Journal of Molecular Biology

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  • Pyruvate Formate-Lyase Interacts Directly with the Formate Channel FocA to Regulate Formate Translocation
    [Jun 2014]

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

    Author(s): Claudia Doberenz , Michael Zorn , Dörte Falke , David Nannemann , Doreen Hunger , Lydia Beyer , Christian H. Ihling , Jens Meiler , Andrea Sinz , R. Gary Sawers

    The formate-nitrite transporters (FNT) form a superfamily of pentameric membrane channels that translocate monovalent anions across biological membranes. FocA translocates formate bidirectionally but the mechanism underlying how translocation of formate is controlled and what governs substrate specificity remain unclear. Here we demonstrate that the normally soluble dimeric enzyme pyruvate formate-lyase (PflB), which is responsible for intracellular formate generation in enterobacteria and other microbes, interacts specifically with FocA. Association of PflB with the cytoplasmic membrane was shown to be FocA-dependent and purified, Strep-tagged FocA specifically retrieved PflB from Eschericha coli crude extracts. Using a bacterial two-hybrid system it could be shown that the N-terminus of FocA and the central domain of PflB were involved in the interaction. This finding was confirmed by chemical cross-linking experiments. Using constraints imposed by the amino acid residues identified in the cross-linking study we provide for the first time a model for the FocA-PflB complex. The model suggests that the N-terminus of FocA is important for interaction with PflB. An in vivo assay developed to monitor changes in formate levels in the cytoplasm revealed the importance of the interaction with PflB for optimal tranlocation of formate by FocA. This system represents a paradigm for the control of activity of FNT channel proteins.
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    Categories: Journal Articles
  • The F420-reducing [NiFe]-hydrogenase complex from Methanothermobacter marburgensis, the first X-ray structure of a group 3 family member
    [Jun 2014]

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

    Author(s): Stella Vitt , Kesen Ma , Eberhard Warkentin , Johanna Moll , Antonio Pierik , Seigo Shima , Ulrich Ermler

    The reversible redox reaction between coenzyme F420 and H2 to F420H2 is catalyzed by a F420-reducing [NiFe]-hydrogenase (FrhABG) which is an enzyme of the energy metabolism of methanogenic archaea. FrhABG is a group 3 [NiFe]-hydrogenase with a dodecameric quaternary structure of 1.25 MDa as recently revealed by high resolution cryo electron microscopy. We report on the crystal structure of FrhABG from Methanothermobacter marburgensis at 1.7Å resolution and compare it with the structures of group 1 [NiFe]-hydrogenases, the only group structurally characterized yet. FrhA is similar to the large subunit of group 1 [NiFe]-hydrogenases regarding its core structure and the embedded [NiFe]-center but different because of the truncation of ca. 160 residues which results in similar but modified H2- and proton- transport pathways and in suitable interfaces for oligomerization. The small subunit FrhG is composed of a N-terminal domain related to group 1 enzymes and a new C-terminal ferredoxin-like domain carrying the distal and medial [4Fe-4S] clusters. FrhB adopts a novel fold, binds one [4Fe-4S] cluster as well as one FAD in a U-shaped conformation and provides the F420-binding site at the Si-face of the isoalloxazine ring. Similar electrochemical potentials of both catalytic reactions and the electron-transferring [4Fe-4S] clusters, determined to be E°’ ≈ –400mV, are in full agreement with the equalized forward and backward rates of the FrhABG reaction. The protein might contribute to balanced redox potentials by the aspartate coordination of the proximal [4Fe-4S] cluster, the new ferredoxin module and a rather negatively charged FAD surrounding.
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    Categories: Journal Articles
  • Editorial Board
    [Jun 2014]

    Publication date: 29 May 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 11









    Categories: Journal Articles
  • Contents List
    [Jun 2014]

    Publication date: 29 May 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 11









    Categories: Journal Articles
  • Separate Molecular Determinants in Amyloidogenic and Antimicrobial Peptides
    [Jun 2014]

    Publication date: 29 May 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 11

    Author(s): Michael Landreh , Jan Johansson , Hans Jörnvall

    Several amyloid-forming and antimicrobial peptides (AMYs and AMPs) have the ability to bind to and damage cell membranes. In addition, some AMYs possess antimicrobial activity and some AMPs form amyloid-like fibrils, relating the two peptide types and their properties. However, a comparison of their sequence characteristics reveals important differences. The high β-strand and aggregation propensities typical of AMYs are largely absent in α-helix-forming AMPs, which are instead marked by a strong amphipathic moment not generally found in AMYs. Although a few peptides, for example, islet amyloid polypeptide and dermaseptin S9, combine some determinants of both groups, the structural distinctions suggest that antimicrobial activity and amyloid formation are separate features not generally associated.
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  • Identification of the Active Sites in the Methyltransferases of a Transcribing dsRNA Virus
    [Jun 2014]

    Publication date: 29 May 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 11

    Author(s): Bin Zhu , Chongwen Yang , Hongrong Liu , Lingpeng Cheng , Feng Song , Songjun Zeng , Xiaojun Huang , Gang Ji , Ping Zhu

    Many double-stranded RNA (dsRNA) viruses are capable of transcribing and capping RNA within a stable icosahedral viral capsid. The turret of turreted dsRNA viruses belonging to the family Reoviridae is formed by five copies of the turret protein, which contains domains with both 7-N-methyltransferase and 2′-O-methyltransferase activities, and serves to catalyze the methylation reactions during RNA capping. Cypovirus of the family Reoviridae provides a good model system for studying the methylation reactions in dsRNA viruses. Here, we present the structure of a transcribing cypovirus to a resolution of ~3.8Å by cryo-electron microscopy. The binding sites for both S-adenosyl-l-methionine and RNA in the two methyltransferases of the turret were identified. Structural analysis of the turret in complex with RNA revealed a pathway through which the RNA molecule reaches the active sites of the two methyltransferases before it is released into the cytoplasm. The pathway shows that RNA capping reactions occur in the active sites of different turret protein monomers, suggesting that RNA capping requires concerted efforts by at least three turret protein monomers. Thus, the turret structure provides novel insights into the precise mechanisms of RNA methylation.
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    Categories: Journal Articles
  • Post-Translational Regulation of CD133 by ATase1/ATase2-Mediated Lysine Acetylation
    [Jun 2014]

    Publication date: 29 May 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 11

    Author(s): Anthony B. Mak , Mariana Pehar , Allison M.L. Nixon , Rashida A. Williams , Andrea C. Uetrecht , Luigi Puglielli , Jason Moffat

    The CD133 cell-surface protein expresses the AC133 epitope that is associated with cancer progenitor cells and cancer resistance to traditional anticancer therapies. We report that the endoplasmic reticulum Golgi intermediate compartment residing acetyltransferases, ATase1 (NAT8B) and ATase2 (NAT8), can physically interact with CD133 to acetylate the protein on three lysine residues predicted to reside on the first extracellular loop of CD133. Site-directed mutagenesis of these residues mimicking a loss of acetylation and downregulation or inhibition of ATase1/ATase2 resulted in near-complete abolishment of CD133 protein expression. We also demonstrate that targeting ATase1/ATase2 results in apoptosis of CD133 expressing acute lymphoblastic leukemia cells. Taken together, we suggest that lysine acetylation on predicted extracellular residues plays a key role in expression and trafficking of CD133 protein to the cell surface and can be targeted to disrupt CD133 regulation and function.
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    Categories: Journal Articles
  • APE1 Incision Activity at Abasic Sites in Tandem Repeat Sequences
    [Jun 2014]

    Publication date: 29 May 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 11

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

    Repetitive DNA sequences, such as those present in microsatellites and minisatellites, telomeres, and trinucleotide repeats (linked to fragile X syndrome, Huntington disease, etc.), account for nearly 30% of the human genome. These domains exhibit enhanced susceptibility to oxidative attack to yield base modifications, strand breaks, and abasic sites; have a propensity to adopt non-canonical DNA forms modulated by the positions of the lesions; and, when not properly processed, can contribute to genome instability that underlies aging and disease development. Knowledge on the repair efficiencies of DNA damage within such repetitive sequences is therefore crucial for understanding the impact of such domains on genomic integrity. In the present study, using strategically designed oligonucleotide substrates, we determined the ability of human apurinic/apyrimidinic endonuclease 1 (APE1) to cleave at apurinic/apyrimidinic (AP) sites in a collection of tandem DNA repeat landscapes involving telomeric and CAG/CTG repeat sequences. Our studies reveal the differential influence of domain sequence, conformation, and AP site location/relative positioning on the efficiency of APE1 binding and strand incision. Intriguingly, our data demonstrate that APE1 endonuclease efficiency correlates with the thermodynamic stability of the DNA substrate. We discuss how these results have both predictive and mechanistic consequences for understanding the success and failure of repair protein activity associated with such oxidatively sensitive, conformationally plastic/dynamic repetitive DNA domains.
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    Categories: Journal Articles
  • Mitochondrial DNA Variant in COX1 Subunit Significantly Alters Energy Metabolism of Geographically Divergent Wild Isolates in Caenorhabditis elegans
    [Jun 2014]

    Publication date: 29 May 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 11

    Author(s): Stephen D. Dingley , Erzsebet Polyak , Julian Ostrovsky , Satish Srinivasan , Icksoo Lee , Amy B. Rosenfeld , Mai Tsukikawa , Rui Xiao , Mary A. Selak , Joshua J. Coon , Alexander S. Hebert , Paul A. Grimsrud , Young Joon Kwon , David J. Pagliarini , Xiaowu Gai , Theodore G. Schurr , Maik Hüttemann , Eiko Nakamaru-Ogiso , Marni J. Falk

    Mitochondrial DNA (mtDNA) sequence variation can influence the penetrance of complex diseases and climatic adaptation. While studies in geographically defined human populations suggest that mtDNA mutations become fixed when they have conferred metabolic capabilities optimally suited for a specific environment, it has been challenging to definitively assign adaptive functions to specific mtDNA sequence variants in mammals. We investigated whether mtDNA genome variation functionally influences Caenorhabditis elegans wild isolates of distinct mtDNA lineages and geographic origins. We found that, relative to N2 (England) wild-type nematodes, CB4856 wild isolates from a warmer native climate (Hawaii) had a unique p.A12S amino acid substitution in the mtDNA-encoded COX1 core catalytic subunit of mitochondrial complex IV (CIV). Relative to N2, CB4856 worms grown at 20°C had significantly increased CIV enzyme activity, mitochondrial matrix oxidant burden, and sensitivity to oxidative stress but had significantly reduced lifespan and mitochondrial membrane potential. Interestingly, mitochondrial membrane potential was significantly increased in CB4856 grown at its native temperature of 25°C. A transmitochondrial cybrid worm strain, chpIR (M, CB4856>N2), was bred as homoplasmic for the CB4856 mtDNA genome in the N2 nuclear background. The cybrid strain also displayed significantly increased CIV activity, demonstrating that this difference results from the mtDNA-encoded p.A12S variant. However, chpIR (M, CB4856>N2) worms had significantly reduced median and maximal lifespan relative to CB4856, which may relate to their nuclear–mtDNA genome mismatch. Overall, these data suggest that C. elegans wild isolates of varying geographic origins may adapt to environmental challenges through mtDNA variation to modulate critical aspects of mitochondrial energy metabolism.
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    Categories: Journal Articles
  • Assembly of Robust Bacterial Microcompartment Shells Using Building Blocks from an Organelle of Unknown Function
    [Jun 2014]

    Publication date: 29 May 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 11

    Author(s): Jonathan K. Lassila , Susan L. Bernstein , James N. Kinney , Seth D. Axen , Cheryl A. Kerfeld

    Bacterial microcompartments (BMCs) sequester enzymes from the cytoplasmic environment by encapsulation inside a selectively permeable protein shell. Bioinformatic analyses indicate that many bacteria encode BMC clusters of unknown function and with diverse combinations of shell proteins. The genome of the halophilic myxobacterium Haliangium ochraceum encodes one of the most atypical sets of shell proteins in terms of composition and primary structure. We found that microcompartment shells could be purified in high yield when all seven H. ochraceum BMC shell genes were expressed from a synthetic operon in Escherichia coli. These shells differ substantially from previously isolated shell systems in that they are considerably smaller and more homogeneous, with measured diameters of 39±2nm. The size and nearly uniform geometry allowed the development of a structural model for the shells composed of 260 hexagonal units and 13 hexagons per icosahedral face. We found that new proteins could be recruited to the shells by fusion to a predicted targeting peptide sequence, setting the stage for the use of these remarkably homogeneous shells for applications such as three-dimensional scaffolding and the construction of synthetic BMCs. Our results demonstrate the value of selecting from the diversity of BMC shell building blocks found in genomic sequence data for the construction of novel compartments.
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    Categories: Journal Articles
  • Architectures of Whole-Module and Bimodular Proteins from the 6-Deoxyerythronolide B Synthase
    [Jun 2014]

    Publication date: 29 May 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 11

    Author(s): Andrea L. Edwards , Tsutomu Matsui , Thomas M. Weiss , Chaitan Khosla

    The 6-deoxyerythronolide B synthase (DEBS) is a prototypical assembly line polyketide synthase produced by the actinomycete Saccharopolyspora erythraea that synthesizes the macrocyclic core of the antibiotic erythromycin 6-deoxyerythronolide B. The megasynthase is a 2-MDa trimeric complex composed of three unique homodimers assembled from the gene products DEBS1, DEBS2, and DEBS3, which are housed within the erythromycin biosynthetic gene cluster. Each homodimer contains two clusters of catalytically independent enzymatic domains, each referred to as a module, which catalyzes one round of polyketide chain extension and modification. Modules are named sequentially to indicate the order in which they are utilized during synthesis of 6-deoxyerythronolide B. We report small-angle X-ray scattering (SAXS) analyses of a whole module and a bimodule from DEBS, as well as a set of domains for which high-resolution structures are available. In all cases, the solution state was probed under previously established conditions ensuring that each protein is catalytically active. SAXS data are consistent with atomic-resolution structures of DEBS fragments. Therefore, we used the available high-resolution structures of DEBS domains to model the architectures of the larger protein assemblies using rigid-body refinement. Our data support a model in which the third module of DEBS forms a disc-shaped structure capable of caging the acyl carrier protein domain proximal to each active site. The molecular envelope of DEBS3 is a thin elongated ellipsoid, and the results of rigid-body modeling suggest that modules 5 and 6 stack collinearly along the 2-fold axis of symmetry.
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    Categories: Journal Articles
  • Why Have Small Multidrug Resistance Proteins Not Evolved into Fused, Internally Duplicated Structures?
    [Jun 2014]

    Publication date: 29 May 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 11

    Author(s): Pilar Lloris-Garcerá , Susanna Seppälä , Joanna S.G. Slusky , Mikaela Rapp , Gunnar von Heijne

    The increasing number of solved membrane protein structures has led to the recognition of a common feature in a large fraction of the small-molecule transporters: inverted repeat structures, formed by two fused homologous membrane domains with opposite orientation in the membrane. An evolutionary pathway in which the ancestral state is a single gene encoding a dual-topology membrane protein capable of forming antiparallel homodimers has been posited. A gene duplication event enables the evolution of two oppositely orientated proteins that form antiparallel heterodimers. Finally, fusion of the two genes generates an internally duplicated transporter with two oppositely orientated membrane domains. Strikingly, however, in the small multidrug resistance (SMR) family of transporters, no fused, internally duplicated proteins have been found to date. Here, we have analyzed fused versions of the dual-topology transporter EmrE, a member of the SMR family, by blue-native PAGE and in vivo activity measurements. We find that fused constructs give rise to both intramolecular inverted repeat structures and competing intermolecular dimers of varying activity. The formation of several intramolecularly and intermolecularly paired species indicates that a gene fusion event may lower the overall amount of active protein, possibly explaining the apparent absence of fused SMR proteins in nature.
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    Categories: Journal Articles
  • Systematic Detection of Internal Symmetry in Proteins Using CE-Symm
    [Jun 2014]

    Publication date: 29 May 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 11

    Author(s): Douglas Myers-Turnbull , Spencer E. Bliven , Peter W. Rose , Zaid K. Aziz , Philippe Youkharibache , Philip E. Bourne , Andreas Prlić

    Symmetry is an important feature of protein tertiary and quaternary structures that has been associated with protein folding, function, evolution, and stability. Its emergence and ensuing prevalence has been attributed to gene duplications, fusion events, and subsequent evolutionary drift in sequence. This process maintains structural similarity and is further supported by this study. To further investigate the question of how internal symmetry evolved, how symmetry and function are related, and the overall frequency of internal symmetry, we developed an algorithm, CE-Symm, to detect pseudo-symmetry within the tertiary structure of protein chains. Using a large manually curated benchmark of 1007 protein domains, we show that CE-Symm performs significantly better than previous approaches. We use CE-Symm to build a census of symmetry among domain superfamilies in SCOP and note that 18% of all superfamilies are pseudo-symmetric. Our results indicate that more domains are pseudo-symmetric than previously estimated. We establish a number of recurring types of symmetry–function relationships and describe several characteristic cases in detail. With the use of the Enzyme Commission classification, symmetry was found to be enriched in some enzyme classes but depleted in others. CE-Symm thus provides a methodology for a more complete and detailed study of the role of symmetry in tertiary protein structure [availability: CE-Symm can be run from the Web at http://source.rcsb.org/jfatcatserver/symmetry.jsp. Source code and software binaries are also available under the GNU Lesser General Public License (version 2.1) at https://github.com/rcsb/symmetry. An interactive census of domains identified as symmetric by CE-Symm is available from http://source.rcsb.org/jfatcatserver/scopResults.jsp].
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    Categories: Journal Articles
  • Molecular conformation of the full-length tumor suppressor NF2/Merlin—a small angle neutron scattering study
    [Jun 2014]

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

    Author(s): Jahan Ali Khajeh , Jeong Ho Ju , Moussoubaou Atchiba , Marc Allaire , Christopher Stanley , William T. Heller , David J.E. Callaway , Zimei Bu

    The tumor suppressor protein Merlin inhibits cell proliferation upon establishing cell-cell contacts. Because Merlin has high sequence similarity to the Ezrin-Radixin-Moesin (ERM) family of proteins, the structural model of ERM protein autoinhibition and cycling between closed/resting and open/active conformational states is often employed to explain Merlin function. However, recent biochemical studies suggest alternative molecular models of Merlin function. Here, we have determined the low resolution molecular structure and binding activity of Merlin and a Merlin(S518D) mutant that mimics the inactivating phosphorylation at S518 using small angle neutron scattering (SANS) and binding experiments. SANS shows that in solution both Merlin and Merlin(S518D) adopt a closed conformation, but binding experiments indicate that a significant fraction of either Merlin or Merlin(S518D) is capable of binding to the target protein NHERF1. Upon binding to the phosphatidylinositol 4,5-bisphosphate lipid, the wild-type Merlin adopts a more open conformation than in solution, but Merlin(S518D) remains in a closed conformation. This study supports a rheostat model of Merlin in NHERF1 binding, and contributes to resolve a controversy about the molecular conformation and binding activity of Merlin.
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    Categories: Journal Articles
  • Specific Inhibition of p97/VCP ATPase and Kinetic Analysis Demonstrate Interaction between D1 and D2 ATPase domains
    [Jun 2014]

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

    Author(s): Tsui-Fen Chou , Stacie L. Bulfer , Conrad C. Weihl , Kelin Li , Lev G. Lis , Michael A. Walters , Frank J. Schoenen , Henry J. Lin , Raymond J. Deshaies , Michelle R. Arkin

    The p97 AAA (ATPase associated with diverse cellular activities), also called VCP (valosin-containing protein), is an important therapeutic target for cancer and neurodegenerative diseases. p97 forms a hexamer composed of two AAA domains (D1 and D2) that form two stacked rings, and an N-terminal domain that binds numerous cofactor proteins. The interplay between the three domains in p97 is complex, and a deeper biochemical understanding is needed in order to design selective p97 inhibitors as therapeutic agents. It is clear that the D2 ATPase domain hydrolyzes ATP in vitro, but whether D1 contributes to ATPase activity is controversial. Here, we use Walker A and B mutants to demonstrate that D1 is capable of hydrolyzing ATP, and show for the first time that nucleotide binding in the D2 domain increases the catalytic efficiency (kcat /Km) of D1 ATP hydrolysis 280-fold, by increasing kcat 7-fold and decreasing Km about 40-fold. We further show that an ND1 construct lacking D2 but including the linker between D1 and D2 is catalytically active, resolving a conflict in the literature. Applying enzymatic observations to small-molecule inhibitors, we show that four p97 inhibitors (DBeQ, ML240, ML241, and NMS-873) have differential responses to Walker A and B mutations, to disease-causing IBMPFD mutations, and to the presence of the N-domain binding cofactor protein p47. These differential effects provide the first evidence that p97 cofactors and disease mutations can alter p97 inhibitor potency and suggest the possibility of developing context-dependent inhibitors of p97.
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    Categories: Journal Articles
  • Assembly of Helicobacter pylori initiation complex is determined by sequence-specific and topology-sensitive DnaA-oriC interactions
    [Jun 2014]

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

    Author(s): Rafał Donczew , Thorsten Mielke , Paweł Jaworski , Jolanta Zakrzewska-Czerwińska , Anna Zawilak-Pawlik

    In bacteria, chromosome replication is initiated by binding of the DnaA initiator protein to DnaA boxes located in the origin of chromosomal replication (oriC). This leads to DNA helix opening within the DNA-unwinding element (DUE). Helicobacter pylori oriC, the first bipartite origin identified in Gram-negative bacteria, contains two subregions, oriC1 and oriC2, flanking the dnaA gene. The DUE region is localised in the oriC2 subregion downstream of dnaA. Surprisingly, oriC2-DnaA interactions were shown to depend on DNA topology, which is unusual in bacteria but similar to initiator-origin interactions observed in higher organisms. In this work, we identified three DnaA boxes in the oriC2 subregion, two of which were bound only as supercoiled DNA. We found that all three DnaA boxes play important roles in orisome assembly and subsequent DNA unwinding, but different functions can be assigned to individual boxes. This suggests that the H. pylori oriC may be functionally divided, similar to what was described recently for Escherichia coli oriC. On the basis of these results, we propose a model of initiation complex formation in H. pylori.
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    Categories: Journal Articles
  • Systematic Exploration of Ubiquitin Sequence, E1 Activation Efficiency, and Experimental Fitness in Yeast
    [Jun 2014]

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

    Author(s): Benjamin P. Roscoe , Daniel N.A. Bolon

    The complexity of biological interaction networks poses a challenge to understanding the function of individual connections in the overall network. To address this challenge, we developed a high-throughput reverse engineering strategy to analyze how thousands of specific perturbations (encompassing all point mutations in a central gene) impact both a specific edge (interaction to a directly connected node) and an overall network function. We analyzed the effects of ubiquitin mutations on activation by the E1 enzyme and compared these to effects on yeast growth rate. Using this approach, we delineated ubiquitin mutations that selectively impacted the ubiquitin-E1 edge. We find that the elasticity function relating the efficiency of ubiquitin-E1 interaction to growth rate is non-linear and that a greater than 50-fold decrease in E1 activation efficiency is required to reduce growth rate by 2-fold. Despite the robustness of fitness to decreases in E1 activation efficiency, the effects of most ubiquitin mutations on E1 activation paralleled the effects on growth rate. Our observations indicate that most ubiquitin mutations that disrupt E1 activation also disrupt other functions. The structurally characterized ubiquitin-E1 interface encompasses the interfaces of ubiquitin with most other known binding partners, and we propose that this enables E1 in wild-type cells to selectively activate ubiquitin protein molecules capable of binding to other partners from the cytoplasmic pool of ubiquitin protein that will include molecules with chemical damage and/or errors from transcription and translation.
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    Categories: Journal Articles
  • Competition as a Way of Life for H+-Coupled Antiporters
    [Jun 2014]

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

    Author(s): Shimon Schuldiner

    Antiporters are ubiquitous membrane proteins that catalyze obligatory exchange between two or more substrates across a membrane in opposite directions. Some utilize proton electrochemical gradients generated by primary pumps by coupling the downhill movement of one or more protons to the movement of a substrate. Since the direction of the proton gradient usually favors proton movement toward the cytoplasm, their function results in removal of substrates other than protons from the cytoplasm, either into acidic intracellular compartments or out to the medium. H+-coupled antiporters play central roles in living organisms, for example, storage of neurotransmitter and other small molecules, resistance to antibiotics, homeostasis of ionic content and more. Biochemical and structural data support a general mechanism for H+-coupled antiporters whereby the substrate and the protons cannot bind simultaneously to the protein. In several cases, it was shown that the binding sites overlap, and therefore, there is a direct competition between the protons and the substrate. In others, the “competition” seems to be indirect and it is most likely achieved by allosteric mechanisms. The pK a of one or more carboxyls in the protein must be tuned appropriately in order to ensure the feasibility of such a mechanism. In this review, I discuss in detail the case of EmrE, a multidrug transporter from Escherichia coli and evaluate the information available for other H+-coupled antiporters.
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    Categories: Journal Articles
  • Binding of argtx-636 in the NMDA receptor ion channel
    [Jun 2014]

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

    Author(s): Mette H. Poulsen , Jacob Andersen , Rune Christensen , Kasper B. Hansen , Stephen F. Traynelis , Kristian Strømgaard , Anders Skov Kristensen

    The N-methyl-D-aspartate receptors (NMDARs) constitute an important class of ligand-gated cation channels that are involved in the majority of excitatory neurotransmission in the human brain. Compounds that bind in the NMDAR ion channel and act as blockers, are use- and voltage-dependent inhibitors of NMDAR activity and have therapeutic potential for treatment of a variety of brain diseases or as pharmacological tools for studies of the neurobiological role of NMDARs. We have performed a kinetic analysis of the blocking mechanism of the prototypical polyamine toxin NMDAR ion channel blocker argiotoxin-636 (ArgTX-636) at recombinant GluN1/2A receptors to provide detailed information on the mechanism of block. The predicted binding site of ArgTX-636 is in the pore region of the NMDAR ion channel formed by residues in the transmembrane M3 and the M2 pore-loop segments of the GluN1 and GluN2A subunits. To assess the predicted binding mode in further detail, we performed an alanine- and glycine-scanning mutational analysis of this pore-loop segment to systematically probe the role of pore-lining M2 residues in GluN1 and GluN2A in the channel block by ArgTX-636. Comparison of M2 positions in GluN1 and GluN2A where mutation influences ArgTX-636 potency suggests differential contribution of the M2-loops of GluN1 and GluN2A to binding of ArgTX-636. The results of the mutational analysis are highly relevant for the future structure-based development of argiotoxin-derived NMDAR channel blockers.
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    Categories: Journal Articles
  • Glycan Specificity of the Vibrio vulnificus Hemolysin Lectin Outlines Evolutionary History of Membrane Targeting by a Toxin Family
    [Jun 2014]

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

    Author(s): Katherine Kaus , Jeffrey W. Lary , James L. Cole , Rich Olson

    Pore-forming toxins (PFTs) are a class of pathogen-secreted molecules that oligomerize to form transmembrane channels in cellular membranes. Determining the mechanism for how PFTs bind membranes is important in understanding their role in disease and for developing possible ways to block their action. Vibrio vulnificus, an aquatic pathogen responsible for severe food poisoning and septicemia in humans, secretes a PFT called V. vulnificus hemolysin (VVH), which contains a single C-terminal targeting domain predicted to resemble a β-trefoil lectin fold. In order to understand the selectivity of the lectin for glycan motifs, we expressed the isolated VVH β-trefoil domain and used glycan-chip screening to identify that VVH displays a preference for terminal galactosyl groups including N-acetyl-d-galactosamine and N-acetyl-d-lactosamine. The X-ray crystal structure of the VVH lectin domain solved to 2.0Å resolution reveals a heptameric ring arrangement similar to the oligomeric form of the related, but inactive, lectin from Vibrio cholerae cytolysin. Structures bound to glycerol, N-acetyl-d-galactosamine, and N-acetyl-d-lactosamine outline a common and versatile mode of recognition allowing VVH to target a wide variety of cell-surface ligands. Sequence analysis in light of our structural and functional data suggests that VVH may represent an earlier step in the evolution of Vibrio PFTs.
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    Categories: Journal Articles