Cellularly active N-hydroxyurea FEN1 inhibitors block substrate entry to the active site

August 15th, 2016 by Jack C Exell

Nature Chemical Biology 12, 815 (2016). doi:10.1038/nchembio.2148

Authors: Jack C Exell, Mark J Thompson, L David Finger, Steven J Shaw, Judit Debreczeni, Thomas A Ward, Claire McWhirter, Catrine L B Siöberg, Daniel Martinez Molina, W Mark Abbott, Clifford D Jones, J Willem M Nissink, Stephen T Durant & Jane A Grasby

  • Posted in Nat Chem Biol, Publications
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Structural basis of laminin binding to the LARGE glycans on dystroglycan

August 15th, 2016 by David C Briggs

Nature Chemical Biology 12, 810 (2016). doi:10.1038/nchembio.2146

Authors: David C Briggs, Takako Yoshida-Moriguchi, Tianqing Zheng, David Venzke, Mary E Anderson, Andrea Strazzulli, Marco Moracci, Liping Yu, Erhard Hohenester & Kevin P Campbell

  • Posted in Nat Chem Biol, Publications
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TALE proteins search DNA using a rotationally decoupled mechanism

August 15th, 2016 by Luke Cuculis

Nature Chemical Biology 12, 831 (2016). doi:10.1038/nchembio.2152

Authors: Luke Cuculis, Zhanar Abil, Huimin Zhao & Charles M Schroeder

Discovery of allosteric modulators for GABAA receptors by ligand-directed chemistry

August 15th, 2016 by Kei Yamaura

Nature Chemical Biology 12, 822 (2016). doi:10.1038/nchembio.2150

Authors: Kei Yamaura, Shigeki Kiyonaka, Tomohiro Numata, Ryuji Inoue & Itaru Hamachi

  • Posted in Nat Chem Biol, Publications
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A Glutaredoxin-BolA Complex Serves as an Iron-Sulfur Cluster Chaperone for the Cytosolic Cluster Assembly Machinery [Metabolism]

August 12th, 2016 by Frey, A. G., Palenchar, D. J., Wildemann, J. D., Philpott, C. C.

Cells contain hundreds of proteins that require iron cofactors for activity. Iron cofactors are synthesized in the cell, but the pathways involved in distributing heme, iron-sulfur clusters, and ferrous/ferric ions to apo-proteins remain incompletely defined. In particular, cytosolic monothiol glutaredoxins and BolA-like proteins have been identified as [2Fe-2S]-coordinating complexes in vitro and iron-regulatory proteins in fungi, but it is not clear how these proteins function in mammalian systems or how this complex might affect Fe-S proteins or the cytosolic Fe-S assembly machinery. To explore these questions, we use quantitative immunoprecipitation and live-cell, proximity-dependent biotinylation, to monitor interactions between Glrx3, BolA2, and components of the cytosolic iron-sulfur cluster assembly system. We characterize cytosolic Glrx3-BolA2 as a [2Fe-2S] chaperone complex in human cells. Unlike complexes formed by fungal orthologs, human Glrx3-BolA2 interaction required the coordination of Fe-S clusters, while Glrx3 homodimer formation did not. Cellular Glrx3-BolA2 complexes increased 6-8-fold in response to increasing iron, forming a rapidly-expandable pool of Fe-S clusters. Fe-S coordination by Glrx3-BolA2 did not depend on Ciapin1 or Ciao1, proteins that bind Glrx3 and are involved in cytosolic Fe-S cluster assembly and distribution. Instead, Glrx3 and BolA2 bound and facilitated Fe-S incorporation into Ciapin1, a [2Fe-2S] protein functioning early in the cytosolic Fe-S assembly pathway. Thus, Glrx3-BolA is a [2Fe-2S] chaperone complex capable of transferring [2Fe-2S] clusters to apo-proteins in human cells.

DFT Study on Enzyme Turnover Including Proton and Electron Transfers of Copper-Containing Nitrite Reductase

August 9th, 2016 by Masami Lintuluoto and Juha M. Lintuluoto

TOC Graphic

Biochemistry
DOI: 10.1021/acs.biochem.6b00423

Allostery Wiring Map for Kinesin Energy Transduction and its Evolution [Enzymology]

August 8th, 2016 by Richard, J., Kim, E. D., Nguyen, H., Kim, C. D., Kim, S.

How signals between the kinesin active- and cytoskeletal-binding sites are transmitted is an open question and an allosteric question. By extracting correlated evolutionary changes within 700+ sequences, we built a model of residues that are energetically coupled and that define molecular routes for signal transmission. Typically, these coupled residues are located at multiple distal sites and, thus, are predicted to form a complex, nonlinear network that wires together different functional sites in the protein. Of note, our model connected the site for ATP hydrolysis with sites that ultimately utilize its free energy, such as the microtubule-binding site, drug-binding loop-5, and necklinker. To confirm the calculated energetic connectivity between non-adjacent residues, double-mutant cycle analysis was conducted with 22 kinesin mutants. There was a direct correlation between thermodynamic coupling in experiment and evolutionarily-derived energetic coupling. We conclude that energy transduction is coordinated by multiple distal sites in the protein, rather than only being relayed through adjacent residues. Moreover, this allosteric map forecasts how energetic orchestration gives rise to different nanomotor behaviors within the superfamily.

Different Divalent Cations Alter the Kinetics and Fidelity of DNA Polymerases [DNA and Chromosomes]

August 8th, 2016 by Vashishtha, A. K., Wang, J., Konigsberg, W. H.

Divalent metal ions are essential components of DNA polymerases both for catalysis of the nucleotidyl transfer reaction and for base excision. They occupy two sites, A and B for DNA synthesis. Recently, a third metal ion was shown to be essential for phosphoryl transfer reaction. The metal ion in the A site is coordinated by the carboxylate of two highly conserved acidic residues, water molecules, and the 3' hydroxyl group of the primer so that the A metal is in an octahedral complex. Its catalytic function is to lower the pKa of the hydroxyl group making it a highly effective nucleophile that can attack the alpha phosphorous atom of the incoming dNTP. The metal ion in the B site is coordinated by the same two carboxylates that are affixed to the A metal ion as well as the non-bridging oxygen atoms of the incoming dNTP. The carboxyl oxygen of an adjacent peptide bond serves as the sixth ligand that completes the octahedral coordination geometry of the B metal ion. Similarly two metal ions are required for proof-reading, one helps to lower the pKa of the attacking water molecule and the other helps to stabilize the transition state for nucleotide excision. The role of different divalent cations are discussed in relation to these two activities as well as their influence on base selectivity and misincorporation by DNA polymerases. Some, but not all of the effects of these different metal ions can be rationalized based on their intrinsic properties which are tabulated in this review.

Rice endosperm starch phosphorylase (Pho1) assembles with disproportionating enzyme (Dpe1) to form a protein complex that enhances synthesis of malto-oligosaccharides [Metabolism]

August 8th, 2016 by Hwang, S.-K., Koper, K., Satoh, H., Okita, T. W.

Starch synthesis in cereal grain endosperm is dependent on the concerted actions of many enzymes. The starch plastidial phosphorylase (Pho1) plays an important role in the initiation of starch synthesis and in the maturation of starch granule in developing rice seeds. Prior evidence has suggested that the rice enzyme, OsPho1, may have a physical/functional interaction with other starch biosynthetic enzymes. Pull-down experiments showed that OsPho1 as well as OsPho1 devoid of its L80 region, a peptide unique to higher plant phosphorylases, captures disproportionating enzyme (OsDpe1). Interaction of the latter enzyme form with OsDpe1 indicates that the putative regulatory L80 is not responsible for multi-enzyme assembly. This heterotypic enzyme complex, determined at a molar ratio of 1:1, was validated by reciprocal co-immunoprecipitation studies of native seed proteins and by co-elution chromatographic and co-migration electrophoretic patterns of these enzymes in rice seed extracts. The OsPho1-OsDpe1 complex utilized a broader range of substrates for enhanced synthesis of larger malto-oligosaccharides than each individual enzyme and significantly elevated the substrate affinities of OsPho1 at 30oC. Moreover, the assembly with OsDpe1 enables OsPho1 to utilize products of transglycosylation reactions involving G1 and G3, sugars that it cannot catalyze directly.
  • Posted in Journal of Biological Chemistry, Publications
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Functional Annotation of a Presumed Nitronate Monooxygenase Reveals a New Class of NADH:quinone Reductases [Enzymology]

August 8th, 2016 by Ball, J., Salvi, F., Gadda, G.

The protein PA1024 from Pseudomonas aeruginosa PAO1 is currently classified as 2-nitropropane dioxygenase, the previous name for nitronate monooxygenase in the GenBankTM and PDB databases, but the enzyme was not kinetically characterized. In this study, PA1024 was purified to high levels and the enzymatic activity was investigated by spectroscopic and polarographic techniques. Purified PA1024 did not exhibit nitronate monooxygenase activity; however, it displayed NADH:quinone reductase and a small NADH:oxidase activity. The enzyme preferred NADH to NADPH as a reducing substrate. PA1024 could reduce a broad spectrum of quinone substrates via a Ping Pong Bi-Bi steady-state kinetic mechanism, generating the corresponding hydroquinones. The reductive half reaction with NADH showed a kred value of 24 s-1 and an apparent Kd value estimated in the low μM range. The enzyme was not able to reduce the azo dye methyl red, routinely used in the kinetic characterization of azoreductases. Finally, we revisited and modified the existing six conserved motifs of PA1024, which define a new class of NADH:quinone reductases and are present in more than 490 hypothetical proteins in the GenBankTM, the vast majority of which are currently misannotated as nitronate monooxygenase.