25.7.14 poster final version (portrait)
TRANSCRIPT
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
INTRODUCTION
It is hypothesised that metal selectivity of bacterial DNA-binding metal-sensing
transcriptional regulators may be dictated by relative Kmetal and/or relative
DGCmetal-sensor·DNA 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.
RESULTS
FrmR binds Zn(II), Co(II), and Cu(I)
Figure 1. B, Tyrosine fluorescence emission of 10 µM apo-FrmR (inset) and quenching monitored at 303 nm upon titration with ZnCl2. C, Apo-subtracted difference spectra of FrmR (61.9 µM) 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 µM) 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 µM) and FrmR (10.8 µM, protomer) with ZnCl2. B, Zn(II)-mag-fura-2 absorbance upon titration of mag-fura-2 (12.2 µM) and FrmR (20.4 µM, protomer) with ZnCl2. C, Co(II)-fura-2 fluorescence emission upon titration of 10 µM fura-2 with CoCl2, in the absence (solid symbols) or presence of 10 µM FrmR protomer (open symbols). D, Cu(I)-BCA absorbance upon titration of 40 µM BCA with CuCl2 in presence of 10 µM 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. β-galactosidase activity following growth of SL1344ΔfrmR, 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 type
Figure 9. A, Zn(II)-quin-2 absorbance upon titration of quin-2 (9.39 µM) and E64HFrmR (10.9 µM, protomer) with ZnCl2. B, Zn(II)-mag-fura-2 absorbance upon titration of mag-fura-2 (11.5 µM) and E6HFrmR (24.4 µM, protomer) with ZnCl2. C, Apo-subtracted difference spectra of E64HFrmR (48.4 µM) upon titration with CoCl2. D, Co(II)-fura-2 fluorescence emission upon titration of 10 µM fura-2 with CoCl2, in the absence (solid symbols) or presence of 10 µM E64HFrmR protomer (open symbols). E, Cu(I)-BCA absorbance upon titration of 40 µM BCA with CuCl2 in presence of 10 µM 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 µM ZnCl2 (open symbols).
CONCLUSIONS AND NEXT STEPS
FrmR is responsive to metals in vitro but not in vivo
Figure 5. A-C, Elution profile of Zn and protein following heparin affinity chromatography of either 40 μM FrmR (A) or 20 μM ZntR (B) equilibrated with 10 μM 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 μM FrmR (D) or 20 μM CueR (E) equilibrated with 10 μM 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 μM Co(II) to 62 μM RcnR (black line), 56 μM FrmR (dashed line) or a mixture of 62 μM RcnR and 56 μM 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 cell
Figure 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 µM ZnCl2 (red symbols) or Cu(I)-FrmR (green symbols). B, β-galactosidase activity following growth of SL1344ΔfrmR, 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, β-galactosidase activity following growth of SL1344ΔfrmR, 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 protomer
1:1 Co(II):FrmR protomer
• Interaction 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-sensor·DNA 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: [1]Jefferson et al., (1990) Anal Biochem. 187: 328-336; [2] Xiao et al., (2011) J. Biol Chem. 286: 11047-11055; [3] 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 interaction
Switching FrmR to a metal sensor
Exploring E64HFrmR metal and DNA binding properties to determine how the metal sensing has been established
DB
Exploring the determinants of a metal-sensory network using a non-sensing family member
W
Z
Y
X
W X Y Z A B C CsoR - C H C Y E - RcnR H C H H - - D InrS H C H C - E E STy-FrmR H C H E P E R
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).
[Zn] (M)
0 10 20 30
A 261 nm
0.1
0.2
0.3
A
[Zn] (M)
0 10 20 30 40
A 366 nm
0.0
0.1
0.2
0.3B
[Co(II)] (M)0 5 10 15 20 25
(f i-f 0)/(f 0-f f)
0.0
0.2
0.4
0.6
0.8
1.0
C
1:1 Zn(II):FrmR protomer
10-5 > 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
HCOH
PfrmRA
CH2OH
Helix 1, Monomer 2
Helix 2, Monomer 1
NH
Pro2
Pro34 Cys35 Leu36
SCH2
N
Pro2His3
Pro34 Cys35 Leu36
S
- H2O
Helix 2, Monomer 1
Helix 1, Monomer 2
Helix 2, Monomer 1
Pro34 Cys35 Leu36
SH
O
H
H
ΔGZn(II) (kcal/kmol) = 2.03 ±0.08
ΔGCu(I) (kcal/kmol) = 1.1 ±0.12
log[wt-FrmR] (nM)
1 10 100 1000 10000D
r obs
0.00
0.01
0.02
0.03 Apo- FrmRCu(I)Zn(II)
2:1 FrmR tetramer:DNAA
-galactosidase activity (nm
ols ON
P min
-1 m
g protein-1
)
0
5000
10000
15000
20000
-galactosidase activity (nm
ols ON
P min
-1 m
g protein-1
)
0
500
1000
1500
- + - + 0M
nFe C
oN
i Cu
Zn SeTe
Formaldehyde
-galactosidase activity (nm
ols ON
P min
-1 m
g protein-1
)
0
500
1000
1500
2000
HC
OH
in vitro in vivo
A
B
Fraction
5 10 15 20
Zn (M)
-5
0
5
10
15
ZntR
Fraction
5 10 15 20
Zn (M)
-5
0
5
10
15
FrmR
Fraction
5 10 15 20
Zn (M)
-5
0
5
10
15
ZntRFrmR
Fraction
5 10 15 20
Cu (
M
)
-2.5
0.0
2.5
5.0
7.5
FrmR
Fraction
5 10 15 20
Cu (
M
)
-2.5
0.0
2.5
5.0
7.5
CueR
Fraction
5 10 15 20
Cu (
M
)
-2.5
0.0
2.5
5.0
7.5
CueRFrmR
Wavelength (nm)
550 600 650 700
Absorbance
0.000
0.002
0.004
0.006
Metal Metal sensor
Competition against KMe (M)
Zn(II) ZntR quin-2 [1](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 M
Cu(I) CueR BCS [2](β2 = 1019.8 M-2)
KCu1 = 3.35 ± 0.67x 10-19 M
Co(II) RcnR Fura2 [3](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
Time (min)
0 20 40 60 80 100 120
-galactosidase activity
(nmol O
NP m
in-1
mg protein
-1)
0
500
1000
1500
2000HCOH
Metals
wtFrmR
Time (min)
0 20 40 60 80 100 120
-galactosidase activity
(nmol O
NP m
in-1
mg protein
-1)
0
1000
2000
3000
4000
5000
E64HFrmRHCOH
Co(II)
Zn(II)
A B
[Zn] (M)
0 10 20 30 40
A 366 nm
0.0
0.1
0.2
0.3
[Zn] (M)
0 10 20 30
A 261 nm
0.1
0.2
0.3
(nm)
300 400 500 600 700
(x10
3 M-1
cm-1
)
0.0
0.5
1.0
1.5
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 M
1:1 Zn(II):FrmR protomer
2:1 FrmR tetramer:DNA
1:1 Zn(II):FrmR protomer
[Co(II)]/[FrmR]
0.0 0.5 1.0 1.5 2.0
(x103 M
-1 cm
-1)
0.0
0.5
1.0
1.5
1:1 Co(II):FrmR protomer
[Co(II)] (M)0 5 10 15 20 25
(f i-f 0)/(f 0-f f)
0.0
0.2
0.4
0.6
0.8
1.0
1:1 Co(II):FrmR protomer
[E64HFrmR] (nM)
1 10 100 1000 10000
Dr obs
0.00
0.01
0.02
0.03
Kapo = 106.59 ± 6.78 nM
ΔGZn(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 M
Kapo = 99.39 ± 3.10 nM
[Cu(I)] M
0 2e-5 4e-5 6e-5
Abs
562nm
0.00
0.04
0.08
0.12
K1-4 = 3.26 ± 0.69 x 10-13 MK5-8 = 1.55 ± 0.37 x 10-10 M
2:1 Cu(I):FrmR protomer
[Cu(I)] M
0 2e-5 4e-5 6e-5
Abs
562nm
0.00
0.04
0.08
0.12
K1-4 = 1.41 ± 1.1 x 10-14 M
K5-8 = 1.3 ± 0.79 x 10-11 M
2:1 Cu(I):FrmR protomer
A
[Zn]/[FrmR]
0.0 0.5 1.0 1.5 2.0 2.5Fluorescence emission (
= 303 nm
)
350
400
450
500
W avelength (nm)300 320 340 360 380 400
Fluorescence emisison
0
100
200
300
400
500
↓ [Zn]↑
(nm)
300 400 500 600 700
(x103 M
-1cm
-1)
0.0
0.5
1.0
1.5
[Co(II)]/[FrmR]
0.0 0.5 1.0 1.5 2.0
(x103 M
-1 cm
-1)
0.0
0.5
1.0
1.5
(nm)
250 300 350 400 450
(x 10
3 M-1
cm-1
)
0
2
4
6
8
B C D
D
B CA B C
D E F
A B C
D E F
G