Generating a Metal-responsive Transcriptional Regulator to Test What Confers Metal Sensing in Cells.
Bottom Line: Unexpectedly, FrmR was found to already bind Co(II), Zn(II), and Cu(I), and moreover metals, as well as formaldehyde, trigger an allosteric response that weakens DNA affinity.Counter-intuitively, the allosteric coupling free energy for Zn(II) is smaller in metal-sensing FrmRE64H compared with nonsensing FrmR.By determining the copies of FrmR and FrmRE64H tetramers per cell, then estimating promoter occupancy as a function of intracellular Zn(II) concentration, we show how a modest tightening of Zn(II) affinity, plus weakened DNA affinity of the apoprotein, conspires to make the relative properties of FrmRE64H (compared with ZntR and Zur) sufficient to sense Zn(II) inside cells.
Affiliation: From the School of Biological and Biomedical Sciences and Department of Chemistry, Durham University, Durham DH1 3LE, United Kingdom.Show MeSH
Mentions: Fractional occupancy of the tightest metal-binding site of a sensor with metal as a function of buffered [metal], was determined using the following: (θ) = [metal]buffered/(Kmetal + [metal]buffered). Kmetal = KD (tightest site) of sensor for metal, experimentally determined (Kmetalsensor) (Table 1) (48). For FrmR (and variants), Kmetal was additionally calculated for the DNA-bound form (Kmetalsensor·DNA) from the coupling constant (KC) (Fig. 10E). The concentration of apo- and Zn(II)-protein at a given [Zn(II)] was calculated using the number of tetramers per cell (FrmR and variants; Fig. 9K), and a cell volume of 1 fl. Fractional DNA occupancies with apo- and Zn(II)-protein over a range of protein concentrations were modeled using Dynafit (43) (1:1 binding of tetramer/DNA; assuming the binding of one tetramer conferred repression) with KDNA (from Table 2) and [PfrmRA] as fixed parameters (sample Dynafit script is also shown in the supplemental material). [PfrmRA] was calculated assuming 15 copies cell−1 (due to the presence on low copy number reporter plasmid) and a cell volume of 1 fl. The response was set at 1/[PfrmRA]. The fractional occupancy of PfrmRA with apo- and Zn(II)-protein was summed to give fractional occupancy of PfrmRA at any given buffered [Zn(II)].
Affiliation: From the School of Biological and Biomedical Sciences and Department of Chemistry, Durham University, Durham DH1 3LE, United Kingdom.