ABSTRACT
NikR from Escherichia coli is a nickel-responsive transcription factor that regulates the expression of a nickel ion transporter. Metal analysis reveals that NikR can bind a variety of divalent transition metals, including Ni(II), Cu(II), Zn(II), Co(II), and Cd(II). The selectivity of metal binding to NikR was investigated by using electronic absorption spectroscopy and small-molecule competitors. The relative affinities, Mn(II) < Co(II) < Ni(II) < Cu(II) > or = Zn(II), follow the Irving-Williams series of metal-complex stabilities. Similar metal affinities were measured for the isolated metal-binding domain of NikR. To determine if any of these metal ions confer a differential effect on NikR, the stability of the metal-bound complexes was examined. In both thermal and chemical denaturation experiments, nickel binding stabilizes the protein more than any of the other metals tested. Thermal denaturation experiments indicate that metal dissociation occurs after loss of secondary structure, but there was no evidence for metal binding to unfolded protein following reversible chemical denaturation. These experiments demonstrate that, although several different metals can bind to NikR, nickel exerts a selective allosteric effect. The implications of these experiments on the in vivo role of NikR as a nickel metalloregulator are discussed.
Subject(s)
Escherichia coli Proteins/chemistry , Metals, Heavy/chemistry , Repressor Proteins/chemistry , Circular Dichroism , Copper/chemistry , Copper/metabolism , Escherichia coli Proteins/metabolism , Guanidine/chemistry , Metals, Heavy/metabolism , Nickel/chemistry , Nickel/metabolism , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Protein Binding , Protein Conformation , Protein Denaturation , Protein Folding , Protein Structure, Tertiary , Repressor Proteins/metabolism , Spectrophotometry, Ultraviolet , Spectrum Analysis , Thermodynamics , X-RaysABSTRACT
The NikR transcription factor from Escherichia coli is a Ni(II)-dependent repressor that regulates production of the nickel ion transporter encoded by the nik operon. In the previous paper in this issue (Wang, S. C., Dias, A., Bloom, S. L., and Zamble, D. B. (2004) Selectivity of Metal Binding and Metal-Induced Stability of Escherichia coli NikR, Biochemistry 43, 10018-10028) we demonstrated that NikR can bind 1 equiv of Ni(II) or several other divalent transition metals with similar affinities, but that the Ni(II)-loaded protein is less susceptible to thermal or chemical denaturation than other divalent metal complexes. Here, we investigate the metal selectivity of the DNA-binding activity of NikR. Stoichiometric nickel induces binding of nanomolar NikR to the recognition sequence in the nik promoter, but single equivalents of other divalent metals such as Cd(II), Co(II), and Cu(II) also induce a similar DNA-binding affinity. In the presence of excess nickel, DNA-binding experiments indicate that NikR binds to the nik promoter as a tetramer with much higher affinity (20 pM), and it is this response that is selective for nickel. The DNA binding induced by an excess of other divalent metals is weaker, and is enhanced by the addition of stoichiometric nickel. Nickel titrations into a DNA-binding assay reveal a nickel affinity of 30 nM for a second metal-binding site, and in the presence of 30 nM metal only nickel induces detectable DNA binding by Ni(II)-NikR. These experiments support the hypothesis that there are two metal-binding sites and that both contribute to the nickel-selective DNA-binding response. A model for the in vivo activity of NikR is discussed.
