The response of Escherichia coli NikR to nickel: a second nickel-binding site.
The Escherichia coli transcription factor NikR mediates two levels of regulatory control of Ni(II) uptake in response to changes in the levels of available nickel. Despite the evidence that metal binding to two distinct sites on NikR, referred to as the high- and low-affinity Ni(II) sites, is required for Ni(II)-selective DNA binding by the protein, the role of the latter set of Ni(II) ions in the activation of NikR remains controversial, and the position of the putative low-affinity Ni(II)-binding site(s) on NikR has not been determined. In this study we confirm that NikR has a high-affinity Ni(II)-binding site that is maintained upon DNA binding. The ligands of the low-affinity Ni(II)-binding site were examined by using selective chemical modification and mass spectrometry performed in the presence of excess Ni(II) and DNA. We localized this Ni(II) site to a region at the interface between the metal- and DNA-binding domains and identified His48 and His110 as residues that participate in the low-affinity Ni(II)-binding response. Mutation of His48 and His110 to asparagines reduces significantly both NikR's tendency to precipitate in the presence of excess Ni(II) and the affinity of the DNA-bound complex in the presence of excess Ni(II). A complete scheme involving all of the metal-binding sites that contribute to the regulatory function of E. coli NikR in nickel homeostasis is described.
Wang
Sheila C
SC
Department of Chemistry, University of Toronto, Toronto, Ontario, Canada M5S 3H6.
Li
Yanjie
Y
Ho
ManYing
M
Bernal
Maria-Elena
ME
Sydor
Andrew M
AM
Kagzi
Wasim R
WR
Zamble
Deborah B
DB
D019943:Amino Acid Substitution; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D006706:Homeostasis; D008024:Ligands; D009532:Nickel / Q000378:metabolism*; D012097:Repressor Proteins / Q000378:metabolism*
D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D008024:Ligands; C402409:NikR protein, E coli; D012097:Repressor Proteins; D009532:Nickel