IF 12.595) Pub Date : 2017-09-07
    Rakesh Pathak, Robert Feil

    Oocyte-derived histone H3 lysine 27 methylation controls gene expression in the early embryo Nature Structural & Molecular Biology, Published online: 7 September 2017; doi:10.1038/nsmb.3456 The monoallelic expression of many imprinted genes in mammals depends on DNA methylation marks that originate from the germ cells. Recent studies in mice and fruit flies evoke a novel, transient mode of genomic imprinting in which oocyte-acquired histone H3 Lys27 trimethylation (H3K27me3) marks are transmitted to the zygote and modulate the allele specificity and timing of gene expression in the early embryo.

    更新日期:2017-09-09
  • A tripartite interface that regulates vesicle fusion
    Nat. Struct. Mol. Biol. (IF 12.595) Pub Date : 
    Inês Chen

    A tripartite interface that regulates vesicle fusion Nature Structural & Molecular Biology, Published online: 7 September 2017; doi:10.1038/nsmb.3468

    更新日期:2017-09-09
  • Aging: rewiring the circadian clock
    Nat. Struct. Mol. Biol. (IF 12.595) Pub Date : 2017-09-07
    Rika Ohkubo, Danica Chen

    Aging: rewiring the circadian clock Nature Structural & Molecular Biology, Published online: 7 September 2017; doi:10.1038/nsmb.3461 The robustness of the circadian clock deteriorates with aging. Two new studies show that aging reprograms the circadian transcriptome in a cell-type-dependent manner and that such rewiring can be reversed by caloric restriction.

    更新日期:2017-09-09
  • Identification of an allosteric network that influences assembly and function of group II chaperonins
    Nat. Struct. Mol. Biol. (IF 12.595) Pub Date : 2017-09-07
    Mingliang Jin, Yao Cong

    Identification of an allosteric network that influences assembly and function of group II chaperonins Nature Structural & Molecular Biology, Published online: 7 September 2017; doi:10.1038/nsmb.3459 Group II chaperonins facilitate protein folding by undergoing ATP-driven conformational changes. A recent study reveals a tunable allosteric network in group II chaperonins that includes a residue at the intersubunit interface, which is important for assembly and allosteric coordination. The authors also propose that lower cooperativity allows group II chaperonins to achieve optimal substrate folding over a broad range of ATP concentrations.

    更新日期:2017-09-09
  • Rps26 directs mRNA-specific translation by recognition of Kozak sequence elements
    Nat. Struct. Mol. Biol. (IF 12.595) Pub Date : 2017-07-31
    Max B Ferretti, Homa Ghalei, Ethan A Ward, Elizabeth L Potts, Katrin Karbstein

    We describe a novel approach to separate two ribosome populations from the same cells and use this method in combination with RNA-seq to identify mRNAs bound to Saccharomyces cerevisiae ribosomes with and without Rps26, a protein linked to the pathogenesis of Diamond–Blackfan anemia (DBA). These analyses reveal that Rps26 contributes to mRNA-specific translation by recognition of the Kozak sequence in well-translated mRNAs and that Rps26-deficient ribosomes preferentially translate mRNA from select stress-response pathways. Surprisingly, exposure of yeast to these stresses leads to the formation of Rps26-deficient ribosomes and to the increased translation of their target mRNAs. These results describe a novel paradigm: the production of specialized ribosomes, which play physiological roles in augmenting the well-characterized transcriptional stress response with a heretofore unknown translational response, thereby creating a feed-forward loop in gene expression. Moreover, the simultaneous gain-of-function and loss-of-function phenotypes from Rps26-deficient ribosomes can explain the pathogenesis of DBA.

    更新日期:2017-09-07
  • Decoding the selectivity of eIF2α holophosphatases and PPP1R15A inhibitors
    Nat. Struct. Mol. Biol. (IF 12.595) Pub Date : 2017-07-31
    Marta Carrara, Anna Sigurdardottir, Anne Bertolotti

    The reversible phosphorylation of proteins controls most cellular functions. Protein kinases have been popular drug targets, unlike phosphatases, which remain a drug discovery challenge. Guanabenz and Sephin1 are selective inhibitors of the phosphatase regulatory subunit PPP1R15A (R15A) that prolong the benefit of eIF2α phosphorylation, thereby protecting cells from proteostatic defects. In mice, Sephin1 prevents two neurodegenerative diseases, Charcot–Marie–Tooth 1B (CMT-1B) and SOD1-mediated amyotrophic lateral sclerosis (ALS). However, the molecular basis for R15A inhibition is unknown. Here we reconstituted human recombinant eIF2α holophosphatases, R15A–PP1 and R15B–PP1, whose activity depends on both the catalytic subunit PP1 (protein phosphatase 1) and either R15A or R15B. This system enabled the functional characterization of these holophosphatases and revealed that Guanabenz and Sephin1 induced a selective conformational change in R15A, detected by resistance to limited proteolysis. This altered the recruitment of eIF2α, preventing its dephosphorylation. This work demonstrates that regulatory subunits of phosphatases are valid drug targets and provides the molecular rationale to expand this concept to other phosphatases.

    更新日期:2017-09-07
  • Katanin spiral and ring structures shed light on power stroke for microtubule severing
    Nat. Struct. Mol. Biol. (IF 12.595) Pub Date : 2017-08-07
    Elena Zehr, Agnieszka Szyk, Grzegorz Piszczek, Ewa Szczesna, Xiaobing Zuo, Antonina Roll-Mecak

    Microtubule-severing enzymes katanin, spastin and fidgetin are AAA ATPases important for the biogenesis and maintenance of complex microtubule arrays in axons, spindles and cilia. Because of a lack of known 3D structures for these enzymes, their mechanism of action has remained poorly understood. Here we report the X-ray crystal structure of the monomeric AAA katanin module from Caenorhabditis elegans and cryo-EM reconstructions of the hexamer in two conformations. The structures reveal an unexpected asymmetric arrangement of the AAA domains mediated by structural elements unique to microtubule-severing enzymes and critical for their function. The reconstructions show that katanin cycles between open spiral and closed ring conformations, depending on the ATP occupancy of a gating protomer that tenses or relaxes interprotomer interfaces. Cycling of the hexamer between these conformations would provide the power stroke for microtubule severing.

    更新日期:2017-09-07
  • An information theoretic framework reveals a tunable allosteric network in group II chaperonins
    Nat. Struct. Mol. Biol. (IF 12.595) Pub Date : 2017-07-24
    Tom Lopez, Kevin Dalton, Anthony Tomlinson, Vijay Pande, Judith Frydman

    ATP-dependent allosteric regulation of the ring-shaped group II chaperonins remains ill defined, in part because their complex oligomeric topology has limited the success of structural techniques in suggesting allosteric determinants. Further, their high sequence conservation has hindered the prediction of allosteric networks using mathematical covariation approaches. Here, we develop an information theoretic strategy that is robust to residue conservation and apply it to group II chaperonins. We identify a contiguous network of covarying residues that connects all nucleotide-binding pockets within each chaperonin ring. An interfacial residue between the networks of neighboring subunits controls positive cooperativity by communicating nucleotide occupancy within each ring. Strikingly, chaperonin allostery is tunable through single mutations at this position. Naturally occurring variants at this position that double the extent of positive cooperativity are less prevalent in nature. We propose that being less cooperative than attainable allows chaperonins to support robust folding over a wider range of metabolic conditions.

    更新日期:2017-09-07
  • TRF2 binds branched DNA to safeguard telomere integrity
    Nat. Struct. Mol. Biol. (IF 12.595) Pub Date : 2017-08-14
    Isabelle Schmutz, Leonid Timashev, Wei Xie, Dinshaw J Patel, Titia de Lange

    Although t-loops protect telomeres, they are at risk of cleavage by Holliday junction (HJ) resolvases if branch migration converts the three-way t-loop junction into four-way HJs. T-loop cleavage is repressed by the TRF2 basic domain, which binds three- and four-way junctions and protects HJs in vitro. By replacing the basic domain with bacterial-protein domains binding three- and four-way junctions, we demonstrated the in vivo relevance of branched-DNA binding. Branched-DNA binding also repressed PARP1, presumably by masking the PARP1 site in the t-loop junction. Although PARP1 recruits HJ resolvases and promotes t-loop cleavage, PARP1 activation alone did not result in t-loop cleavage, thus suggesting that the basic domain also prevents formation of HJs. Concordantly, removal of HJs by BLM helicase mitigated t-loop cleavage in response to loss of the basic domain. We propose that TRF2 masks and stabilizes the t-loop three-way junction, thereby protecting telomeres from detrimental deletions and PARP1 activation.

    更新日期:2017-09-07
  • Structural basis of TIR-domain-assembly formation in MAL- and MyD88-dependent TLR4 signaling
    Nat. Struct. Mol. Biol. (IF 12.595) Pub Date : 2017-07-31
    Thomas Ve, Parimala R Vajjhala, Andrew Hedger, Tristan Croll, Frank DiMaio, Shane Horsefield, Xiong Yu, Peter Lavrencic, Zahid Hassan, Garry P Morgan, Ashley Mansell, Mehdi Mobli, Ailis O'Carroll, Brieuc Chauvin, Yann Gambin, Emma Sierecki, Michael J Landsberg, Katryn J Stacey, Edward H Egelman, Bostjan Kobe

    Toll-like receptor (TLR) signaling is a key innate immunity response to pathogens. Recruitment of signaling adapters such as MAL (TIRAP) and MyD88 to the TLRs requires Toll/interleukin-1 receptor (TIR)-domain interactions, which remain structurally elusive. Here we show that MAL TIR domains spontaneously and reversibly form filaments in vitro. They also form cofilaments with TLR4 TIR domains and induce formation of MyD88 assemblies. A 7-Å-resolution cryo-EM structure reveals a stable MAL protofilament consisting of two parallel strands of TIR-domain subunits in a BB-loop-mediated head-to-tail arrangement. Interface residues that are important for the interaction are conserved among different TIR domains. Although large filaments of TLR4, MAL or MyD88 are unlikely to form during cellular signaling, structure-guided mutagenesis, combined with in vivo interaction assays, demonstrated that the MAL interactions defined within the filament represent a template for a conserved mode of TIR-domain interaction involved in both TLR and interleukin-1 receptor signaling.

    更新日期:2017-09-07
  • An antimicrobial peptide that inhibits translation by trapping release factors on the ribosome
    Nat. Struct. Mol. Biol. (IF 12.595) Pub Date : 2017-07-24
    Tanja Florin, Cristina Maracci, Michael Graf, Prajwal Karki, Dorota Klepacki, Otto Berninghausen, Roland Beckmann, Nora Vázquez-Laslop, Daniel N Wilson, Marina V Rodnina, Alexander S Mankin

    Many antibiotics stop bacterial growth by inhibiting different steps of protein synthesis. However, no specific inhibitors of translation termination are known. Proline-rich antimicrobial peptides, a component of the antibacterial defense system of multicellular organisms, interfere with bacterial growth by inhibiting translation. Here we show that Api137, a derivative of the insect-produced antimicrobial peptide apidaecin, arrests terminating ribosomes using a unique mechanism of action. Api137 binds to the Escherichia coli ribosome and traps release factor (RF) RF1 or RF2 subsequent to the release of the nascent polypeptide chain. A high-resolution cryo-EM structure of the ribosome complexed with RF1 and Api137 reveals the molecular interactions that lead to RF trapping. Api137-mediated depletion of the cellular pool of free release factors causes the majority of ribosomes to stall at stop codons before polypeptide release, thereby resulting in a global shutdown of translation termination.

    更新日期:2017-09-07
  • X-ray structures of endothelin ETB receptor bound to clinical antagonist bosentan and its analog
    Nat. Struct. Mol. Biol. (IF 12.595) Pub Date : 
    Wataru Shihoya, Tomohiro Nishizawa, Keitaro Yamashita, Asuka Inoue, Kunio Hirata, Francois Marie Ngako Kadji, Akiko Okuta, Kazutoshi Tani, Junken Aoki, Yoshinori Fujiyoshi, Tomoko Doi, Osamu Nureki

    Endothelin receptors (ETRs) have crucial roles in vascular control and are targets for drugs designed to treat circulatory-system diseases and cancer progression. The nonpeptide dual-ETR antagonist bosentan is the first oral drug approved to treat pulmonary arterial hypertension. Here we report crystal structures of human endothelin ETB receptor bound to bosentan and to the ETB-selective analog K-8794, at 3.6-Å and 2.2-Å resolution, respectively. The K-8794-bound structure reveals the detailed water-mediated hydrogen-bonding network at the transmembrane core, which could account for the weak negative allosteric modulation of ETB by Na+ ions. The bosentan-bound structure reveals detailed interactions with ETB, which are probably conserved in the ETA receptor. A comparison of the two structures shows unexpected similarity between antagonist and agonist binding. Despite this similarity, bosentan sterically prevents the inward movement of transmembrane helix 6 (TM6), and thus exerts its antagonistic activity. These structural insights will facilitate the rational design of new ETR-targeting drugs.

    更新日期:2017-09-07
  • Constraints and consequences of the emergence of amino acid repeats in eukaryotic proteins
    Nat. Struct. Mol. Biol. (IF 12.595) Pub Date : 
    Sreenivas Chavali, Pavithra L Chavali, Guilhem Chalancon, Natalia Sanchez de Groot, Rita Gemayel, Natasha S Latysheva, Elizabeth Ing-Simmons, Kevin J Verstrepen, Santhanam Balaji, M Madan Babu

    Proteins with amino acid homorepeats have the potential to be detrimental to cells and are often associated with human diseases. Why, then, are homorepeats prevalent in eukaryotic proteomes? In yeast, homorepeats are enriched in proteins that are essential and pleiotropic and that buffer environmental insults. The presence of homorepeats increases the functional versatility of proteins by mediating protein interactions and facilitating spatial organization in a repeat-dependent manner. During evolution, homorepeats are preferentially retained in proteins with stringent proteostasis, which might minimize repeat-associated detrimental effects such as unregulated phase separation and protein aggregation. Their presence facilitates rapid protein divergence through accumulation of amino acid substitutions, which often affect linear motifs and post-translational-modification sites. These substitutions may result in rewiring protein interaction and signaling networks. Thus, homorepeats are distinct modules that are often retained in stringently regulated proteins. Their presence facilitates rapid exploration of the genotype–phenotype landscape of a population, thereby contributing to adaptation and fitness.

    更新日期:2017-09-07
  • Hydroxylation of a conserved tRNA modification establishes non-universal genetic code in echinoderm mitochondria
    Nat. Struct. Mol. Biol. (IF 12.595) Pub Date : 2017-08-07
    Asuteka Nagao, Mitsuhiro Ohara, Kenjyo Miyauchi, Shin-ichi Yokobori, Akihiko Yamagishi, Kimitsuna Watanabe, Tsutomu Suzuki

    The genetic code is not frozen but still evolving, which can result in the acquisition of 'dialectal' codons that deviate from the universal genetic code. RNA modifications in the anticodon region of tRNAs play a critical role in establishing such non-universal genetic codes. In echinoderm mitochondria, the AAA codon specifies asparagine instead of lysine. By analyzing mitochondrial (mt-) tRNALys isolated from the sea urchin (Mesocentrotus nudus), we discovered a novel modified nucleoside, hydroxy-N6-threonylcarbamoyladenosine (ht6A), 3′ adjacent to the anticodon (position 37). Biochemical analysis revealed that ht6A37 has the ability to prevent mt-tRNALys from misreading AAA as lysine, thereby indicating that hydroxylation of N6-threonylcarbamoyladenosine (t6A) contributes to the establishment of the non-universal genetic code in echinoderm mitochondria.

    更新日期:2017-09-07
  • Eukaryotic ribosome assembly, transport and quality control
    Nat. Struct. Mol. Biol. (IF 12.595) Pub Date : 
    Cohue Peña, Ed Hurt, Vikram Govind Panse

    Eukaryotic ribosome synthesis is a complex, energy-consuming process that takes place across the nucleolus, nucleoplasm and cytoplasm and requires more than 200 conserved assembly factors. Here, we discuss mechanisms by which the ribosome assembly and nucleocytoplasmic transport machineries collaborate to produce functional ribosomes. We also highlight recent cryo-EM studies that provided unprecedented snapshots of ribosomes during assembly and quality control.

    更新日期:2017-09-07
  • Oocyte-derived histone H3 lysine 27 methylation controls gene expression in the early embryo
    Nat. Struct. Mol. Biol. (IF 12.595) Pub Date : 
    Rakesh Pathak, Robert Feil

    Oocyte-derived histone H3 lysine 27 methylation controls gene expression in the early embryo Nature Structural & Molecular Biology, Published online: 7 September 2017; doi:10.1038/nsmb.3456 The monoallelic expression of many imprinted genes in mammals depends on DNA methylation marks that originate from the germ cells. Recent studies in mice and fruit flies evoke a novel, transient mode of genomic imprinting in which oocyte-acquired histone H3 Lys27 trimethylation (H3K27me3) marks are transmitted to the zygote and modulate the allele specificity and timing of gene expression in the early embryo.

    更新日期:2017-09-07
  • A tripartite interface that regulates vesicle fusion
    Nat. Struct. Mol. Biol. (IF 12.595) Pub Date : 
    Inês Chen

    A tripartite interface that regulates vesicle fusion Nature Structural & Molecular Biology, Published online: 7 September 2017; doi:10.1038/nsmb.3468

    更新日期:2017-09-07
  • Aging: rewiring the circadian clock
    Nat. Struct. Mol. Biol. (IF 12.595) Pub Date : 
    Rika Ohkubo, Danica Chen

    Aging: rewiring the circadian clock Nature Structural & Molecular Biology, Published online: 7 September 2017; doi:10.1038/nsmb.3461 The robustness of the circadian clock deteriorates with aging. Two new studies show that aging reprograms the circadian transcriptome in a cell-type-dependent manner and that such rewiring can be reversed by caloric restriction.

    更新日期:2017-09-07
  • Identification of an allosteric network that influences assembly and function of group II chaperonins
    Nat. Struct. Mol. Biol. (IF 12.595) Pub Date : 
    Mingliang Jin, Yao Cong

    Identification of an allosteric network that influences assembly and function of group II chaperonins Nature Structural & Molecular Biology, Published online: 7 September 2017; doi:10.1038/nsmb.3459 Group II chaperonins facilitate protein folding by undergoing ATP-driven conformational changes. A recent study reveals a tunable allosteric network in group II chaperonins that includes a residue at the intersubunit interface, which is important for assembly and allosteric coordination. The authors also propose that lower cooperativity allows group II chaperonins to achieve optimal substrate folding over a broad range of ATP concentrations.

    更新日期:2017-09-07
  • CNPY2 is a key initiator of the PERK–CHOP pathway of the unfolded protein response
    Nat. Struct. Mol. Biol. (IF 12.595) Pub Date : 2017-09-04
    Feng Hong, Bei Liu, Bill X Wu, Jordan Morreall, Brady Roth, Christopher Davies, Shaoli Sun, J Alan Diehl, Zihai Li

    The unfolded protein response (UPR) in the endoplasmic reticulum (ER) is a highly conserved protein-quality-control mechanism critical for cells to make survival-or-death decisions under ER-stress conditions. However, how UPR sensors are activated remains unclear. Here, we report that ER luminal protein canopy homolog 2 (CNPY2) is released from grp78 upon ER stress. Free CNPY2 then engages protein kinase R-like ER kinase (PERK) to induce expression of the transcription factor C/EBP homologous protein (CHOP), thereby initiating the UPR. Indeed, deletion of CNPY2 blocked the PERK–CHOP pathway and protected mice from UPR-induced liver damage and steatosis. Additionally, CNPY2 is transcriptionally upregulated by CHOP in a forward-feed loop to further enhance UPR signaling. These findings demonstrate the critical roles of CNPY2 in ER stress and suggest that CNPY2 is a potential new therapeutic target for UPR-related diseases such as metabolic disorders, inflammation and cancer.

    更新日期:2017-09-07
  • N6-methyladenosine (m6A) recruits and repels proteins to regulate mRNA homeostasis
    Nat. Struct. Mol. Biol. (IF 12.595) Pub Date : 2017-09-04
    Raghu R Edupuganti, Simon Geiger, Rik G H Lindeboom, Hailing Shi, Phillip J Hsu, Zhike Lu, Shuang-Yin Wang, Marijke P A Baltissen, Pascal W T C Jansen, Martin Rossa, Markus Müller, Hendrik G Stunnenberg, Chuan He, Thomas Carell, Michiel Vermeulen

    N6-methyladenosine (m6A) recruits and repels proteins to regulate mRNA homeostasis Nature Structural & Molecular Biology, Published online: 4 September 2017; doi:10.1038/nsmb.3462 A comprehensive proteomics screen for ‘reader’ proteins that recognize m6A-modified RNA reveals that the modification both promotes and prevents the binding of factors that control mRNA homeostasis in mammalian cells.

    更新日期:2017-09-07
  • Crystal structure of an inactivated mutant mammalian voltage-gated K+ channel
    Nat. Struct. Mol. Biol. (IF 12.595) Pub Date : 2017-08-28
    Victor Pau, Yufeng Zhou, Yajamana Ramu, Yanping Xu, Zhe Lu

    C-type inactivation underlies important roles played by voltage-gated K+ (Kv) channels. Functional studies have provided strong evidence that a common underlying cause of this type of inactivation is an alteration near the extracellular end of the channel's ion-selectivity filter. Unlike N-type inactivation, which is known to reflect occlusion of the channel's intracellular end, the structural mechanism of C-type inactivation remains controversial and may have many detailed variations. Here we report that in voltage-gated Shaker K+ channels lacking N-type inactivation, a mutation enhancing inactivation disrupts the outermost K+ site in the selectivity filter. Furthermore, in a crystal structure of the Kv1.2-2.1 chimeric channel bearing the same mutation, the outermost K+ site, which is formed by eight carbonyl-oxygen atoms, appears to be slightly too small to readily accommodate a K+ ion and in fact exhibits little ion density; this structural finding is consistent with the functional hallmark of C-type inactivation.

    更新日期:2017-09-07
  • NEAT1 scaffolds RNA-binding proteins and the Microprocessor to globally enhance pri-miRNA processing
    Nat. Struct. Mol. Biol. (IF 12.595) Pub Date : 2017-08-28
    Li Jiang, Changwei Shao, Qi-Jia Wu, Geng Chen, Jie Zhou, Bo Yang, Hairi Li, Lan-Tao Gou, Yi Zhang, Yangming Wang, Gene W Yeo, Yu Zhou, Xiang-Dong Fu

    MicroRNA (miRNA) biogenesis is known to be modulated by a variety of RNA-binding proteins (RBPs), but in most cases, individual RBPs appear to influence the processing of a small subset of target miRNAs. Here, we report that the RNA-binding NONO–PSF heterodimer binds a large number of expressed pri-miRNAs in HeLa cells to globally enhance pri-miRNA processing by the Drosha–DGCR8 Microprocessor. NONO and PSF are key components of paraspeckles organized by the long noncoding RNA (lncRNA) NEAT1. We further demonstrate that NEAT1 also has a profound effect on global pri-miRNA processing. Mechanistic dissection reveals that NEAT1 broadly interacts with the NONO–PSF heterodimer as well as many other RBPs and that multiple RNA segments in NEAT1, including a 'pseudo pri-miRNA' near its 3′ end, help attract the Microprocessor. These findings suggest a 'bird nest' model in which an lncRNA orchestrates efficient processing of potentially an entire class of small noncoding RNAs in the nucleus.

    更新日期:2017-09-07
  • p53 pulses lead to distinct patterns of gene expression albeit similar DNA-binding dynamics
    Nat. Struct. Mol. Biol. (IF 12.595) Pub Date : 2017-08-21
    Antonina Hafner, Jacob Stewart-Ornstein, Jeremy E Purvis, William C Forrester, Martha L Bulyk, Galit Lahav

    The dynamics of transcription factors play important roles in a variety of biological systems. However, the mechanisms by which these dynamics are decoded into different transcriptional responses are not well understood. Here we focus on the dynamics of the tumor-suppressor protein p53, which exhibits a series of pulses in response to DNA damage. We performed time course RNA sequencing (RNA-seq) and chromatin immunoprecipitation sequencing (ChIP-seq) measurements to determine how p53 oscillations are linked with gene expression genome wide. We discovered multiple distinct patterns of gene expression in response to p53 pulses. Surprisingly, p53-binding dynamics were uniform across all genomic loci, even for genes that exhibited distinct mRNA dynamics. Using a mathematical model, supported by additional experimental measurements in response to sustained p53 input, we determined that p53 binds to and activates transcription of its target genes uniformly, whereas post-transcriptional mechanisms are responsible for the differences in gene expression dynamics.

    更新日期:2017-09-07
  • Discovery of a cryptic peptide-binding site on PCSK9 and design of antagonists
    Nat. Struct. Mol. Biol. (IF 12.595) Pub Date : 2017-08-21
    Yingnan Zhang, Mark Ultsch, Nicholas J Skelton, Daniel J Burdick, Maureen H Beresini, Wei Li, Monica Kong-Beltran, Andrew Peterson, John Quinn, Cecilia Chiu, Yan Wu, Steven Shia, Paul Moran, Paola Di Lello, Charles Eigenbrot, Daniel Kirchhofer

    Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates plasma LDL cholesterol (LDL-c) levels by promoting the degradation of liver LDL receptors (LDLRs). Antibodies that inhibit PCSK9 binding to the EGF(A) domain of the LDLR are effective in lowering LDL-c. However, the discovery of small-molecule therapeutics is hampered by difficulty in targeting the relatively flat EGF(A)-binding site on PCSK9. Here we demonstrate that it is possible to target this site, based on the finding that the PCSK9 P′ helix displays conformational flexibility. As a consequence, the vacated N-terminal groove of PCSK9, which is adjacent to the EGF(A)-binding site, is in fact accessible to small peptides. In phage-display experiments, the EGF(A)-mimicking peptide Pep2-8 was used as an anchor peptide for the attachment of an extension peptide library directed toward the groove site. Guided by structural information, we further engineered the identified groove-binding peptides into antagonists, which encroach on the EGF(A)-binding site and inhibit LDLR binding.

    更新日期:2017-09-07
  • X-ray structures of endothelin ETB receptor bound to clinical antagonist bosentan and its analog
    Nat. Struct. Mol. Biol. (IF 12.595) Pub Date : 2017-08-14
    Wataru Shihoya, Tomohiro Nishizawa, Keitaro Yamashita, Asuka Inoue, Kunio Hirata, Francois Marie Ngako Kadji, Akiko Okuta, Kazutoshi Tani, Junken Aoki, Yoshinori Fujiyoshi, Tomoko Doi, Osamu Nureki

    Endothelin receptors (ETRs) have crucial roles in vascular control and are targets for drugs designed to treat circulatory-system diseases and cancer progression. The nonpeptide dual-ETR antagonist bosentan is the first oral drug approved to treat pulmonary arterial hypertension. Here we report crystal structures of human endothelin ETB receptor bound to bosentan and to the ETB-selective analog K-8794, at 3.6-Å and 2.2-Å resolution, respectively. The K-8794-bound structure reveals the detailed water-mediated hydrogen-bonding network at the transmembrane core, which could account for the weak negative allosteric modulation of ETB by Na+ ions. The bosentan-bound structure reveals detailed interactions with ETB, which are probably conserved in the ETA receptor. A comparison of the two structures shows unexpected similarity between antagonist and agonist binding. Despite this similarity, bosentan sterically prevents the inward movement of transmembrane helix 6 (TM6), and thus exerts its antagonistic activity. These structural insights will facilitate the rational design of new ETR-targeting drugs.

    更新日期:2017-09-07
  • Constraints and consequences of the emergence of amino acid repeats in eukaryotic proteins
    Nat. Struct. Mol. Biol. (IF 12.595) Pub Date : 2017-08-14
    Sreenivas Chavali, Pavithra L Chavali, Guilhem Chalancon, Natalia Sanchez de Groot, Rita Gemayel, Natasha S Latysheva, Elizabeth Ing-Simmons, Kevin J Verstrepen, Santhanam Balaji, M Madan Babu

    Proteins with amino acid homorepeats have the potential to be detrimental to cells and are often associated with human diseases. Why, then, are homorepeats prevalent in eukaryotic proteomes? In yeast, homorepeats are enriched in proteins that are essential and pleiotropic and that buffer environmental insults. The presence of homorepeats increases the functional versatility of proteins by mediating protein interactions and facilitating spatial organization in a repeat-dependent manner. During evolution, homorepeats are preferentially retained in proteins with stringent proteostasis, which might minimize repeat-associated detrimental effects such as unregulated phase separation and protein aggregation. Their presence facilitates rapid protein divergence through accumulation of amino acid substitutions, which often affect linear motifs and post-translational-modification sites. These substitutions may result in rewiring protein interaction and signaling networks. Thus, homorepeats are distinct modules that are often retained in stringently regulated proteins. Their presence facilitates rapid exploration of the genotype–phenotype landscape of a population, thereby contributing to adaptation and fitness.

    更新日期:2017-09-07
  • Dueling RNA-binding proteins promote translational activation
    Nat. Struct. Mol. Biol. (IF 12.595) Pub Date : 2017-08-03
    Paul Lasko

    Meiotic progression is controlled by cytoplasmic polyadenylation and translational activation of masked, maternal mRNAs. RNA-binding-protein interactions with adjacent cis elements cause local conformational changes to the mRNAs that determine the extent and timing of their activation.

    更新日期:2017-09-06
  • A tetrad of chromatin interactions for chromosome pairing in X inactivation
    Nat. Struct. Mol. Biol. (IF 12.595) Pub Date : 2017-08-03
    Ivan Krivega, Ann Dean

    An unusual pairing of homologous X chromosomes occurs during X inactivation. A new study in mouse embryonic stem cells shows that telomeres and the telomeric RNA PAR-TERRA are responsible for additional pairwise interactions that guide Xic–Xic pairing.

    更新日期:2017-09-06
  • Two chaperones locked in an embrace: structure and function of the ribosome-associated complex RAC
    Nat. Struct. Mol. Biol. (IF 12.595) Pub Date : 2017-08-03
    Ying Zhang, Irmgard Sinning, Sabine Rospert

    Chaperones, which assist protein folding are essential components of every living cell. The yeast ribosome-associated complex (RAC) is a chaperone that is highly conserved in eukaryotic cells. The RAC consists of the J protein Zuo1 and the unconventional Hsp70 homolog Ssz1. The RAC heterodimer stimulates the ATPase activity of the ribosome-bound Hsp70 homolog Ssb, which interacts with nascent polypeptide chains to facilitate de novo protein folding. In addition, the RAC–Ssb system is required to maintain the fidelity of protein translation. Recent work reveals important details of the unique structures of RAC and Ssb and identifies how the chaperones interact with the ribosome. The new findings start to uncover how the exceptional chaperone triad cooperates in protein folding and maintenance of translational fidelity and its connection to extraribosomal functions.

    更新日期:2017-09-06
  • PAR-TERRA directs homologous sex chromosome pairing
    Nat. Struct. Mol. Biol. (IF 12.595) Pub Date : 2017-07-10
    Hsueh-Ping Chu, John E Froberg, Barry Kesner, Hyun Jung Oh, Fei Ji, Ruslan Sadreyev, Stefan F Pinter, Jeannie T Lee

    In mammals, homologous chromosomes rarely pair outside meiosis. One exception is the X chromosome, which transiently pairs during X-chromosome inactivation (XCI). How two chromosomes find each other in 3D space is not known. Here, we reveal a required interaction between the X-inactivation center (Xic) and the telomere in mouse embryonic stem (ES) cells. The subtelomeric, pseudoautosomal regions (PARs) of the two sex chromosomes (X and Y) also undergo pairing in both female and male cells. PARs transcribe a class of telomeric RNA, dubbed PAR-TERRA, which accounts for a vast majority of all TERRA transcripts. PAR-TERRA binds throughout the genome, including to the PAR and Xic. During X-chromosome pairing, PAR-TERRA anchors the Xic to the PAR, creating a 'tetrad' of pairwise homologous interactions (Xic–Xic, PAR–PAR, and Xic–PAR). Xic pairing occurs within the tetrad. Depleting PAR-TERRA abrogates pairing and blocks initiation of XCI, whereas autosomal PAR-TERRA induces ectopic pairing. We propose a 'constrained diffusion model' in which PAR-TERRA creates an interaction hub to guide Xic homology searching during XCI.

    更新日期:2017-09-06
  • Asparagine endopeptidase cleaves α-synuclein and mediates pathologic activities in Parkinson's disease
    Nat. Struct. Mol. Biol. (IF 12.595) Pub Date : 2017-07-03
    Zhentao Zhang, Seong Su Kang, Xia Liu, Eun Hee Ahn, Zhaohui Zhang, Li He, P Michael Iuvone, Duc M Duong, Nicholas T Seyfried, Matthew J Benskey, Fredric P Manfredsson, Lingjing Jin, Yi E Sun, Jian-Zhi Wang, Keqiang Ye

    Aggregated forms of α-synuclein play a crucial role in the pathogenesis of synucleinopathies such as Parkinson's disease (PD). However, the molecular mechanisms underlying the pathogenic effects of α-synuclein are not completely understood. Here we show that asparagine endopeptidase (AEP) cleaves human α-synuclein, triggers its aggregation and escalates its neurotoxicity, thus leading to dopaminergic neuronal loss and motor impairments in a mouse model. AEP is activated and cleaves human α-synuclein at N103 in an age-dependent manner. AEP is highly activated in human brains with PD, and it fragments α-synuclein, which is found aggregated in Lewy bodies. Overexpression of the AEP-cleaved α-synuclein1–103 fragment in the substantia nigra induces both dopaminergic neuronal loss and movement defects in mice. In contrast, inhibition of AEP-mediated cleavage of α-synuclein (wild type and A53T mutant) diminishes α-synuclein's pathologic effects. Together, these findings support AEP's role as a key mediator of α-synuclein-related etiopathological effects in PD.

    更新日期:2017-09-06
  • Regulator-dependent mechanisms of C3b processing by factor I allow differentiation of immune responses
    Nat. Struct. Mol. Biol. (IF 12.595) Pub Date : 2017-07-03
    Xiaoguang Xue, Jin Wu, Daniel Ricklin, Federico Forneris, Patrizia Di Crescenzio, Christoph Q Schmidt, Joke Granneman, Thomas H Sharp, John D Lambris, Piet Gros

    The complement system labels microbes and host debris for clearance. Degradation of surface-bound C3b is pivotal to direct immune responses and protect host cells. How the serine protease factor I (FI), assisted by regulators, cleaves either two or three distant peptide bonds in the CUB domain of C3b remains unclear. We present a crystal structure of C3b in complex with FI and regulator factor H (FH; domains 1–4 with 19–20). FI binds C3b–FH between FH domains 2 and 3 and a reoriented C3b C-terminal domain and docks onto the first scissile bond, while stabilizing its catalytic domain for proteolytic activity. One cleavage in C3b does not affect its overall structure, whereas two cleavages unfold CUB and dislodge the thioester-containing domain (TED), affecting binding of regulators and thereby determining the number of cleavages. These data explain how FI generates late-stage opsonins iC3b or C3dg in a context-dependent manner, to react to foreign, danger or healthy self signals.

    更新日期:2017-09-06
  • Mechanochemical evolution of the giant muscle protein titin as inferred from resurrected proteins
    Nat. Struct. Mol. Biol. (IF 12.595) Pub Date : 2017-07-03
    Aitor Manteca, Jörg Schönfelder, Alvaro Alonso-Caballero, Marie J Fertin, Nerea Barruetabeña, Bruna F Faria, Elias Herrero-Galán, Jorge Alegre-Cebollada, David De Sancho, Raul Perez-Jimenez

    The sarcomere-based structure of muscles is conserved among vertebrates; however, vertebrate muscle physiology is extremely diverse. A molecular explanation for this diversity and its evolution has not been proposed. We use phylogenetic analyses and single-molecule force spectroscopy (smFS) to investigate the mechanochemical evolution of titin, a giant protein responsible for the elasticity of muscle filaments. We resurrect eight-domain fragments of titin corresponding to the common ancestors to mammals, sauropsids, and tetrapods, which lived 105–356 Myr ago, and compare them with titin fragments from some of their modern descendants. We demonstrate that the resurrected titin molecules are rich in disulfide bonds and display high mechanical stability. These mechanochemical elements have changed over time, creating a paleomechanical trend that seems to correlate with animal body size, allowing us to estimate the sizes of extinct species. We hypothesize that mechanical adjustments in titin contributed to physiological changes that allowed the muscular development and diversity of modern tetrapods.

    更新日期:2017-09-06
  • Multi-domain utilization by TUT4 and TUT7 in control of let-7 biogenesis
    Nat. Struct. Mol. Biol. (IF 12.595) Pub Date : 2017-07-03
    Christopher R Faehnle, Jack Walleshauser, Leemor Joshua-Tor

    The uridyl transferases TUT4 and TUT7 (collectively called TUT4(7)) switch between two modes of activity, either promoting expression of let-7 microRNA (monoU) or marking it for degradation (oligoU). Lin28 modulates the switch via recruitment of TUT4(7) to the precursor pre-let-7 in stem cells and human cancers. We found that TUT4(7) utilize two multidomain functional modules during the switch from monoU to oligoU. The catalytic module (CM) is essential for both activities, while the Lin28-interacting module (LIM) is indispensable for oligoU. A TUT7 CM structure trapped in the monoU activity staterevealed a duplex-RNA-binding pocket that orients group II pre-let-7 hairpins to favor monoU addition. Conversely, the switch to oligoU requires the ZK domain of Lin28 to drive the formation of a stable ternary complex between pre-let-7 and the inactive LIM. Finally, ZK2 of TUT4(7) aids oligoU addition by engaging the growing oligoU tail through uracil-specific interactions.

    更新日期:2017-09-06
  • A two-helix motif positions the lysophosphatidic acid acyltransferase active site for catalysis within the membrane bilayer
    Nat. Struct. Mol. Biol. (IF 12.595) Pub Date : 2017-07-17
    Rosanna M Robertson, Jiangwei Yao, Stefan Gajewski, Gyanendra Kumar, Erik W Martin, Charles O Rock, Stephen W White

    Phosphatidic acid (PA), the central intermediate in membrane phospholipid synthesis, is generated by two acyltransferases in a pathway conserved in all life forms. The second step in this pathway is catalyzed by 1-acyl-sn-glycerol-3-phosphate acyltransferase, called PlsC in bacteria. Here we present the crystal structure of PlsC from Thermotoga maritima, revealing an unusual hydrophobic/aromatic N-terminal two-helix motif linked to an acyltransferase αβ-domain that contains the catalytic HX4D motif. PlsC dictates the acyl chain composition of the 2-position of phospholipids, and the acyl chain selectivity 'ruler' is an appropriately placed and closed hydrophobic tunnel. We confirmed this by site-directed mutagenesis and membrane composition analysis of Escherichia coli cells that expressed mutant PlsC. Molecular dynamics (MD) simulations showed that the two-helix motif represents a novel substructure that firmly anchors the protein to one leaflet of the membrane. This binding mode allows the PlsC active site to acylate lysophospholipids within the membrane bilayer by using soluble acyl donors.

    更新日期:2017-09-06
  • Musashi 1 regulates the timing and extent of meiotic mRNA translational activation by promoting the use of specific CPEs
    Nat. Struct. Mol. Biol. (IF 12.595) Pub Date : 2017-07-17
    Laure Weill, Eulàlia Belloc, Chiara Lara Castellazzi, Raúl Méndez

    The translational reactivation of maternal mRNAs encoding meiotic drivers in vertebrates is accomplished mainly by cytoplasmic polyadenylation. The cytoplasmic polyadenylation elements (CPEs) present in the 3′ untranslated regions (3′ UTRs) of these transcripts, together with their cognate CPE-binding proteins (CPEBs), define a combinatorial code that determines the timing and extent of translational activation upon meiosis resumption. In addition, the RNA-binding protein Musashi1 (Msi1) regulates polyadenylation of CPE-containing mRNAs by a yet undefined CPEB-dependent or CPEB-independent mechanism. Here we show that Msi1 alone does not support cytoplasmic polyadenylation, but its binding triggers the remodeling of RNA structure, thereby exposing adjacent CPEs and stimulating polyadenylation. In this way, Msi1 directs the preferential use of specific CPEs, which in turn affects the timing and extent of polyadenylation during meiotic progression. Genome-wide analysis of CPEB1- and Msi1-associated mRNAs identified 491 common targets, thus revealing a new layer of CPE-mediated translational control.

    更新日期:2017-09-06
  • TRF2 binds branched DNA to safeguard telomere integrity
    Nat. Struct. Mol. Biol. (IF 12.595) Pub Date : 2017-08-14
    Isabelle Schmutz, Leonid Timashev, Wei Xie, Dinshaw J Patel, Titia de Lange

    Although t-loops protect telomeres, they are at risk of cleavage by Holliday junction (HJ) resolvases if branch migration converts the three-way t-loop junction into four-way HJs. T-loop cleavage is repressed by the TRF2 basic domain, which binds three- and four-way junctions and protects HJs in vitro. By replacing the basic domain with bacterial-protein domains binding three- and four-way junctions, we demonstrated the in vivo relevance of branched-DNA binding. Branched-DNA binding also repressed PARP1, presumably by masking the PARP1 site in the t-loop junction. Although PARP1 recruits HJ resolvases and promotes t-loop cleavage, PARP1 activation alone did not result in t-loop cleavage, thus suggesting that the basic domain also prevents formation of HJs. Concordantly, removal of HJs by BLM helicase mitigated t-loop cleavage in response to loss of the basic domain. We propose that TRF2 masks and stabilizes the t-loop three-way junction, thereby protecting telomeres from detrimental deletions and PARP1 activation.

    更新日期:2017-09-06
  • Hydroxylation of a conserved tRNA modification establishes non-universal genetic code in echinoderm mitochondria
    Nat. Struct. Mol. Biol. (IF 12.595) Pub Date : 2017-08-07
    Asuteka Nagao, Mitsuhiro Ohara, Kenjyo Miyauchi, Shin-ichi Yokobori, Akihiko Yamagishi, Kimitsuna Watanabe, Tsutomu Suzuki

    The genetic code is not frozen but still evolving, which can result in the acquisition of 'dialectal' codons that deviate from the universal genetic code. RNA modifications in the anticodon region of tRNAs play a critical role in establishing such non-universal genetic codes. In echinoderm mitochondria, the AAA codon specifies asparagine instead of lysine. By analyzing mitochondrial (mt-) tRNALys isolated from the sea urchin (Mesocentrotus nudus), we discovered a novel modified nucleoside, hydroxy-N6-threonylcarbamoyladenosine (ht6A), 3′ adjacent to the anticodon (position 37). Biochemical analysis revealed that ht6A37 has the ability to prevent mt-tRNALys from misreading AAA as lysine, thereby indicating that hydroxylation of N6-threonylcarbamoyladenosine (t6A) contributes to the establishment of the non-universal genetic code in echinoderm mitochondria.

    更新日期:2017-09-06
  • Katanin spiral and ring structures shed light on power stroke for microtubule severing
    Nat. Struct. Mol. Biol. (IF 12.595) Pub Date : 2017-08-07
    Elena Zehr, Agnieszka Szyk, Grzegorz Piszczek, Ewa Szczesna, Xiaobing Zuo, Antonina Roll-Mecak

    Microtubule-severing enzymes katanin, spastin and fidgetin are AAA ATPases important for the biogenesis and maintenance of complex microtubule arrays in axons, spindles and cilia. Because of a lack of known 3D structures for these enzymes, their mechanism of action has remained poorly understood. Here we report the X-ray crystal structure of the monomeric AAA katanin module from Caenorhabditis elegans and cryo-EM reconstructions of the hexamer in two conformations. The structures reveal an unexpected asymmetric arrangement of the AAA domains mediated by structural elements unique to microtubule-severing enzymes and critical for their function. The reconstructions show that katanin cycles between open spiral and closed ring conformations, depending on the ATP occupancy of a gating protomer that tenses or relaxes interprotomer interfaces. Cycling of the hexamer between these conformations would provide the power stroke for microtubule severing.

    更新日期:2017-09-06
  • Decoding the selectivity of eIF2α holophosphatases and PPP1R15A inhibitors
    Nat. Struct. Mol. Biol. (IF 12.595) Pub Date : 2017-07-31
    Marta Carrara, Anna Sigurdardottir, Anne Bertolotti

    The reversible phosphorylation of proteins controls most cellular functions. Protein kinases have been popular drug targets, unlike phosphatases, which remain a drug discovery challenge. Guanabenz and Sephin1 are selective inhibitors of the phosphatase regulatory subunit PPP1R15A (R15A) that prolong the benefit of eIF2α phosphorylation, thereby protecting cells from proteostatic defects. In mice, Sephin1 prevents two neurodegenerative diseases, Charcot–Marie–Tooth 1B (CMT-1B) and SOD1-mediated amyotrophic lateral sclerosis (ALS). However, the molecular basis for R15A inhibition is unknown. Here we reconstituted human recombinant eIF2α holophosphatases, R15A–PP1 and R15B–PP1, whose activity depends on both the catalytic subunit PP1 (protein phosphatase 1) and either R15A or R15B. This system enabled the functional characterization of these holophosphatases and revealed that Guanabenz and Sephin1 induced a selective conformational change in R15A, detected by resistance to limited proteolysis. This altered the recruitment of eIF2α, preventing its dephosphorylation. This work demonstrates that regulatory subunits of phosphatases are valid drug targets and provides the molecular rationale to expand this concept to other phosphatases.

    更新日期:2017-09-06
  • Rps26 directs mRNA-specific translation by recognition of Kozak sequence elements
    Nat. Struct. Mol. Biol. (IF 12.595) Pub Date : 2017-07-31
    Max B Ferretti, Homa Ghalei, Ethan A Ward, Elizabeth L Potts, Katrin Karbstein

    We describe a novel approach to separate two ribosome populations from the same cells and use this method in combination with RNA-seq to identify mRNAs bound to Saccharomyces cerevisiae ribosomes with and without Rps26, a protein linked to the pathogenesis of Diamond–Blackfan anemia (DBA). These analyses reveal that Rps26 contributes to mRNA-specific translation by recognition of the Kozak sequence in well-translated mRNAs and that Rps26-deficient ribosomes preferentially translate mRNA from select stress-response pathways. Surprisingly, exposure of yeast to these stresses leads to the formation of Rps26-deficient ribosomes and to the increased translation of their target mRNAs. These results describe a novel paradigm: the production of specialized ribosomes, which play physiological roles in augmenting the well-characterized transcriptional stress response with a heretofore unknown translational response, thereby creating a feed-forward loop in gene expression. Moreover, the simultaneous gain-of-function and loss-of-function phenotypes from Rps26-deficient ribosomes can explain the pathogenesis of DBA.

    更新日期:2017-09-06
  • Structural basis of TIR-domain-assembly formation in MAL- and MyD88-dependent TLR4 signaling
    Nat. Struct. Mol. Biol. (IF 12.595) Pub Date : 2017-07-31
    Thomas Ve, Parimala R Vajjhala, Andrew Hedger, Tristan Croll, Frank DiMaio, Shane Horsefield, Xiong Yu, Peter Lavrencic, Zahid Hassan, Garry P Morgan, Ashley Mansell, Mehdi Mobli, Ailis O'Carroll, Brieuc Chauvin, Yann Gambin, Emma Sierecki, Michael J Landsberg, Katryn J Stacey, Edward H Egelman, Bostjan Kobe

    Toll-like receptor (TLR) signaling is a key innate immunity response to pathogens. Recruitment of signaling adapters such as MAL (TIRAP) and MyD88 to the TLRs requires Toll/interleukin-1 receptor (TIR)-domain interactions, which remain structurally elusive. Here we show that MAL TIR domains spontaneously and reversibly form filaments in vitro. They also form cofilaments with TLR4 TIR domains and induce formation of MyD88 assemblies. A 7-Å-resolution cryo-EM structure reveals a stable MAL protofilament consisting of two parallel strands of TIR-domain subunits in a BB-loop-mediated head-to-tail arrangement. Interface residues that are important for the interaction are conserved among different TIR domains. Although large filaments of TLR4, MAL or MyD88 are unlikely to form during cellular signaling, structure-guided mutagenesis, combined with in vivo interaction assays, demonstrated that the MAL interactions defined within the filament represent a template for a conserved mode of TIR-domain interaction involved in both TLR and interleukin-1 receptor signaling.

    更新日期:2017-09-06
  • An antimicrobial peptide that inhibits translation by trapping release factors on the ribosome
    Nat. Struct. Mol. Biol. (IF 12.595) Pub Date : 2017-07-24
    Tanja Florin, Cristina Maracci, Michael Graf, Prajwal Karki, Dorota Klepacki, Otto Berninghausen, Roland Beckmann, Nora Vázquez-Laslop, Daniel N Wilson, Marina V Rodnina, Alexander S Mankin

    Many antibiotics stop bacterial growth by inhibiting different steps of protein synthesis. However, no specific inhibitors of translation termination are known. Proline-rich antimicrobial peptides, a component of the antibacterial defense system of multicellular organisms, interfere with bacterial growth by inhibiting translation. Here we show that Api137, a derivative of the insect-produced antimicrobial peptide apidaecin, arrests terminating ribosomes using a unique mechanism of action. Api137 binds to the Escherichia coli ribosome and traps release factor (RF) RF1 or RF2 subsequent to the release of the nascent polypeptide chain. A high-resolution cryo-EM structure of the ribosome complexed with RF1 and Api137 reveals the molecular interactions that lead to RF trapping. Api137-mediated depletion of the cellular pool of free release factors causes the majority of ribosomes to stall at stop codons before polypeptide release, thereby resulting in a global shutdown of translation termination.

    更新日期:2017-09-06
  • An information theoretic framework reveals a tunable allosteric network in group II chaperonins
    Nat. Struct. Mol. Biol. (IF 12.595) Pub Date : 2017-07-24
    Tom Lopez, Kevin Dalton, Anthony Tomlinson, Vijay Pande, Judith Frydman

    ATP-dependent allosteric regulation of the ring-shaped group II chaperonins remains ill defined, in part because their complex oligomeric topology has limited the success of structural techniques in suggesting allosteric determinants. Further, their high sequence conservation has hindered the prediction of allosteric networks using mathematical covariation approaches. Here, we develop an information theoretic strategy that is robust to residue conservation and apply it to group II chaperonins. We identify a contiguous network of covarying residues that connects all nucleotide-binding pockets within each chaperonin ring. An interfacial residue between the networks of neighboring subunits controls positive cooperativity by communicating nucleotide occupancy within each ring. Strikingly, chaperonin allostery is tunable through single mutations at this position. Naturally occurring variants at this position that double the extent of positive cooperativity are less prevalent in nature. We propose that being less cooperative than attainable allows chaperonins to support robust folding over a wider range of metabolic conditions.

    更新日期:2017-09-06
Some contents have been Reproduced with permission of the American Chemical Society.
Some contents have been Reproduced by permission of The Royal Society of Chemistry.
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