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Determining the mechanism of allosteric regulation of NikR binding to DNA activated by Ni 2+
Author(s) -
Bradley Michael John,
Chivers Peter,
Baker Nathan
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.4.a489-c
Subject(s) - chemistry , dna , repressor , homotetramer , dna binding domain , binding site , protein–dna interaction , binding domain , biophysics , cooperative binding , dna binding protein , biochemistry , transcription factor , biology , protein subunit , gene
E. coli NikR is a nickel and DNA binding transcriptional repressor protein that responds to elevated levels of intracellular nickel by binding to the nikABCDE operator and repressing nickel permease production. NikR is a homotetramer composed of two distinct domains. The two N‐terminal ribbon‐helix‐helix (RHH) domains are made up of intertwined dimers and bind to DNA while the C‐terminal regulatory domain (an ACT type fold) is responsible for tetramerization and contains one high affinity nickel binding site per chain (1 Ni 2+ /monomer, K d ~ 1 pM). Metal binding controls the DNA‐binding capability of NikR. One Ni 2+ per monomer activates NikR tetramer binding to nikABCDE operator DNA with ~5nM affinity. The mechanism by which nickel modulates DNA binding affinity is not known. Specific DNA binding also causes a change in high‐affinity Ni 2+ coordination geometry (4‐ to 6‐ coordinate), suggesting a conformational connection between nickel and DNA binding sites. We have utilized equilibrium molecular dynamics (MD) analyses and conformational clustering strategies to explore the structural and energetic connections between NikR domains. In addition we have used coarse‐grained modeling techniques to elucidate large‐scale NikR structural transitions. We have also applied hydrogen/deuterium (H/D) exchange measured by mass spectrometry to experimentally measure conformational changes caused by metal and DNA binding. These three strategies are combined to suggest residues that are important for transducing the Ni 2+ binding signal into activation of the DNA binding competent state of NikR. Site‐directed mutagenesis of E. coli NikR and subsequent biophysical characterization will reveal the role of these residues in allosteric activation of this regulatory protein.