title: Metal binding at the Deinococcus radiodurans Dps-1 N-terminal metal site controls dodecameric assembly and DNA binding.
abstract: The prokaryotic DNA protection during starvation (Dps) proteins typically protect macromolecules against damaging agents via physical association with DNA and by oxidizing and sequestering iron. However, Deinococcus radiodurans Dps-1, which binds DNA with high affinity, fails to protect DNA against hydroxyl radicals due to iron leakage from the core, raising the question of how (•)OH-mediated damage to Dps-1-bound DNA is avoided. As shown here, Mn(II) inhibits ferroxidase activity, suggesting that ferroxidation may be prevented in vivo as D. radiodurans accumulates a high ratio of Mn:Fe. Dps-1 has an N-terminal extension with a unique metal-binding site, an extension that has been proposed to be important for DNA binding and dodecameric assembly. Electrophoretic mobility shift assays show that Mn(II) restores DNA binding to bipyridyl-treated Dps-1, whereas Fe(II) fails to do so in the presence of H(2)O(2), thus preventing DNA binding under conditions of ongoing ferroxidase activity. We also show that disruption of the N-terminal metal site leads to a significant reduction in DNA binding and to compromised oligomeric assembly, with the mutant protein assembling into a hexamer in the presence of divalent metal. We propose that securing the N-terminal loop by metal binding is required to initiate dodecameric assembly by contacting the neighboring dimer and that the absence of such optimal contacts results in formation of a hexameric assembly intermediate in which three dimers associate about one of the 3-fold axes. Once dodecameric Dps-1 is assembled, metal binding no longer affects oligomeric state; instead, differential metal binding controls DNA interaction under conditions of oxidative stress.
authors: Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803, USA.
authors: Khoa Huynh
authors: KH
authors: Nguyen
authors: Anne
authors: A
authors: Grove
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry*; D001665:Binding Sites; D002570:Ceruloplasmin / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry*; D034301:Deinococcus / Q000737:chemistry; D005296:Ferrous Compounds / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000494:pharmacology; D017665:Hydroxyl Radical / Q000378:metabolism; D007501:Iron / Q000494:pharmacology*; D008345:Manganese / Q000494:pharmacology*; D008958:Models, Molecular; D018384:Oxidative Stress / Q000502:physiology; D055503:Protein Multimerization; D016415:Sequence Alignment
chemical_list: D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C083911:DPS protein, Bacteria; D005296:Ferrous Compounds; D017665:Hydroxyl Radical; D008345:Manganese; D006861:Hydrogen Peroxide; D007501:Iron; D002570:Ceruloplasmin