Limits...
The Effectors and Sensory Sites of Formaldehyde-responsive Regulator FrmR and Metal-sensing Variant *

View Article: PubMed Central - PubMed

ABSTRACT

The DUF156 family of DNA-binding transcriptional regulators includes metal sensors that respond to cobalt and/or nickel (RcnR, InrS) or copper (CsoR) plus CstR, which responds to persulfide, and formaldehyde-responsive FrmR. Unexpectedly, the allosteric mechanism of FrmR from Salmonella enterica serovar Typhimurium is triggered by metals in vitro, and variant FrmRE64H gains responsiveness to Zn(II) and cobalt in vivo. Here we establish that the allosteric mechanism of FrmR is triggered directly by formaldehyde in vitro. Sensitivity to formaldehyde requires a cysteine (Cys35 in FrmR) conserved in all DUF156 proteins. A crystal structure of metal- and formaldehyde-sensing FrmRE64H reveals that an FrmR-specific amino-terminal Pro2 is proximal to Cys35, and these residues form the deduced formaldehyde-sensing site. Evidence is presented that implies that residues spatially close to the conserved cysteine tune the sensitivities of DUF156 proteins above or below critical thresholds for different effectors, generating the semblance of specificity within cells. Relative to FrmR, RcnR is less responsive to formaldehyde in vitro, and RcnR does not sense formaldehyde in vivo, but reciprocal mutations FrmRP2S and RcnRS2P, respectively, impair and enhance formaldehyde reactivity in vitro. Formaldehyde detoxification by FrmA requires S-(hydroxymethyl)glutathione, yet glutathione inhibits formaldehyde detection by FrmR in vivo and in vitro. Quantifying the number of FrmR molecules per cell and modeling formaldehyde modification as a function of [formaldehyde] demonstrates that FrmR reactivity is optimized such that FrmR is modified and frmRA is derepressed at lower [formaldehyde] than required to generate S-(hydroxymethyl)glutathione. Expression of FrmA is thereby coordinated with the accumulation of its substrate.

No MeSH data available.


Related in: MedlinePlus

Structure of FrmRE64H and inferred Zn(II)/Co(II)-sensing site.A, ribbon representation of the 2.19 Å resolution crystal structure of FrmRE64H tetramer (Protein Data Bank code 5LCY; see Table 2 for a summary of the crystallographic data). Each monomer is colored differently, and secondary structural units are labeled on the cyan monomer. B, electrostatic surface potential of FrmRE64H tetramer using Chimera (103). The color scale is from −10 (negative potential; red) to +10 (positive potential; blue) kcal/mol·e. C, anisotropy change upon titration of a limiting concentration (10 nm) of frmRAPro (solid symbols) or frmRAPro* (half-site defined in Fig. 2D; open symbols) with FrmR (circles) or FrmRE64H (triangles) in the presence of 5 mm EDTA. The lines are fits of the data to a model describing a 2:1 protein tetramer (nondissociable)/DNA stoichiometry (binding with equal affinity) (50, 86). D, expansion of the dimeric interface with backbone helices from two different monomers shaded green and cyan (the same colors as used in A). The inferred Zn(II)/Co(II)-binding site comprises Cys35 from α2′, and His60 and His64 from α2 (belonging to the XYZ motif required for metal binding in DUF156 members CsoR, RcnR, and InrS (39, 46, 68), with His3 from α1 (position W (46, 61)) and Asp63 presenting candidate fourth ligands. E, analysis of fractions (0.5 ml) for protein by Bradford assay (open circles) and metal by inductively coupled plasma MS (filled circles) following size exclusion chromatography of FrmR, FrmRE64H, FrmRC35A (50 μm, monomer), or FrmRH60L (in this case, [monomer] = 32.5 μm), preincubated with 150 μm ZnCl2.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5016687&req=5

Figure 4: Structure of FrmRE64H and inferred Zn(II)/Co(II)-sensing site.A, ribbon representation of the 2.19 Å resolution crystal structure of FrmRE64H tetramer (Protein Data Bank code 5LCY; see Table 2 for a summary of the crystallographic data). Each monomer is colored differently, and secondary structural units are labeled on the cyan monomer. B, electrostatic surface potential of FrmRE64H tetramer using Chimera (103). The color scale is from −10 (negative potential; red) to +10 (positive potential; blue) kcal/mol·e. C, anisotropy change upon titration of a limiting concentration (10 nm) of frmRAPro (solid symbols) or frmRAPro* (half-site defined in Fig. 2D; open symbols) with FrmR (circles) or FrmRE64H (triangles) in the presence of 5 mm EDTA. The lines are fits of the data to a model describing a 2:1 protein tetramer (nondissociable)/DNA stoichiometry (binding with equal affinity) (50, 86). D, expansion of the dimeric interface with backbone helices from two different monomers shaded green and cyan (the same colors as used in A). The inferred Zn(II)/Co(II)-binding site comprises Cys35 from α2′, and His60 and His64 from α2 (belonging to the XYZ motif required for metal binding in DUF156 members CsoR, RcnR, and InrS (39, 46, 68), with His3 from α1 (position W (46, 61)) and Asp63 presenting candidate fourth ligands. E, analysis of fractions (0.5 ml) for protein by Bradford assay (open circles) and metal by inductively coupled plasma MS (filled circles) following size exclusion chromatography of FrmR, FrmRE64H, FrmRC35A (50 μm, monomer), or FrmRH60L (in this case, [monomer] = 32.5 μm), preincubated with 150 μm ZnCl2.

Mentions: Repression by FrmR (and FrmRE64H) is alleviated by exogenous formaldehyde in vivo (Fig. 2, E and F), but DNA binding to the target frmRA operator-promoter (frmRAPro) (Fig. 2D) is weakened by Zn(II) (and Cu(I)) in vitro (50). To explore whether the in vivo response might be transduced by metals during formaldehyde stress or whether formaldehyde is able to act directly on FrmR, fluorescence anisotropy was used to monitor the interaction of FrmR with fluorescently labeled frmRAPro in the presence of formaldehyde (Fig. 3A). FrmR has previously been shown to bind frmRAPro with a stoichiometry of two tetramers per DNA molecule and a KDNA of 9.9 ± 0.3 × 10−8m for each tetramer, in the absence of effector (50) (also confirmed here in Fig. 4C). Consequently, a limiting concentration (10 nm) of frmRAPro was used for titration with FrmR in the presence of 10 or 20 μm formaldehyde, concentrations chosen to minimize nonspecific formaldehyde cross-linking, which is likely at higher formaldehyde concentrations (60). EDTA was included as a metal chelator to eliminate any effect that may arise due to the presence of (allosterically effective) trace metals. The anisotropy data were fit to a model describing the binding of two non-dissociable FrmR tetramers per DNA molecule and revealed that DNA binding of FrmR to frmRAPro was weakened by ∼6.5-fold and ≥70-fold (compared with the published value (50); Table 1) in the presence of 10 and 20 μm formaldehyde, respectively (Fig. 3A). This identifies formaldehyde as a direct allosteric effector of FrmR.


The Effectors and Sensory Sites of Formaldehyde-responsive Regulator FrmR and Metal-sensing Variant *
Structure of FrmRE64H and inferred Zn(II)/Co(II)-sensing site.A, ribbon representation of the 2.19 Å resolution crystal structure of FrmRE64H tetramer (Protein Data Bank code 5LCY; see Table 2 for a summary of the crystallographic data). Each monomer is colored differently, and secondary structural units are labeled on the cyan monomer. B, electrostatic surface potential of FrmRE64H tetramer using Chimera (103). The color scale is from −10 (negative potential; red) to +10 (positive potential; blue) kcal/mol·e. C, anisotropy change upon titration of a limiting concentration (10 nm) of frmRAPro (solid symbols) or frmRAPro* (half-site defined in Fig. 2D; open symbols) with FrmR (circles) or FrmRE64H (triangles) in the presence of 5 mm EDTA. The lines are fits of the data to a model describing a 2:1 protein tetramer (nondissociable)/DNA stoichiometry (binding with equal affinity) (50, 86). D, expansion of the dimeric interface with backbone helices from two different monomers shaded green and cyan (the same colors as used in A). The inferred Zn(II)/Co(II)-binding site comprises Cys35 from α2′, and His60 and His64 from α2 (belonging to the XYZ motif required for metal binding in DUF156 members CsoR, RcnR, and InrS (39, 46, 68), with His3 from α1 (position W (46, 61)) and Asp63 presenting candidate fourth ligands. E, analysis of fractions (0.5 ml) for protein by Bradford assay (open circles) and metal by inductively coupled plasma MS (filled circles) following size exclusion chromatography of FrmR, FrmRE64H, FrmRC35A (50 μm, monomer), or FrmRH60L (in this case, [monomer] = 32.5 μm), preincubated with 150 μm ZnCl2.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5016687&req=5

Figure 4: Structure of FrmRE64H and inferred Zn(II)/Co(II)-sensing site.A, ribbon representation of the 2.19 Å resolution crystal structure of FrmRE64H tetramer (Protein Data Bank code 5LCY; see Table 2 for a summary of the crystallographic data). Each monomer is colored differently, and secondary structural units are labeled on the cyan monomer. B, electrostatic surface potential of FrmRE64H tetramer using Chimera (103). The color scale is from −10 (negative potential; red) to +10 (positive potential; blue) kcal/mol·e. C, anisotropy change upon titration of a limiting concentration (10 nm) of frmRAPro (solid symbols) or frmRAPro* (half-site defined in Fig. 2D; open symbols) with FrmR (circles) or FrmRE64H (triangles) in the presence of 5 mm EDTA. The lines are fits of the data to a model describing a 2:1 protein tetramer (nondissociable)/DNA stoichiometry (binding with equal affinity) (50, 86). D, expansion of the dimeric interface with backbone helices from two different monomers shaded green and cyan (the same colors as used in A). The inferred Zn(II)/Co(II)-binding site comprises Cys35 from α2′, and His60 and His64 from α2 (belonging to the XYZ motif required for metal binding in DUF156 members CsoR, RcnR, and InrS (39, 46, 68), with His3 from α1 (position W (46, 61)) and Asp63 presenting candidate fourth ligands. E, analysis of fractions (0.5 ml) for protein by Bradford assay (open circles) and metal by inductively coupled plasma MS (filled circles) following size exclusion chromatography of FrmR, FrmRE64H, FrmRC35A (50 μm, monomer), or FrmRH60L (in this case, [monomer] = 32.5 μm), preincubated with 150 μm ZnCl2.
Mentions: Repression by FrmR (and FrmRE64H) is alleviated by exogenous formaldehyde in vivo (Fig. 2, E and F), but DNA binding to the target frmRA operator-promoter (frmRAPro) (Fig. 2D) is weakened by Zn(II) (and Cu(I)) in vitro (50). To explore whether the in vivo response might be transduced by metals during formaldehyde stress or whether formaldehyde is able to act directly on FrmR, fluorescence anisotropy was used to monitor the interaction of FrmR with fluorescently labeled frmRAPro in the presence of formaldehyde (Fig. 3A). FrmR has previously been shown to bind frmRAPro with a stoichiometry of two tetramers per DNA molecule and a KDNA of 9.9 ± 0.3 × 10−8m for each tetramer, in the absence of effector (50) (also confirmed here in Fig. 4C). Consequently, a limiting concentration (10 nm) of frmRAPro was used for titration with FrmR in the presence of 10 or 20 μm formaldehyde, concentrations chosen to minimize nonspecific formaldehyde cross-linking, which is likely at higher formaldehyde concentrations (60). EDTA was included as a metal chelator to eliminate any effect that may arise due to the presence of (allosterically effective) trace metals. The anisotropy data were fit to a model describing the binding of two non-dissociable FrmR tetramers per DNA molecule and revealed that DNA binding of FrmR to frmRAPro was weakened by ∼6.5-fold and ≥70-fold (compared with the published value (50); Table 1) in the presence of 10 and 20 μm formaldehyde, respectively (Fig. 3A). This identifies formaldehyde as a direct allosteric effector of FrmR.

View Article: PubMed Central - PubMed

ABSTRACT

The DUF156 family of DNA-binding transcriptional regulators includes metal sensors that respond to cobalt and/or nickel (RcnR, InrS) or copper (CsoR) plus CstR, which responds to persulfide, and formaldehyde-responsive FrmR. Unexpectedly, the allosteric mechanism of FrmR from Salmonella enterica serovar Typhimurium is triggered by metals in vitro, and variant FrmRE64H gains responsiveness to Zn(II) and cobalt in vivo. Here we establish that the allosteric mechanism of FrmR is triggered directly by formaldehyde in vitro. Sensitivity to formaldehyde requires a cysteine (Cys35 in FrmR) conserved in all DUF156 proteins. A crystal structure of metal- and formaldehyde-sensing FrmRE64H reveals that an FrmR-specific amino-terminal Pro2 is proximal to Cys35, and these residues form the deduced formaldehyde-sensing site. Evidence is presented that implies that residues spatially close to the conserved cysteine tune the sensitivities of DUF156 proteins above or below critical thresholds for different effectors, generating the semblance of specificity within cells. Relative to FrmR, RcnR is less responsive to formaldehyde in vitro, and RcnR does not sense formaldehyde in vivo, but reciprocal mutations FrmRP2S and RcnRS2P, respectively, impair and enhance formaldehyde reactivity in vitro. Formaldehyde detoxification by FrmA requires S-(hydroxymethyl)glutathione, yet glutathione inhibits formaldehyde detection by FrmR in vivo and in vitro. Quantifying the number of FrmR molecules per cell and modeling formaldehyde modification as a function of [formaldehyde] demonstrates that FrmR reactivity is optimized such that FrmR is modified and frmRA is derepressed at lower [formaldehyde] than required to generate S-(hydroxymethyl)glutathione. Expression of FrmA is thereby coordinated with the accumulation of its substrate.

No MeSH data available.


Related in: MedlinePlus