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Negative cooperativity of uric acid binding to the transcriptional regulator HucR from Deinococcus radiodurans.
J Mol Biol. 2005 Jul 22; 350(4):617-30.JM

Abstract

Members of the MarR family of winged helix transcriptional regulators have been shown to regulate multidrug and oxidative stress response, pathogenesis, and catabolism of aromatic compounds. Many respond to anionic lipophilic compounds in their capacity to bind DNA, and the co-crystal structure of MarR bound to salicylate revealed two ligand-binding pockets, SAL-A and SAL-B. The MarR homolog, HucR, from Deinococcus radiodurans has been shown to repress expression of a predicted uricase, and DNA-binding by HucR is antagonized by uric acid, the substrate of uricase. We provide a biochemical investigation of DNA-binding and uric acid-binding by HucR. Equilibrium analytical ultracentrifugation indicates that HucR exists as a dimer. Intrinsic fluorescence spectra suggest that the association of the HucR dimer with its cognate DNA involves conformational flexibility in the globular interior and/or dimerization domain of the protein, and near-UV circular dichroism spectra indicate a concomitant change in the helical twist of the DNA duplex. DNA-binding affinity, measured by electrophoretic mobility-shift assays, for HucR mutants bearing single amino acid substitutions suggests the importance of the beta-hairpin "wing" in DNA binding. Analysis of intrinsic fluorescence spectra demonstrates that uric acid induces conformational changes in HucR and binds with an apparent K(d)=11.6(+/-3.7)muM and a Hill coefficient of 0.7+/-0.1, indicating negative cooperativity. Fluorescence and DNA-binding properties of the HucR variants indicate that SAL-A is a low-affinity, uric acid-binding site and that negative cooperativity exists between homologous, high-affinity sites. The conservation of residues comprising site SAL-A suggests that it is a low-affinity, ligand-binding site in MarR homologs. Mechanistic considerations suggest that HucR is regulated by uric acid to maintain optimal cellular levels of this scavenger of free radicals in response to oxidative stress and DNA damage.

Authors+Show Affiliations

Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803, USA. swilki2@lsu.eduNo affiliation info available

Pub Type(s)

Journal Article
Research Support, U.S. Gov't, Non-P.H.S.

Language

eng

PubMed ID

15967460

Citation

Wilkinson, Steven P., and Anne Grove. "Negative Cooperativity of Uric Acid Binding to the Transcriptional Regulator HucR From Deinococcus Radiodurans." Journal of Molecular Biology, vol. 350, no. 4, 2005, pp. 617-30.
Wilkinson SP, Grove A. Negative cooperativity of uric acid binding to the transcriptional regulator HucR from Deinococcus radiodurans. J Mol Biol. 2005;350(4):617-30.
Wilkinson, S. P., & Grove, A. (2005). Negative cooperativity of uric acid binding to the transcriptional regulator HucR from Deinococcus radiodurans. Journal of Molecular Biology, 350(4), 617-30.
Wilkinson SP, Grove A. Negative Cooperativity of Uric Acid Binding to the Transcriptional Regulator HucR From Deinococcus Radiodurans. J Mol Biol. 2005 Jul 22;350(4):617-30. PubMed PMID: 15967460.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Negative cooperativity of uric acid binding to the transcriptional regulator HucR from Deinococcus radiodurans. AU - Wilkinson,Steven P, AU - Grove,Anne, PY - 2005/03/28/received PY - 2005/05/09/revised PY - 2005/05/12/accepted PY - 2005/6/22/pubmed PY - 2005/8/16/medline PY - 2005/6/22/entrez SP - 617 EP - 30 JF - Journal of molecular biology JO - J Mol Biol VL - 350 IS - 4 N2 - Members of the MarR family of winged helix transcriptional regulators have been shown to regulate multidrug and oxidative stress response, pathogenesis, and catabolism of aromatic compounds. Many respond to anionic lipophilic compounds in their capacity to bind DNA, and the co-crystal structure of MarR bound to salicylate revealed two ligand-binding pockets, SAL-A and SAL-B. The MarR homolog, HucR, from Deinococcus radiodurans has been shown to repress expression of a predicted uricase, and DNA-binding by HucR is antagonized by uric acid, the substrate of uricase. We provide a biochemical investigation of DNA-binding and uric acid-binding by HucR. Equilibrium analytical ultracentrifugation indicates that HucR exists as a dimer. Intrinsic fluorescence spectra suggest that the association of the HucR dimer with its cognate DNA involves conformational flexibility in the globular interior and/or dimerization domain of the protein, and near-UV circular dichroism spectra indicate a concomitant change in the helical twist of the DNA duplex. DNA-binding affinity, measured by electrophoretic mobility-shift assays, for HucR mutants bearing single amino acid substitutions suggests the importance of the beta-hairpin "wing" in DNA binding. Analysis of intrinsic fluorescence spectra demonstrates that uric acid induces conformational changes in HucR and binds with an apparent K(d)=11.6(+/-3.7)muM and a Hill coefficient of 0.7+/-0.1, indicating negative cooperativity. Fluorescence and DNA-binding properties of the HucR variants indicate that SAL-A is a low-affinity, uric acid-binding site and that negative cooperativity exists between homologous, high-affinity sites. The conservation of residues comprising site SAL-A suggests that it is a low-affinity, ligand-binding site in MarR homologs. Mechanistic considerations suggest that HucR is regulated by uric acid to maintain optimal cellular levels of this scavenger of free radicals in response to oxidative stress and DNA damage. SN - 0022-2836 UR - https://www.unboundmedicine.com/medline/citation/15967460/Negative_cooperativity_of_uric_acid_binding_to_the_transcriptional_regulator_HucR_from_Deinococcus_radiodurans_ DB - PRIME DP - Unbound Medicine ER -