Journal of Molecular Biology

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  • Editorial Board
    [Aug 2014]

    Publication date: 12 August 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 16









    Categories: Journal Articles
  • Contents List
    [Aug 2014]

    Publication date: 12 August 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 16









    Categories: Journal Articles
  • Conformational Dynamics of Thermus aquaticus DNA Polymerase I during Catalysis
    [Aug 2014]

    Publication date: 12 August 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 16

    Author(s): Cuiling Xu , Brian A. Maxwell , Zucai Suo

    Despite the fact that DNA polymerases have been investigated for many years and are commonly used as tools in a number of molecular biology assays, many details of the kinetic mechanism they use to catalyze DNA synthesis remain unclear. Structural and kinetic studies have characterized a rapid, pre-catalytic open-to-close conformational change of the Finger domain during nucleotide binding for many DNA polymerases including Thermus aquaticus DNA polymerase I (Taq Pol), a thermostable enzyme commonly used for DNA amplification in PCR. However, little has been performed to characterize the motions of other structural domains of Taq Pol or any other DNA polymerase during catalysis. Here, we used stopped-flow Förster resonance energy transfer to investigate the conformational dynamics of all five structural domains of the full-length Taq Pol relative to the DNA substrate during nucleotide binding and incorporation. Our study provides evidence for a rapid conformational change step induced by dNTP binding and a subsequent global conformational transition involving all domains of Taq Pol during catalysis. Additionally, our study shows that the rate of the global transition was greatly increased with the truncated form of Taq Pol lacking the N-terminal domain. Finally, we utilized a mutant of Taq Pol containing a de novo disulfide bond to demonstrate that limiting protein conformational flexibility greatly reduced the polymerization activity of Taq Pol.
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    Categories: Journal Articles
  • Undesigned Selection for Replication Termination of Bacterial Chromosomes
    [Aug 2014]

    Publication date: 12 August 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 16

    Author(s): Nobuaki Kono , Kazuharu Arakawa , Mitsuru Sato , Hirofumi Yoshikawa , Masaru Tomita , Mitsuhiro Itaya

    The oriC DNA replication origin in bacterial chromosomes, the location of which appears to be physically identified, is genetically regulated by relevant molecular machinery. In contrast, the location of the terminus remains obscure for many bacterial replicons, except for terC, the proposed and well-studied chromosome termination site in certain bacteria. The terC locus, which is composed of specific sequences for its binding protein, is located at a site opposite from oriC, exhibiting a symmetric structure around the oriC–terC axis. Here, we investigated Bacillus subtilis 168 strains whose axes were hindered and found that the native terC function was robust. However, eradication of terminus region specific binding protein resulted in the natural terC sites not being used for termination; instead, new termini were selected at a location exactly opposite to oriC. We concluded that replication generally terminates at the loci where the two approaching replisomes meet. This site was automatically selected, and two replisomes moving at the same rate supported symmetrical chromosome structures relative to oriC. The rule, which was even validated by artificial chromosomes irrespective of oriC, should be general for replicons administered by two replisomes.
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    Categories: Journal Articles
  • Sus1p Facilitates Pre-Initiation Complex Formation at the SAGA-Regulated Genes Independently of Histone H2B De-Ubiquitylation
    [Aug 2014]

    Publication date: 12 August 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 16

    Author(s): Geetha Durairaj , Rwik Sen , Bhawana Uprety , Abhijit Shukla , Sukesh R. Bhaumik

    Sus1p is a common component of transcriptional co-activator, SAGA (Spt-Ada-Gcn5-Acetyltransferase), and mRNA export complex, TREX-2 (Transcription-export 2), and is involved in promoting transcription and mRNA export. However, it is not clearly understood how Sus1p promotes transcription. Here, we show that Sus1p is predominantly recruited to the upstream activating sequence of a SAGA-dependent gene, GAL1, under transcriptionally active conditions as a component of SAGA to promote the formation of pre-initiation complex (PIC) at the core promoter and, consequently, transcriptional initiation. Likewise, Sus1p promotes the PIC formation at other SAGA-dependent genes and hence transcriptional initiation. Such function of Sus1p in promoting PIC formation and transcriptional initiation is not mediated via its role in regulation of SAGA's histone H2B de-ubiquitylation activity. However, Sus1p's function in regulation of histone H2B ubiquitylation is associated with transcriptional elongation, DNA repair and replication. Collectively, our results support that Sus1p promotes PIC formation (and hence transcriptional initiation) at the SAGA-regulated genes independently of histone H2B de-ubiquitylation and further controls transcriptional elongation, DNA repair and replication via orchestration of histone H2B ubiquitylation, thus providing distinct functional insights of Sus1p in regulation of DNA transacting processes.
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    Categories: Journal Articles
  • Topology, Dimerization, and Stability of the Single-Span Membrane Protein CadC
    [Aug 2014]

    Publication date: 12 August 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 16

    Author(s): Eric Lindner , Stephen H. White

    Under acid stress, Escherichia coli induce expression of CadA (lysine decarboxylase) and CadB (lysine/cadaverine antiporter) in a lysine-rich environment. The ToxR-like transcriptional activator CadC controls expression of the cadBA operon. Using a novel signal peptidase I (SPase I) cleavage assay, we show that CadC is a type II single-span membrane protein (S-SMP) with a cytoplasmic DNA-binding domain and a periplasmic sensor domain. We further show that, as long assumed, dimerization of the sensor domain is required for activating the cadBA operon. We prove this using a chimera in which the periplasmic domain of RodZ—a type II membrane protein involved in the maintenance of the rod shape of E. coli—replaces the CadC sensor domain. Because the RodZ periplasmic domain cannot dimerize, the chimera cannot activate the operon. However, replacement of the transmembrane (TM) domain of the chimera with the glycophorin A TM domain causes intramembrane dimerization and consequently operon activation. Using a low-expression protocol that eliminates extraneous TM helix dimerization signals arising from protein over-expression, we enhanced dramatically the dynamic range of the β-galactosidase assay for cadBA activation. Consequently, the strength of the intramembrane dimerization of the glycophorin A domain could be compared quantitatively with the strength of the much stronger periplasmic dimerization of CadC. For the signal peptidase assay, we inserted an SPase I cleavage site (AAA or AQA) at the periplasmic end of the TM helix. Cleavage occurred with high efficiency for all TM and periplasmic domains tested, thus eliminating the need for the cumbersome spheroplast-proteinase K method for topology determinations.
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    Categories: Journal Articles
  • NMR Model of PrgI–SipD Interaction and Its Implications in the Needle-Tip Assembly of the Salmonella Type III Secretion System
    [Aug 2014]

    Publication date: 12 August 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 16

    Author(s): Thenmalarchelvi Rathinavelan , Maria Lara-Tejero , Matthew Lefebre , Srirupa Chatterjee , Andrew C. McShan , Da-Chuan Guo , Chun Tang , Jorge E. Galan , Roberto N. De Guzman

    Salmonella and other pathogenic bacteria use the type III secretion system (T3SS) to inject virulence proteins into human cells to initiate infections. The structural component of the T3SS contains a needle and a needle tip. The needle is assembled from PrgI needle protomers and the needle tip is capped with several copies of the SipD tip protein. How a tip protein docks on the needle is unclear. A crystal structure of a PrgI–SipD fusion protein docked on the PrgI needle results in steric clash of SipD at the needle tip when modeled on the recent atomic structure of the needle. Thus, there is currently no good model of how SipD is docked on the PrgI needle tip. Previously, we showed by NMR paramagnetic relaxation enhancement (PRE) methods that a specific region in the SipD coiled coil is the binding site for PrgI. Others have hypothesized that a domain of the tip protein—the N-terminal α-helical hairpin—has to swing away during the assembly of the needle apparatus. Here, we show by PRE methods that a truncated form of SipD lacking the α-helical hairpin domain binds more tightly to PrgI. Further, PRE-based structure calculations revealed multiple PrgI binding sites on the SipD coiled coil. Our PRE results together with the recent NMR-derived atomic structure of the Salmonella needle suggest a possible model of how SipD might dock at the PrgI needle tip. SipD and PrgI are conserved in other bacterial T3SSs; thus, our results have wider implication in understanding other needle-tip complexes.
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    Categories: Journal Articles
  • Rtr1 Is a Dual Specificity Phosphatase That Dephosphorylates Tyr1 and Ser5 on the RNA Polymerase II CTD
    [Aug 2014]

    Publication date: 12 August 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 16

    Author(s): Peter L. Hsu , Fan Yang , Whitney Smith-Kinnaman , Wen Yang , Jae-Eun Song , Amber L. Mosley , Gabriele Varani

    The phosphorylation state of heptapeptide repeats within the C-terminal domain (CTD) of the largest subunit of RNA polymerase II (PolII) controls the transcription cycle and is maintained by the competing action of kinases and phosphatases. Rtr1 was recently proposed to be the enzyme responsible for the transition of PolII into the elongation and termination phases of transcription by removing the phosphate marker on serine 5, but this attribution was questioned by the apparent lack of enzymatic activity. Here we demonstrate that Rtr1 is a phosphatase of new structure that is auto-inhibited by its own C-terminus. The enzymatic activity of the protein in vitro is functionally important in vivo as well: a single amino acid mutation that reduces activity leads to the same phenotype in vivo as deletion of the protein-coding gene from yeast. Surprisingly, Rtr1 dephosphorylates not only serine 5 on the CTD but also the newly described anti-termination tyrosine 1 marker, supporting the hypothesis that Rtr1 and its homologs promote the transition from transcription to termination.
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    Categories: Journal Articles
  • The Positive Inside Rule Is Stronger When Followed by a Transmembrane Helix
    [Aug 2014]

    Publication date: 12 August 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 16

    Author(s): Minttu T. Virkki , Christoph Peters , Daniel Nilsson , Therese Sörensen , Susana Cristobal , Björn Wallner , Arne Elofsson

    The translocon recognizes transmembrane helices with sufficient level of hydrophobicity and inserts them into the membrane. However, sometimes less hydrophobic helices are also recognized. Positive inside rule, orientational preferences of and specific interactions with neighboring helices have been shown to aid in the recognition of these helices, at least in artificial systems. To better understand how the translocon inserts marginally hydrophobic helices, we studied three naturally occurring marginally hydrophobic helices, which were previously shown to require the subsequent helix for efficient translocon recognition. We find no evidence for specific interactions when we scan all residues in the subsequent helices. Instead, we identify arginines located at the N-terminal part of the subsequent helices that are crucial for the recognition of the marginally hydrophobic transmembrane helices, indicating that the positive inside rule is important. However, in two of the constructs, these arginines do not aid in the recognition without the rest of the subsequent helix; that is, the positive inside rule alone is not sufficient. Instead, the improved recognition of marginally hydrophobic helices can here be explained as follows: the positive inside rule provides an orientational preference of the subsequent helix, which in turn allows the marginally hydrophobic helix to be inserted; that is, the effect of the positive inside rule is stronger if positively charged residues are followed by a transmembrane helix. Such a mechanism obviously cannot aid C-terminal helices, and consequently, we find that the terminal helices in multi-spanning membrane proteins are more hydrophobic than internal helices.
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    Categories: Journal Articles
  • Hydrophobic Gating in Ion Channels
    [Aug 2014]

    Publication date: Available online 12 August 2014
    Source:Journal of Molecular Biology

    Author(s): Prafulla Aryal , Mark S.P. Sansom , Stephen J. Tucker

    Biological ion channels are nanoscale transmembrane pores. When water and ions are enclosed within the narrow confines of a sub-nanometer hydrophobic pore, they exhibit behavior not evident from macroscopic descriptions. At this nanoscopic level, the unfavorable interaction between the lining of a hydrophobic pore and water may lead to stochastic liquid–vapor transitions. These transient vapor states are “dewetted”, i.e. effectively devoid of water molecules within all or part of the pore, thus leading to an energetic barrier to ion conduction. This process, termed “hydrophobic gating”, was first observed in molecular dynamics simulations of model nanopores, where the principles underlying hydrophobic gating (i.e., changes in diameter, polarity, or transmembrane voltage) have now been extensively validated. Computational, structural, and functional studies now indicate that biological ion channels may also exploit hydrophobic gating to regulate ion flow within their pores. Here we review the evidence for this process and propose that this unusual behavior of water represents an increasingly important element in understanding the relationship between ion channel structure and function.
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    Categories: Journal Articles
  • Erratum for “Specific RNA-Binding Antibodies with a Four-Amino-Acid Code” [J. Mol. Biol. 426 (2014) 2145–2157]
    [Aug 2014]

    Publication date: Available online 10 August 2014
    Source:Journal of Molecular Biology

    Author(s): Eileen M. Sherman , Sean Holmes , Jing-Dong Ye







    Categories: Journal Articles
  • Dynamic and Thermodynamic Response of the Ras Protein Cdc42Hs upon Association with the Effector Domain of PAK3
    [Aug 2014]

    Publication date: Available online 7 August 2014
    Source:Journal of Molecular Biology

    Author(s): Veronica R. Moorman , Kathleen G. Valentine , Sabrina Bédard , Vignesh Kasinath , Jakob Dogan , Fiona M. Love , A. Joshua Wand

    Human cell division cycle protein 42 (Cdc42Hs) is a small, Rho-type guanosine triphosphatase involved in multiple cellular processes through its interactions with downstream effectors. The binding domain of one such effector, the actin cytoskeleton-regulating p21-activated kinase 3, is known as PBD46. Nitrogen-15 backbone and carbon-13 methyl NMR relaxation was measured to investigate the dynamical changes in activated GMPPCP·Cdc42Hs upon PBD46 binding. Changes in internal motion of the Cdc42Hs, as revealed by methyl axis order parameters, were observed not only near the Cdc42Hs–PBD46 interface but also in remote sites on the Cdc42Hs molecule. The binding-induced changes in side-chain dynamics propagate along the long axis of Cdc42Hs away from the site of PBD46 binding with sharp distance dependence. Overall, the binding of the PBD46 effector domain on the dynamics of methyl-bearing side chains of Cdc42Hs results in a modest rigidification, which is estimated to correspond to an unfavorable change in conformational entropy of approximately −10kcalmol−1 at 298K. A cluster of methyl probes closest to the nucleotide-binding pocket of Cdc42Hs becomes more rigid upon binding of PBD46 and is proposed to slow the catalytic hydrolysis of the γ phosphate moiety. An additional cluster of methyl probes surrounding the guanine ring becomes more flexible on binding of PBD46, presumably facilitating nucleotide exchange mediated by a guanosine exchange factor. In addition, the Rho insert helix, which is located at a site remote from the PBD46 binding interface, shows a significant dynamic response to PBD46 binding.
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    Categories: Journal Articles
  • The gene silencing transcription factor REST represses miR-132 expression in hippocampal neurons destined to die
    [Aug 2014]

    Publication date: Available online 6 August 2014
    Source:Journal of Molecular Biology

    Author(s): Jee-Yeon Hwang , Naoki Kaneko , Kyung-Min Noh , Fabrizio Pontarelli , R. Suzanne Zukin

    The gene silencing transcription factor REST/NRSF (Repressor Element-1 (RE1) Silencing Transcription Factor/Neuron-Restrictive Silencer Factor) actively represses a large array of coding and noncoding neuron-specific genes important to synaptic plasticity including miR-132. miR-132 is a neuron-specific microRNA and plays a pivotal role in synaptogenesis, synaptic plasticity and structural remodeling. However, a role for miR-132 in neuronal death is not, as yet, well-delineated. Here we show that ischemic insults promote REST binding and epigenetic remodeling at the miR-132 promoter and silencing of miR-132 expression in selectively-vulnerable hippocampal CA1 neurons. REST occupancy was not altered at the miR-9 or miR-124a promoters despite the presence of RE1 sites, indicating REST target specificity. Ischemia induced a substantial decrease in two marks of active gene transcription, dimethylation of lysine 4 on core histone 3 (H3K4me2) and acetylation of lysine 9 on H3 (H3K9ac) at the miR-132 promoter. RNAi-mediated depletion of REST in vivo blocked ischemia-induced loss of miR-132 in insulted hippocampal neurons, consistent with a causal relation between activation of REST and silencing of miR-132. Overexpression of miR-132 in primary cultures of hippocampal neurons or delivered directly into the CA1 of living rats by means of the lentiviral expression system prior to induction of ischemia afforded robust protection against ischemia-induced neuronal death. These findings document a previously unappreciated role for REST-dependent repression of miR-132 in the neuronal death associated with global ischemia and identify a novel therapeutic target for amelioration of the neurodegeneration and cognitive deficits associated with ischemic stroke.
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    Categories: Journal Articles
  • Identification of a Unique Fe-S Cluster Binding Site in a Glycyl-Radical Type Microcompartment Shell Protein
    [Aug 2014]

    Publication date: Available online 4 August 2014
    Source:Journal of Molecular Biology

    Author(s): Michael C. Thompson , Nicole M. Wheatley , Julien Jorda , Michael R. Sawaya , Soheil D. Gidaniyan , Hoda Ahmed , Zhongyu Yang , Krystal N. McCarty , Julian P. Whitelegge , Todd O. Yeates

    Recently, progress has been made toward understanding the functional diversity of bacterial microcompartment (MCP) systems, which serve as protein-based metabolic organelles in diverse microbes. New types of MCPs have been identified, including the glycyl-radical propanediol (Grp) MCP. Within these elaborate protein complexes, BMC-domain shell proteins [bacterial microcompartment (in reference to the shell protein domain)] assemble to form a polyhedral barrier that encapsulates the enzymatic contents of the MCP. Interestingly, the Grp MCP contains a number of shell proteins with unusual sequence features. GrpU is one such shell protein whose amino acid sequence is particularly divergent from other members of the BMC-domain superfamily of proteins that effectively defines all MCPs. Expression, purification, and subsequent characterization of the protein showed, unexpectedly, that it binds an iron-sulfur cluster. We determined X-ray crystal structures of two GrpU orthologs, providing the first structural insight into the homohexameric BMC-domain shell proteins of the Grp system. The X-ray structures of GrpU, both obtained in the apo form, combined with spectroscopic analyses and computational modeling, show that the metal cluster resides in the central pore of the BMC shell protein at a position of broken 6-fold symmetry. The result is a structurally polymorphic iron-sulfur cluster binding site that appears to be unique among metalloproteins studied to date.
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    Categories: Journal Articles
  • Molecular Analysis of Two Novel Missense Mutations in the GDF5 Proregion That Reduce Protein Activity and Are Associated with Brachydactyly Type C
    [Aug 2014]

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

    Author(s): Katja Stange , Tino Thieme , Karen Hertel , Silke Kuhfahl , Andreas R. Janecke , Hildegunde Piza-Katzer , Maila Penttinen , Marja Hietala , Katarina Dathe , Stefan Mundlos , Elisabeth Schwarz , Petra Seemann

    Growth and differentiation factor 5 (GDF5) plays a central role in bone and cartilage development by regulating the proliferation and differentiation of chondrogenic tissue. GDF5 is synthesized as a preproprotein. The biological function of the proregion comprising 354 residues is undefined. We identified two families with a heterozygosity for the novel missense mutations p.T201P or p.L263P located in the proregion of GDF5. The patients presented with dominant brachydactyly type C characterized by the shortening of skeletal elements in the distal extremities. Both mutations gave rise to decreased biological activity in in vitro analyses. The variants reduced the GDF5-induced activation of SMAD signaling by the GDF5 receptors BMPR1A and BMPR1B. Ectopic expression in micromass cultures yielded relatively low protein levels of the variants and showed diminished chondrogenic activity as compared to wild-type GDF5. Interestingly, stimulation of micromass cells with recombinant human proGDF5T201P and proGDF5L263P revealed their reduced chondrogenic potential compared to the wild-type protein. Limited proteolysis of the mutant recombinant proproteins resulted in a fragment pattern profoundly different from wild-type proGDF5. Modeling of a part of the GDF5 proregion into the known three-dimensional structure of TGFβ1 latency-associated peptide revealed that the homologous positions of both mutations are conserved regions that may be important for the folding of the mature protein or the assembly of dimeric protein complexes. We hypothesize that the missense mutations p.T201P and p.L263P interfere with the protein structure and thereby reduce the amount of fully processed, biologically active GDF5, finally causing the clinical loss of function phenotype.
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    Categories: Journal Articles
  • From Foe to Friend: Using Animal Toxins to Investigate Ion Channel Function
    [Aug 2014]

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

    Author(s): Jeet Kalia , Mirela Milescu , Juan Salvatierra , Jordan Wagner , Julie K. Klint , Glenn F. King , Baldomero M. Olivera , Frank Bosmans

    Ion channels are vital contributors to cellular communication in a wide range of organisms, a distinct feature that renders this ubiquitous family of membrane-spanning proteins a prime target for toxins found in animal venom. For many years, the unique properties of these naturally occurring molecules have enabled researchers to probe the structural and functional features of ion channels and to define their physiological roles in normal and diseased tissues. To illustrate their considerable impact on the ion channel field, this review will highlight fundamental insights into toxin–channel interactions and recently developed toxin screening methods and practical applications of engineered toxins.
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    Categories: Journal Articles
  • Histone Methyltransferase EZH2 Is Transcriptionally Induced by Estradiol as Well as Estrogenic Endocrine Disruptors Bisphenol-A and Diethylstilbestrol
    [Aug 2014]

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

    Author(s): Arunoday Bhan , Imran Hussain , Khairul I. Ansari , Samara A.M. Bobzean , Linda I. Perrotti , Subhrangsu S. Mandal

    Enhancer of Zeste homolog 2 (EZH2), a methyltransferase specific to histone 3 lysine 27, is a critical player in gene silencing and is overexpressed in breast cancer. Our studies demonstrate that EZH2 is transcriptionally induced by estradiol in cultured breast cancer cells and in the mammary glands of ovariectomized rats. EZH2 promoter contains multiple functional estrogen-response elements. Estrogen receptors (ERs) and ER coregulators such as mixed lineage leukemia (MLL) histone methylases (MLL2 and MLL3) and histone acetyltransferase CBP/P300 bind to the EZH2 promoter in the presence of estradiol and regulate estradiol-induced EZH2 expression. EZH2 expression is also increased upon exposure to estrogenic endocrine disrupting chemicals (EDCs) such as bisphenol-A (BPA) and diethylstilbestrol (DES). Similar to estradiol, BPA and DES-induced EZH2 expression is coordinated by ERs, MLLs and CBP/P300. In summary, we demonstrate that EZH2 is transcriptionally regulated by estradiol in vitro and in vivo, and its expression is potentially dysregulated upon exposure to estrogenic EDCs.
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    Categories: Journal Articles
  • A Perspective on the Complexity of Dietary Fiber Structures and Their Potential Effect on the Gut Microbiota
    [Aug 2014]

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

    Author(s): Bruce R. Hamaker , Yunus E. Tuncil

    Even though there are many factors that determine the human colon microbiota composition, diet is an important one because most microorganisms in the colon obtain energy for their growth by degrading complex dietary compounds, particularly dietary fibers. While fiber carbohydrates that escape digestion in the upper gastrointestinal tract are recognized to have a range of structures, the vastness in number of chemical structures from the perspective of the bacteria is not well appreciated. In this article, we introduce the concept of “discrete structure” that is defined as a unique chemical structure, often within a fiber molecule, which aligns with encoded gene clusters in bacterial genomes. The multitude of discrete structures originates from the array of different fiber types coupled with structural variations within types due to genotype and growing environment, anatomical parts of the grain or plant, discrete regions within polymers, and size of oligosaccharides and small polysaccharides. These thousands of discrete structures conceivably could be used to favor bacteria in the competitive colon environment. A global framework needs to be developed to better understand how dietary fibers can be used to obtain predicted changes in microbiota composition for improved health. This will require a multi-disciplinary effort that includes biological scientists, clinicians, and carbohydrate specialists.
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    Categories: Journal Articles
  • Crystal Structure of TIR Domain of TLR6 Reveals Novel Dimeric Interface of TIR–TIR Interaction for Toll-Like Receptor Signaling Pathway
    [Aug 2014]

    Publication date: Available online 31 July 2014
    Source:Journal of Molecular Biology

    Author(s): Tae-ho Jang , Hyun Ho Park

    Toll-like receptors (TLRs) are responsible for recognition of particular pathogens during the innate immune response and cytoplasmic Toll/interleukin-1 receptor (TIR) domain responsible for downstream signaling. TLR6 working with TLR2 can detect bacterial lipoprotein leading signal for nuclear factor-kappaB activation for immune response. To better understand TLR-mediated signaling event in the innate immune system, in this study, we report the first crystal structure of the TIR domain of TLR6 at 2.2Å resolution. Our structure reveals novel homo-dimerization interfaces, which might be a critical for the interaction with TIR-containing adaptor proteins and itself. We also report structural similarities and differences of TLR6 with those of other TIR domains, which may be functionally relevant.
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    Categories: Journal Articles
  • Cu,Zn-Superoxide Dismutase without Zn Is Folded but Catalytically Inactive
    [Aug 2014]

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

    Author(s): Sean Nedd , Rachel L. Redler , Elizabeth A. Proctor , Nikolay V. Dokholyan , Anastassia N. Alexandrova

    Amyotrophic lateral sclerosis has been linked to the gain of aberrant function of superoxide dismutase, Cu,Zn-SOD1 upon protein misfolding. The mechanism of SOD1 misfolding is thought to involve mutations leading to the loss of Zn, followed by protein unfolding and aggregation. We show that the removal of Zn from SOD1 may not lead to an immediate unfolding but immediately deactivates the enzyme through a combination of subtle structural and electronic effects. Using quantum mechanics/discrete molecular dynamics, we showed that both Zn-less wild-type (WT)-SOD1 and its D124N mutant that does not bind Zn have at least metastable folded states. In those states, the reduction potential of Cu increases, leading to the presence of detectable amounts of Cu(I) instead of Cu(II) in the active site, as confirmed experimentally. The Cu(I) protein cannot participate in the catalytic Cu(I)–Cu(II) cycle. However, even without the full reduction to Cu(I), the Cu site in the Zn-less variants of SOD1 is shown to be catalytically incompetent: unable to bind superoxide in a way comparable to the WT-SOD1. The changes are more radical and different in the D124N Zn-less mutant than in the Zn-less WT-SOD1, suggesting D124N being perhaps not the most adequate model for Zn-less SOD1. Overall, Zn in SOD1 appears to be influencing the Cu site directly by adjusting its reduction potential and geometry. Thus, the role of Zn in SOD1 is not just structural, as was previously thought; it is a vital part of the catalytic machinery.
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    Categories: Journal Articles