Functional Complementation Studies Reveal Different Interaction Partners of Escherichia coli IscS and Human NFS1

August 16th, 2017 by Martin Bühning, Martin Friemel and Silke Leimkühler

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Biochemistry
DOI: 10.1021/acs.biochem.7b00627

Structural analyses of the bacterial primosomal protein DnaB reveal that it is a tetramer and forms a complex with a primosomal re-initiation protein [Protein Structure and Folding]

August 14th, 2017 by Yi-Ching Li, Vankadari Naveen, Min-Guan Lin, Chwan-Deng Hsiao

The DnaB primosomal protein from Gram-positive bacteria plays a key role in DNA replication and restart as a loader protein for the recruitment of replisome cascade proteins. Previous investigations have established that DnaB is composed of an N-terminal domain, a middle domain, and a C-terminal domain. However, structural evidence for how DnaB functions at the atomic level is lacking. Here, we report the crystal structure of DnaB, encompassing the N-terminal and middle domains (residues 1-300), from Geobacillus stearothermophilus (GstDnaB1-300) at 2.8 Å resolution. Our structure revealed that GstDnaB1-300 forms a tetramer with two basket-like architecture, a finding s consistent with those from solution studies using analytical ultracentrifugation. Furthermore, our results from both GST pull-down assays and analytical ultracentrifugation show that GstDnaB1-300 is sufficient to form a complex with PriA, the primosomal re-initiation protein. Moreover, with the aid of small angle X-ray scattering (SAXS) experiments, we also determined the structural envelope of full-length DnaB (GstDnaBFL) in solution. These SAXS studies indicated that GstDnaBFL has an elongated conformation and that the protruding density envelopes originating from GstDnaB1-300 could completely accommodate the GstDnaB C-terminal domain (residues 301-461) . Taken together with biochemical assays, our results suggest that GstDnaB uses different domains to distinguish the PriA-interaction and ssDNA-binding. This finding can further extend our understanding of primosomal assembly in replication restart.
  • Posted in Journal of Biological Chemistry, Publications
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Small-molecule studies identify CDK8 as a regulator of IL-10 in myeloid cells

August 14th, 2017 by Liv Johannessen

Nature Chemical Biology 13, 1102 (2017). doi:10.1038/nchembio.2458

Authors: Liv Johannessen, Thomas B Sundberg, Daniel J O'Connell, Raivo Kolde, James Berstler, Katelyn J Billings, Bernard Khor, Brinton Seashore-Ludlow, Anne Fassl, Caitlin N Russell, Isabel J Latorre, Baishan Jiang, Daniel B Graham, Jose R Perez, Piotr Sicinski, Andrew J Phillips, Stuart L Schreiber, Nathanael S Gray, Alykhan F Shamji & Ramnik J Xavier

  • Posted in Nat Chem Biol, Publications
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Optogenetic control of kinetochore function

August 14th, 2017 by Huaiying Zhang

Nature Chemical Biology 13, 1096 (2017). doi:10.1038/nchembio.2456

Authors: Huaiying Zhang, Chanat Aonbangkhen, Ekaterina V Tarasovetc, Edward R Ballister, David M Chenoweth & Michael A Lampson

Kinetochore regulation: Let there be light

August 14th, 2017 by Ana C Figueiredo

Nature Chemical Biology 13, 1058 (2017). doi:10.1038/nchembio.2464

Authors: Ana C Figueiredo & Helder Maiato

Kinetochores form the critical interface with spindle microtubules that accounts for chromosome movement and segregation fidelity during mitosis. Spatial and temporal control of motor protein and checkpoint signaling at kinetochores is now possible with a new set of optogenetic tools.

Engineering Aromatic–Aromatic Interactions To Nucleate Folding in Intrinsically Disordered Regions of Proteins

August 11th, 2017 by Swati Balakrishnan and Siddhartha P. Sarma

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Biochemistry
DOI: 10.1021/acs.biochem.7b00437

Fibronectin Conformation and Assembly: Analysis of Fibronectin Deletion Mutants and Fibronectin Glomerulopathy (GFND) Mutants

August 10th, 2017 by Tomoo Ohashi, Christopher A. Lemmon and Harold P. Erickson

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Biochemistry
DOI: 10.1021/acs.biochem.7b00589

Mapping Functionally Important Residues in the Na+/Dicarboxylate Cotransporter, NaDC1

August 10th, 2017 by Claire Colas, Avner Schlessinger and Ana M. Pajor

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Biochemistry
DOI: 10.1021/acs.biochem.7b00503

Efficient reduction of CO2 by the molybdenum-containing formate dehydrogenase from Cupriavidus necator (Ralstonia eutropha). [Molecular Biophysics]

August 7th, 2017 by Xuejun Yu, Dimitri Niks, Ashok Mulchandani, Russ Hille

The ability of the FdsABG formate dehydrogenase from Cupriavidus necator (formerly known as Ralstonia eutropha) to catalyze the reverse of the physiological reaction, the reduction of CO2 to formate utilizing NADH as electron donor, has been investigated. Contrary to previous studies of this enzyme, we demonstrate that it is in fact effective in catalyzing the reverse reaction, with a kcat of 11 ± 0.4 s-1. We also quantify the stoichiometric accumulation of formic acid as the product of the reaction and demonstrate that the observed kinetic parameters for catalysis in the forward and reverse reaction are thermodynamically consistent, complying with the expected Haldane relationships. Finally, we demonstrate the reaction conditions necessary for gauging the ability of a given formate dehydrogenase or other CO2-utilizing enzyme to catalyze the reverse direction so as to avoid false negative results. In conjunction with our earlier studies on the reaction mechanism of this enzyme (Niks et al. (2016) J. Biol. Chem. 291, 1162- 1174), and on the basis of the present work we conclude that all molybdenum- and tungsten-containing formate dehydrogenases and related enzymes likely operate via a simple hydride transfer mechanism and are effective in catalysing the reversible interconversion of CO2 and formate under the appropriate experimental conditions.
  • Posted in Journal of Biological Chemistry, Publications
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Colibactin assembly line enzymes use S-adenosylmethionine to build a cyclopropane ring

August 7th, 2017 by Li Zha

Nature Chemical Biology 13, 1063 (2017). doi:10.1038/nchembio.2448

Authors: Li Zha, Yindi Jiang, Matthew T Henke, Matthew R Wilson, Jennifer X Wang, Neil L Kelleher & Emily P Balskus

Despite containing an α-amino acid, the versatile cofactor S-adenosylmethionine (SAM) is not a known building block for nonribosomal peptide synthetase (NRPS) assembly lines. Here we report an unusual NRPS module from colibactin biosynthesis that uses SAM for amide bond formation and subsequent cyclopropanation. Our findings showcase a new use for SAM and reveal a novel biosynthetic route to a functional group that likely mediates colibactin's genotoxicity.

  • Posted in Nat Chem Biol, Publications
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