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Generating a Metal-responsive Transcriptional Regulator to Test What Confers Metal Sensing in Cells.

Osman D, Piergentili C, Chen J, Chakrabarti B, Foster AW, Lurie-Luke E, Huggins TG, Robinson NJ - J. Biol. Chem. (2015)

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.

View Article: PubMed Central - PubMed

Affiliation: From the School of Biological and Biomedical Sciences and Department of Chemistry, Durham University, Durham DH1 3LE, United Kingdom.

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Zn(II) weakens KDNA of FrmR and FrmRE64H and its effect on DNA occupancy. Anisotropy change upon titration of a high concentration of frmRAPro (2.5 μm) with FrmR (A), FrmRE64H (B), or a limiting concentration of frmRAPro (10 nm) (C) with apo-FrmR in the presence of 5 mm EDTA (closed symbols) or Zn(II)-FrmR in the presence of 5 μm ZnCl2 (open symbols). D, as C but using FrmRE64H. Symbol shapes represent individual experiments. Data were fit to a model describing a 2:1 protein tetramer (nondissociable):DNA stoichiometry (binding with equal affinity), and lines represent simulated curves produced from the average KDNA determined across the experimental replicas shown. E, coupled thermodynamic equilibria (assuming a closed system) describing the relationship between FrmR tetramer (P), Zn(II) (Z), and PfrmRA (D) (9, 65, 66). The coupling constant (KC) is determined from the ratio K4/K3 (KDNAZn(II)·FrmR/KDNAFrmR) (Equation 1) and used to calculate K2 (the Zn(II) affinity of the DNA-bound protein, KZn(II)FrmR·DNA) from K1 (KZn(II)FrmR) (Equation 2). F, calculated fractional occupancy of PfrmRA with FrmR (filled circles) and FrmRE64H (open circles) as a function of (buffered) [Zn(II)], which incorporates the determined FrmR or FrmRE64H abundance, KZn(II)sensor (off DNA), and KDNA (Table 1). Additional lines represent hypothetical fractional occupancy of PfrmRA with FrmRE64H but substituting KZn(II) (dotted) or KDNA (dashed) for that of FrmR. G, as F but using the determined abundance for FrmRDOWN (solid symbols) and FrmRE64HUP (open symbols). H and I, as F and G, respectively, except using KZn(II)sensor·DNA (on-DNA) (calculated using the equations in E).
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Figure 10: Zn(II) weakens KDNA of FrmR and FrmRE64H and its effect on DNA occupancy. Anisotropy change upon titration of a high concentration of frmRAPro (2.5 μm) with FrmR (A), FrmRE64H (B), or a limiting concentration of frmRAPro (10 nm) (C) with apo-FrmR in the presence of 5 mm EDTA (closed symbols) or Zn(II)-FrmR in the presence of 5 μm ZnCl2 (open symbols). D, as C but using FrmRE64H. Symbol shapes represent individual experiments. Data were fit to a model describing a 2:1 protein tetramer (nondissociable):DNA stoichiometry (binding with equal affinity), and lines represent simulated curves produced from the average KDNA determined across the experimental replicas shown. E, coupled thermodynamic equilibria (assuming a closed system) describing the relationship between FrmR tetramer (P), Zn(II) (Z), and PfrmRA (D) (9, 65, 66). The coupling constant (KC) is determined from the ratio K4/K3 (KDNAZn(II)·FrmR/KDNAFrmR) (Equation 1) and used to calculate K2 (the Zn(II) affinity of the DNA-bound protein, KZn(II)FrmR·DNA) from K1 (KZn(II)FrmR) (Equation 2). F, calculated fractional occupancy of PfrmRA with FrmR (filled circles) and FrmRE64H (open circles) as a function of (buffered) [Zn(II)], which incorporates the determined FrmR or FrmRE64H abundance, KZn(II)sensor (off DNA), and KDNA (Table 1). Additional lines represent hypothetical fractional occupancy of PfrmRA with FrmRE64H but substituting KZn(II) (dotted) or KDNA (dashed) for that of FrmR. G, as F but using the determined abundance for FrmRDOWN (solid symbols) and FrmRE64HUP (open symbols). H and I, as F and G, respectively, except using KZn(II)sensor·DNA (on-DNA) (calculated using the equations in E).

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)].


Generating a Metal-responsive Transcriptional Regulator to Test What Confers Metal Sensing in Cells.

Osman D, Piergentili C, Chen J, Chakrabarti B, Foster AW, Lurie-Luke E, Huggins TG, Robinson NJ - J. Biol. Chem. (2015)

Zn(II) weakens KDNA of FrmR and FrmRE64H and its effect on DNA occupancy. Anisotropy change upon titration of a high concentration of frmRAPro (2.5 μm) with FrmR (A), FrmRE64H (B), or a limiting concentration of frmRAPro (10 nm) (C) with apo-FrmR in the presence of 5 mm EDTA (closed symbols) or Zn(II)-FrmR in the presence of 5 μm ZnCl2 (open symbols). D, as C but using FrmRE64H. Symbol shapes represent individual experiments. Data were fit to a model describing a 2:1 protein tetramer (nondissociable):DNA stoichiometry (binding with equal affinity), and lines represent simulated curves produced from the average KDNA determined across the experimental replicas shown. E, coupled thermodynamic equilibria (assuming a closed system) describing the relationship between FrmR tetramer (P), Zn(II) (Z), and PfrmRA (D) (9, 65, 66). The coupling constant (KC) is determined from the ratio K4/K3 (KDNAZn(II)·FrmR/KDNAFrmR) (Equation 1) and used to calculate K2 (the Zn(II) affinity of the DNA-bound protein, KZn(II)FrmR·DNA) from K1 (KZn(II)FrmR) (Equation 2). F, calculated fractional occupancy of PfrmRA with FrmR (filled circles) and FrmRE64H (open circles) as a function of (buffered) [Zn(II)], which incorporates the determined FrmR or FrmRE64H abundance, KZn(II)sensor (off DNA), and KDNA (Table 1). Additional lines represent hypothetical fractional occupancy of PfrmRA with FrmRE64H but substituting KZn(II) (dotted) or KDNA (dashed) for that of FrmR. G, as F but using the determined abundance for FrmRDOWN (solid symbols) and FrmRE64HUP (open symbols). H and I, as F and G, respectively, except using KZn(II)sensor·DNA (on-DNA) (calculated using the equations in E).
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Figure 10: Zn(II) weakens KDNA of FrmR and FrmRE64H and its effect on DNA occupancy. Anisotropy change upon titration of a high concentration of frmRAPro (2.5 μm) with FrmR (A), FrmRE64H (B), or a limiting concentration of frmRAPro (10 nm) (C) with apo-FrmR in the presence of 5 mm EDTA (closed symbols) or Zn(II)-FrmR in the presence of 5 μm ZnCl2 (open symbols). D, as C but using FrmRE64H. Symbol shapes represent individual experiments. Data were fit to a model describing a 2:1 protein tetramer (nondissociable):DNA stoichiometry (binding with equal affinity), and lines represent simulated curves produced from the average KDNA determined across the experimental replicas shown. E, coupled thermodynamic equilibria (assuming a closed system) describing the relationship between FrmR tetramer (P), Zn(II) (Z), and PfrmRA (D) (9, 65, 66). The coupling constant (KC) is determined from the ratio K4/K3 (KDNAZn(II)·FrmR/KDNAFrmR) (Equation 1) and used to calculate K2 (the Zn(II) affinity of the DNA-bound protein, KZn(II)FrmR·DNA) from K1 (KZn(II)FrmR) (Equation 2). F, calculated fractional occupancy of PfrmRA with FrmR (filled circles) and FrmRE64H (open circles) as a function of (buffered) [Zn(II)], which incorporates the determined FrmR or FrmRE64H abundance, KZn(II)sensor (off DNA), and KDNA (Table 1). Additional lines represent hypothetical fractional occupancy of PfrmRA with FrmRE64H but substituting KZn(II) (dotted) or KDNA (dashed) for that of FrmR. G, as F but using the determined abundance for FrmRDOWN (solid symbols) and FrmRE64HUP (open symbols). H and I, as F and G, respectively, except using KZn(II)sensor·DNA (on-DNA) (calculated using the equations in E).
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)].

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.

View Article: PubMed Central - PubMed

Affiliation: From the School of Biological and Biomedical Sciences and Department of Chemistry, Durham University, Durham DH1 3LE, United Kingdom.

Show MeSH