title: Nonspecific interactions between Escherichia coli NikR and DNA are critical for nickel-activated DNA binding.
abstract: The Escherichia coli transcription factor NikR is responsible for nickel-mediated repression of the operon encoding the Nik uptake transporter. The crystal structure of Ni(II)-NikR bound to the nik operator sequence revealed that residues in the loop preceding helix α3 in the metal-binding domain, which becomes structurally ordered upon stoichiometric nickel binding, interact with the DNA backbone. Here, we show that mutating both of these residues that make the nonspecific contacts, K64 and R65, abolishes DNA binding in vitro and nickel-responsive transcriptional repression of the nik promoter in vivo. In contrast, mutation of Q118, which forms a bridge between R65 and a potassium site, does not impact the activities of NikR. These data support the model that the nonspecific interactions between the metal-binding domain of the protein and the DNA phosphodiester backbone are critical for the Ni(II)-responsive activity of E. coli NikR.
authors.affiliation: Department of Chemistry, University of Toronto, Toronto, Ontario, Canada M5S 3H6.
authors.forename: Sandra
authors.initials: S
authors.lastname: Krecisz
authors.forename: Michael D
authors.initials: MD
authors.lastname: Jones
authors.forename: Deborah B
authors.initials: DB
authors.lastname: Zamble
mesh_terms: D002942:Circular Dichroism; D018983:DNA Footprinting; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D003851:Deoxyribonucleases; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation; D009532:Nickel / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D013054:Spectrophotometry, Atomic; D001616:beta-Galactosidase
chemical_list: D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; C402409:NikR protein, E coli; D012097:Repressor Proteins; D009532:Nickel; D003851:Deoxyribonucleases; D001616:beta-Galactosidase