25.7.14 poster final version (portrait)
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Cecilia Piergentili1, Deenah Osman1, Nigel Robinson1, Elena Lurie-Luke2, Junjun Chen3, Tanuja Chaudary 3, Tom Huggins 3,
1Biophysical Sciences Institute, Department of Chemistry, School of Biological and Biomedical Sciences, Durham University, DH1 3LE, UK 2Procter & Gamble, London Innovation Centre, Whitehall Lane, Egham, Surrey, TW20 9NW, UK 3Procter & Gamble, 8700 Mason Montgomery Rd Mason, OH 45040
INTRODUCTIONIt is hypothesised that metal selectivity of bacterial DNA-binding metal-sensing transcriptional regulators may be dictated by relative Kmetal and/or relative DGCmetal-sensorDNA across a set of metal-sensors. Salmonella Typhimurium FrmR (STy-FrmR) is a member of the CsoR/RcnR family of DNA-binding transcriptional repressors and responds to formaldehyde in vivo, regulating expression of frmA glutathione-dependant-formaldehyde dehydrogenase. STy-FrmR contains three of four ligands consistent with metal binding of Ni(II)/Co(II)-sensing RcnR (the so-called W-X-Y-Z motif). We demonstrate that FrmR binds Cu(I), Zn(II), and Co(II) in vitro and Zn(II) is able to impair Sty-FrmR binding to the frmA operator-promoter. However, STy-FrmR does not respond to zinc (or copper and cobalt) in vivo.
OBJECTIVEFrmR metal- and DNA-binding properties have been determined to understand why this formaldehyde sensor is not able to sense metals in vivo. Relative metal affinities within the set of Salmonella metal sensors have been explored. Moreover substitution of Glu64 with His (residue Z of the motif) conferred a gain-of-function to Sty-FrmR which now responds to Zn(II) and Co(II) in vivo.
RESULTSFrmR binds Zn(II), Co(II), and Cu(I)Figure 1. B, Tyrosine fluorescence emission of 10 M apo-FrmR (inset) and quenching monitored at 303 nm upon titration with ZnCl2. C, Apo-subtracted difference spectra of FrmR (61.9 M) upon titration with CoCl2 and binding isotherms (inset) at 294 nm (circles), 334 nm (triangles), and 614 nm (squares). D, Apo-subtracted difference spectra of FrmR (20 M) upon titration with CuCl2 and binding isotherm (inset) at 240 nm. Figure 2. A, Representative (n=4) Zn(II)-quin-2 absorbance upon titration of quin-2 (9.39 M) and FrmR (10.8 M, protomer) with ZnCl2. B, Zn(II)-mag-fura-2 absorbance upon titration of mag-fura-2 (12.2 M) and FrmR (20.4 M, protomer) with ZnCl2. C, Co(II)-fura-2 fluorescence emission upon titration of 10 M fura-2 with CoCl2, in the absence (solid symbols) or presence of 10 M FrmR protomer (open symbols). D, Cu(I)-BCA absorbance upon titration of 40 M BCA with CuCl2 in presence of 10 M FrmR protomer.
A gain-of-function mutation allows Glu64HisFrmR to respond to Zn and Co in vivo. Single point mutation involves residue Z in the W-X-Y-Z motif Figure 6. -galactosidase activity following growth of SL1344frmR, harbouring either PfrmRA-frmR::lacZ (A) or PfrmRA-frmRE64H::lacZ (B), to mid-exponential phase in M9 minimal media followed by continued incubation in M9 (solid circles) or exposure to MNICs of Mn (open circles), Fe (solid squares), Co (open squares), Ni (solid diamonds), Cu (open diamonds), Zn (solid triangles) or formaldehyde (open triangles). E64HFrmR has similar metal- and DNA-binding properties to wild typeFigure 9. A, Zn(II)-quin-2 absorbance upon titration of quin-2 (9.39 M) and E64HFrmR (10.9 M, protomer) with ZnCl2. B, Zn(II)-mag-fura-2 absorbance upon titration of mag-fura-2 (11.5 M) and E6HFrmR (24.4 M, protomer) with ZnCl2. C, Apo-subtracted difference spectra of E64HFrmR (48.4 M) upon titration with CoCl2. D, Co(II)-fura-2 fluorescence emission upon titration of 10 M fura-2 with CoCl2, in the absence (solid symbols) or presence of 10 M E64HFrmR protomer (open symbols). E, Cu(I)-BCA absorbance upon titration of 40 M BCA with CuCl2 in presence of 10 M E64HFrmR protomer. F, Anisotropy change of PfrmRA (10 nM), upon titration with either apo-E64HFrmR in the presence of 5 mM EDTA (closed symbols) or Zn(II)-E64HFrmR in the presence of 5 M ZnCl2 (open symbols).
CONCLUSIONS AND NEXT STEPS
FrmR is responsive to metals in vitro but not in vivoFigure 5. A-C, Elution profile of Zn and protein following heparin affinity chromatography of either 40 M FrmR (A) or 20 M ZntR (B) equilibrated with 10 M Zn(II), or after Zn(II)-loading of FrmR and subsequent addition of ZntR (C). D-F, Elution profile of Cu and protein following heparin affinity chromatography of either 40 M FrmR (D) or 20 M CueR (E) equilibrated with 10 M Cu(I), or after Cu(I)-loading of FrmR and subsequent addition of CueR (F). G Apo-subtracted difference spectra following addition and 15 h incubation of 14 M Co(II) to 62 M RcnR (black line), 56 M FrmR (dashed line) or a mixture of 62 M RcnR and 56 M FrmR (red). Table, Metal affinities determined by competition with chelators of known metal affinities.
Competition between FrmR and cognate metal sensors shows how relative Kmetal is a key parameter in the regulation of metal selectivity within the cellFigure 4. A, Anisotropy change of a fluorescently labelled 33-bp oligonucleotide region, PfrmRA (10 nM), upon titration with either apo-FrmR in the presence of 5 mM EDTA (orange symbols), Zn(II)-FrmR in the presence of 5 M ZnCl2 (red symbols) or Cu(I)-FrmR (green symbols). B, -galactosidase activity following growth of SL1344frmR, harbouring a construct containing either PfrmRA::lacZ (left) or PfrmRA-frmR::lacZ (right), in M9 minimal media in the absence or presence of minimum non-inhibitory concentrations (MNICs) of formaldehyde. C, -galactosidase activity following growth of SL1344frmR, harbouring a PfrmRA-frmR::lacZ construct, in M9 minimal media in the absence or presence of MNICs of MnCl2, FeC6H8O7, CoCl2, NiSO4, CuSO4, ZnCl2, SeO3Na2, TeO3Na2 and formaldehyde. Figure 3. A, Genomic region of frmR and frmA (to scale) and deduced transcriptional regulator binding site (palindromic region rich in CG) upstream of frmRA. The -10 sequence is underlined. B, Proposed mechanism of formaldehyde binding to FrmR by modification of Cys35 and Pro2.
1:1 Zn(II):FrmR protomer1:1 Co(II):FrmR protomerInteraction of FrmR towards its target DNA sequence has been studied along with the effect that formaldehyde and metals have on this interaction. The affinity of STy-FrmR for Cu(I), Zn(II) and Co(II) is weaker than the affinity of the cognate sensors of these metal ions, CueR for Cu(I), Zur and ZntR for Zn(II), and RcnR for Co(II), consistent with relative Kmetal being a parameter of metal selectivity within the cell. Mutation of Glu64 into His (residue Z of the motif) confers a gain-of-function to Sty-FrmR which now responds to Zn(II) and Co(II) in vivo. The successful switching of a non-metal into a metal sensor can add important insights in understanding how a metallo-regulator can respond to the correct metals in the cell, illustrating the complexity underlying metal discrimination in a network of sensors.Metal affinities and Allosteric Coupling Free Energies DGCmetal-sensorDNA have been determined for wild type and E64HFrmR without showing a significant difference in the behaviours of the two species. The question about how the ability to sense Zn(II) and Co(II) has been established remains open.Future work involves determination of wild type and mutant FrmR abundances in the cell with the aim to explore a potential mass-action effect.
References: Jefferson et al., (1990) Anal Biochem. 187: 328-336;  Xiao et al., (2011) J. Biol Chem. 286: 11047-11055;  Kwan and Putney (1990) J. Biol Chem. 265: 678-684;
Exploring FrmR metal binding properties FrmR has a weaker Zn(II), Co(II) and Cu(I) affinity than cognate sensors In vivo FrmR directly binds to PfrmRA repressing transcription in the absence of formaldehyde stress. In vitro also Zn(II) and Cu(I) are able to disrupt the DNA-protein interactionSwitching FrmR to a metal sensorExploring E64HFrmR metal and DNA binding properties to determine how the metal sensing has been established DBExploring the determinants of a metal-sensory network using a non-sensing family member
Figure 1. A, M. tuberculosis CsoR (pdb2hh7) with FrmR residues at positions X , Y and Z and metal ion in yellow (Cu(I) for CsoR).
C1:1 Zn(II):FrmR protomer10-5 > K1, K2 > 8.64 x 10-8 M K1-2 = 5.01 0.96 x 10-11 MK3 = 1.29 0.27 x 10-9 MK4 = 3.78 0.38 x 10-6 M
GZn(II) (kcal/kmol) = 2.03 0.08GCu(I) (kcal/kmol) = 1.1 0.12
Apo- FrmRCu(I)Zn(II)2:1 FrmR tetramer:DNAA
- +- +
HCOHin vitro in vivoAB
MetalMetal sensorCompetition againstKMe (M)Zn(II)ZntRquin-2 (KZn(II) = 3.7 x 10-12 M)KZn1 = 3.20 0.73 x 10-12 M
KZn2 = 2.68 0.73 x 10-11 MCu(I)CueRBCS (2 = 1019.8 M-2)KCu1 = 3.35 0.67x 10-19 MCo(II)RcnRFura2 (at pH 7 KCo(II)= 8.64 x 10-9 M) KCo1-2 = 5.06 0.86 x 10-10 M
KCo3 = 1.18 0.90 x 10-7 M
K1-2 = 3.61 0.79 x 10-11 M
K3 = 6.44 0.34 x 10-10 M
K4 = 5.85 2.13 x 10-6 M1:1 Zn(II):FrmR protomer 2:1 FrmR tetramer:DNA1:1 Zn(II):FrmR protomer
1:1 Co(II):FrmR protomer
1:1 Co(II):FrmR protomer
Kapo = 106.59 6.78 nM
GZn(II) =1.92 0.09kcal/kmol
Acknowledgements: This work is supported by BBSRC and Procter and Gamble (METALSP&G Industrial Parternship Award)
10-5 > K1, K2 > 8.64 x 10-8 MKapo = 99.39 3.10 nM
K1-4 = 3.26 0.69 x 10-13 MK5-8 = 1.55 0.37 x 10-10 M2:1 Cu(I):FrmR protomer
K1-4 = 1.41 1.1 x 10-14 M
K5-8 = 1.3 0.79 x 10-11 M2:1 Cu(I):FrmR protomer A