Supplementary information
Distinct functions of serial metal binding domains in the Escherichia coli P1B-ATPase CopA
Steffen L. Drees, Dominik F. Beyer, Christina Lomscher, Mathias Lübben
List of Contents
Supplementary Tables
Table S1. Co-complementation of E. coli DC 194 with variants of copA and mbd1/mbd2/copZBsu.
Table S2. PCR Primers used for cloning work.
Table S3. Outline of the generated strains/plasmid combinations.
Supplementary Figures
Figure S1. SDS PAGE survey of protein purifications.
Figure S2. Copper dependent steady state kinetics of N-terminally truncated variants of CopA.
Figure S3. Growth of the used E. coli strains in LB medium with CuSO4
Figure S4. Determination of MBD affinities to copper - complete.
Figure S5. Analytical gel filtrations for copper transfer between MBDs and ΔN1/2CopA.
Figure S6. Activation of ΔN1/2CopA in response to the addition of copper providing ligands.
Figure S3. pAC-BAD-CopA expression plasmid.
Supplementary Experimental Procedures
Determination of reduced protein content
Supplementary References
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Table S1. Co-complementation of the copA defective E. coli strain DC194 expressing variants of copA
(pAC-BAD vector) with pBAD based vectors producing the MBD1 fragment, C14/17S MBD1 or the copper
chaperone CopZ from Bacillus subtilis. Cultures were inoculated to an OD of approx 0.005 and
cultivated in LB medium with antibiotics and copper supplementation at 37°C for 12 hours.
OD600 after 12h cultivation
pBAD (mock)
a
pBAD::mbd1
pBAD::C14/17S-mbd1
-Cu
+ Cu c
-Cu
+ Cu c
-Cu
+ Cu c
-Cu
+ Cu c
pACBAD::(mock) b
pAC-BAD::copA
2.550
±0.186
0.161
±0.039
2.628
±0.183
0.138
±0.082
2.622
±0.240
0.161
±0.106
2.422
±0.262
0.239
±0.205
2.554
±0.164
2.057
±0.382
2.567
±0.182
2.376
±0.646
2.665
±0.182
1.977
±0.454
2.577
±0.216
2.542
±0.263
pAC-BAD::∆N1copA
pAC-BAD::∆N1
C110/113S-copA
pACBAD::∆N1/2copA
2.615
±0.179
0.523
±0.137
2.569
±0.224
1.996
±0.615
2.617
±0.178
0.625
±0.422
2.471
±0.195
2.504
±0.230
2.644
±0.205
0.411
±0.095
2.642
±0.186
0.611
±0.250
2.582
±0.270
0.473
±0.275
-
-
2.542
±0.222
0.291
±0.102
2.604
±0.164
0.537
±0.284
2.608
±0.181
0.350
±0.284
-
-
a, b
c
pBAD::copZBsu
control plasmid constructs, without gene inserted to expression cassette
CuSO4 was present at a concentration of 3 mM
Table S2. PCR Primers used for cloning work.
Name
5’copA
3’copA
5’N1copA
5’N1/2copA
5’C14/17S
3’C14/17S
5’C110/113S
3’C110/113S
3’mbd1
5’mbd2
3’mbd2
5’BAD-trans-NsiI
3’BAD-trans-NsiI
5’CopZ
5’CopZ
2
sequence 5’->3’
gcgcgcccatggctagctggagccac
gcgcgcggatccttattccttcggtttaaaccgcagc
gccatggcgaaaccgctggcggagtc
gccatggcgaaaccgctggcggagtc
ctgtccagcggtcacagcg
cgctgtgaccgctggacag
gagcagcgccagcagtgtc
gacactgctggcgctgctc
gcgggatccgcggttaagcctttgggtggc
ccatggatgacagccagcagttgc
aagcttaatcttcaatcgcttccgcgcc
gcgcatgcatcccgccattcagagaagaaacc
cgcgatgcatgtcgacggagctcgaattcgg
gcccatggaacaaaaaacattgcaag
caagcttacttggctacgtcatagc
Table S3. Outline of the generated strains/plasmid combinations.
Strain name
DC194
DC194-CopA
DC194-N1CopA
DC194-C110/113SN1CopA
DC194-N1/2CopA
DC194-MBD1
DC194-CopA-MBD1
DC194-N1CopAMBD1
DC194-C110/113SN1CopA-MBD1
DC194-N1/2CopA-MBD1
DC194-CS-MBD1
DC194-CopA-CS-MBD1
DC194-N1CopA-CS-MBD1
DC194-C110/113SN1CopA-CS-MBD1
DC194-N1/2CopA-CS-MBD1
DC194-CS-MBD1
DC194-CopZ
DC194-CopA-CopZ
DC194-N1CopA-CopZ
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plasmid 1
pBAD-His-MycA
pBAD-His-MycA
pBAD-His-MycA
pBAD-His-MycA
pBAD-His-MycA
pBAD-His-MBD1
pBAD-His-MBD1
pBAD-His-MBD1
pBAD-His-MBD1
pBAD-His-MBD1
pBAD-His-C14/17SMBD1
pBAD-His-C14/17SMBD1
pBAD-His-C14/17SMBD1
pBAD-His-C14/17SMBD1
pBAD-His-C14/17SMBD1
pBAD-His-C14/17SMBD1
pBAD-His-CopZ
pBAD-His-CopZ
pBAD-His-CopZ
plasmid 2
pAC-BAD-empty
pAC-BAD-CopA
pAC-BAD-N1CopA
pAC-BAD-C110/113SN1CopA
pAC-BAD-N1/2CopA
pAC-BAD-empty
pAC-BAD-CopA
pAC-BAD-N1CopA
pAC-BAD-C110/113SN1CopA
pAC-BAD-N1/2CopA
pAC-BAD-empty
pAC-BAD-CopA
pAC-BAD-N1CopA
pAC-BAD-C110/113SN1CopA
pAC-BAD-N1/2CopA
pAC-BAD-empty
pAC-BAD-empty
pAC-BAD-CopA
pAC-BAD-N1CopA
Fig. S1. SDS PAGE survey of protein purifications. A ΔN1/2CopA with membranes (5), detergent solubilized
membrans (6), membrane proteins (7) and isolated ΔN 1/2CopA (8). B Purified CopA variants, wt CopA (1),
ΔN1CopA (2) and ΔN1/2CopA (3). C Purification of MBD1 with cell extracts, pellets and supernatants (3-6), after
strep-chromatography (7) and after gel filtration (8).
Fig. S2. Copper dependent steady state kinetics of the N-terminally truncated variants of CopA. KM values were
for 5.4±0.6 µM for CopA, 8.7±0.6 µM for N1CopA and 31.9±5. µM for N1/2CopA. Curve fitting was performed
according to the Michaelis Menten model. Due to the atypical decrease of ATP hydrolysis activity at high copper
concentrations, data were weighted for the curve fitting (linear decrease from 100 % at 40 µM to to 20 % at 200
µM). vmax and ½ vmax are indicated by grey lines. Squares/dashed line – wild type CopA, triangles/dash-dotted line
- ΔN1CopA, circles/solid line - ΔN1/2CopA. Error bars denote standard errors over three measurements.
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Fig. S3. Growth of the used E. coli strains in LB medium supplied with the denoted concentrations of CuSO 4. OD
was measured after overnight growth, strain DC194 pAC-BAD::copA was supplemented with 0.0002 % arabinose.
Fig. S4. Determination of MBD affinities to copper by competitive titration against the high affinity chelator BCS.
Charts display exemplary titration data. Titrations against 600 µM BCS (A), 400 µM BCS (B), 200 µM BCS (C) and
800 µM BCS (D) were fitted after an appropriate model.
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Fig. S5. Analytical gel filtrations for estimation of copper transfer between MBDs and ΔN 1/2CopA. Charts in A and
B display UV absorption and conductivity readouts of chromatographies. Shadings indicate pointwise standard
errors from four experiment repeats. A copper transfer from MBD2 to ΔN1/2CopA, B copper transfer from MBD2
to ΔN1/2CopA, C SDS-PAGE analysis of A. As shown, high salt conditions seem to suffice to suppress interactions
of ΔN1/2CopA and MBDs.
Fig. S6. Activation of ΔN1/2CopA in response to the addition of copper providing ligands. Sample compositions
are denoted in the plot. ATP hydrolysis was monitored by HPLC quantification of ADP. Errorbars reflect standard
errors from triplicate determination.
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Fig. S7. pAC-BAD-CopA expression plasmid. Significant features are highlighted and labelled within the scheme.
Supplementary Experimental Procedures
Determination of reduced protein content
An accurate determination of protein concentrations is a crucial prerequisite for the reliability of
bioanalytical assays. Metal binding proteins with surface-exposed CxxC motifs are known to be
susceptible for oxidative disulphide formation so that, in such cases, the determination of reduced
CxxC content is pivotal. For this application, an assay based on the reaction of free thiols with Ellman’s
reagent (DTNB) and subsequent colorimetric detection was suggested and can be considered the
reference method (Xiao and Wedd, 2010). We propose an alternative method that utilizes the high
affinity copper binding characteristic of our reduced MBD1 or MBD2 domains.
Prior to our experiments, we discovered that, due to their high affinities, both MBDs remove copper
completely from a [Cu(I)-BCA2]3- complex. Thus, in an equimolar mixture of BCA and either MBD with
a substoichiometric amount of Cu(I), more than 99% of the copper is bound to the respective protein.
As a consequence, this allows measuring the protein concentration by photometrically detecting the
decay of [Cu(I)-BCA2]3- absorption in a premixed solution after addition of a defined volume of protein
solution. We compared our test to the established DTNB assay and came up with insignificant
deviations. Also, due to the high absorption coefficient of ε358nm = 42000 M-1 cm-1, detection via BCA
is more sensitive than a regular Ellman assay.
In addition, we used the assay to monitor the oxidation of protein over the course of our titration
experiment and came up with oxidation levels of less than 5 %.
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Supplementary References
Xiao, Z., and Wedd, A.G. (2010) The challenges of determining metal-protein affinities. Nat Prod Rep
27: 768–89.
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