Metal-binding stoichiometry and selectivity of the copper chaperoneAgathe Urvoas1, Mireille Moutiez1, Cle´ment Estienne1, Joe¨l Couprie1, Elisabeth Mintz2 and Loı¨c Le Clainche1 1 De´par
Trang 1Metal-binding stoichiometry and selectivity of the copper chaperone
Agathe Urvoas1, Mireille Moutiez1, Cle´ment Estienne1, Joe¨l Couprie1, Elisabeth Mintz2
and Loı¨c Le Clainche1
1
De´partement d’Inge´nierie et d’Etudes des Prote´ines, Direction des Sciences du Vivant, CEA Saclay, Gif sur Yvette, France;
2
Laboratoire de Biophysique Mole´culaire et Cellulaire, UMR 5090, CFA-CNRS, Universite Joseph Fourier, Direction des
Sciences du Vivant, CEA Grenoble, France
We studied the interaction of several metal ions with the
copper chaperone from Enterococcus hirae (EhCopZ) We
show that the stoichiometry of the protein–metal complex
varies with the experimental conditions used At high
con-centration of the protein in a noncoordinatingbuffer, a
dimer, (EhCopZ)2–metal, was formed The presence of a
potentially coordinatingmolecule L in the solution leads to
the formation of a monomeric ternary complex, EhCopZ–
Cu–L, where L can be a buffer or a
coordinatingmole-cule (glutathione, tris(2-carboxyethyl)phosphine) This was
demonstrated in the presence of glutathione by electrospray
ionization MS The presence of a tyrosine close to the
metal-bindingsite allowed us to follow the bindingof cadmium to
EhCopZ by fluorescence spectroscopy and to determine the correspondingdissociation constant (Kd¼ 30 nM) Com-petition experiments were performed with mercury, copper and cobalt, and the correspondingdissociation constants were calculated A high preference for copper was found, with an upper limit for the dissociation constant of 10)12M These results confirm the capacity of EhCopZ to bind cop-per at very low concentrations in livingcells and may provide new clues in the determination of the mechanism of the uptake and transport of copper by the chaperone EhCopZ Keywords: copper transport; CopZ; metal binding; metal-lochaperone; selectivity
Copper is a first-row transition metal, which plays a
fundamental role in livingorganisms Although it is
involved in the catalytic active site of several enzymes [1],
its redox properties can also generate highly toxic hydroxy
radicals in cells [2] Therefore, its intracellular concentration
has to be tightly regulated Copper chaperones have recently
been reported to be key proteins in the uptake and transport
of copper in cells, and in the transfer of the metal ion
to appropriate partners [3–5] Many recent studies have
provided data on their biological function, and an
increas-ingnumber of 3D structures have been resolved for many
members of this family both in the apo and
metal-loaded state (vide infra)
In this study, we focused on the protein CopZ, which
has been reported to be involved in copper homoeostasis
in Enterococcus hirae (hereafter referred to as EhCopZ)
[6,7] It belongs to the cop operon, which also encodes
two copper ATPases, CopA and CopB, and a repressor
CopY EhCopZ has been shown to transfer two copper
ions to CopY [8,9], thereby controllingthe expression of the cop operon The 3D NMR structure of apo EhCopZ has been resolved [10] EhCopZ exhibits a ferredoxin-like fold (babbab) in which the four b-strands and the two a-helices are connected by loop regions exposed to the solvent The metal-bindingsite is located at the C-terminal extremity of the first loop and on the first turn of helix a1 (Fig 1) Its sequence is highly conserved in the family and consists of a consensus motif MXCXXC The bindingof the metal ion is mainly accomplished via the two sulfur atoms of the side chain of the two cysteine residues Cys11 and Cys14 Surprisingly, various stoichiometries have been reported so far for metal–chaperone complexes (Table 1) Monomeric compounds have been found in the case of BsCopZ–Cu (CopZ from Bacillus subtilis) [11] and with the homologous proteins MerP–Hg [12], Atx1–Cu [13] and Atx1–Hg[14], whereas dimers have been reported in the case of EhCopZ–Cu [10] and BsCopZ–Cu [15,16] and with the homologous protein HAH1 loaded with Cu, Cd
or Hg[17] Therefore, it would be interestingto determine the stoichiometry in solution of copper-loaded EhCopZ
as it may offer a molecular basis for the copper-transfer mechanism from the copper chaperone to the target protein
Another relevant question is the selectivity of these metallochaperones for different metals As it is well known that the MXCXXC motif can bind various metals [18], the determinants of the selectivity of these proteins for a specific ion remain poorly understood For example, in vitro studies have shown that MNKr2, a copper-bindingsubdomain of the Menkes ATPase, is able to bind Ag(I) or Cu(I) but
Correspondence to L Le Clainche, De´partement d’Inge´nierie et
d’Etudes des Prote´ines, Direction des Sciences du Vivant,
CEA Saclay, 91191 Gif sur Yvette Cedex, France.
Fax: + 33 0169089071, Tel.: + 33 0169084215,
E-mail: leclainche@dsvidf.cea.fr
Abbreviations: EhCopZ, copper chaperone from Enterococcus hirae;
BsCopZ, copper chaperone from Bacillus subtilis; TCEP,
Tris(2-carboxyethyl)phosphine.
(Received 27 November 2003, revised 15 January 2004,
accepted 19 January 2004)
Trang 2cannot bind a larger ion such as Cd(II), as is the case for the
protein HAH1 [19–21] However, the
metal-bindingaffinit-ies of any copper chaperone for metal ions have not been
reported so far Therefore, a study of the strength of
interaction of EhCopZ with different metals and a
com-parison with other proteins could provide a better
under-standingof this selectivity
Here we describe a study of the bindingof several metal
ions to the protein EhCopZ The stoichiometry of the
metal-loaded chaperone was found to depend on the experimental
conditions used, especially the concentration of the protein
and the presence of an exogenous coordinating molecule
The dissociation constants for cadmium, mercury and
cobalt were determined usingfluorescence spectroscopy,
and the upper limit of the dissociation constant for copper
was also determined
Materials and methods
Primer design for the synthetic geneEhcopz
Oligonucleotides (60-mer) were designed from the sequence
of the gene Ehcopz (E hirae copZ) with optimized codons
for Escherichia coli; they were synthesized and purified by MWG-Biotech The six oligonucleotides used for the syn-thetic gene construction were: copz-p1, (5¢-GGGCCGGC GGCCATGGCTAAACAGGAATTCTCGGTTAAAGG TATGTCTTGCAAC-3¢); copz-ap2, (5¢-GATACGACC AACAGCTTCTTCGATACGAGCAACGCAGTGGT TGCAAGACATACCTTTAAC-3¢); copz-p3, (5¢-GAA GCTGTTGGTCGTATCTCTGGTGTTAAAAAAGTT AAAGTTCAGCTGAAGAAAGAAAAG-3¢); copz-ap4, (5¢-GGTAGCCTGAACGTTAGCTTCGTCGAATTTAA CAACAGCCTTTTCTTTCTTCAGCTGAAC-3¢); copz-p5, (5¢-GAAGCTAACGTTCAGGCTACCGAAATCTG CCAGGCTATCAACGAACTGGGTTACCAGGCT-3¢); copz-ap6, (5¢-GGGCCGGCGCGGTTAGATCTAAGCT TAGATAACTTCAGCCTGGTAACCCAGTTCGTT-3¢) Primers 1, 3 and 5 corresponded to the codingstrand, respectively, for positions 1–54, 76–135, 154–213 Primers 2, 4 and 6 corresponded to the complementary strand, respect-ively, for positions 34–93, 115–174, 193–220 of the coding strand Each primer overlapped the followingone by 27 bases Restriction sites for NcoI and BglII were introduced, respectively, in the N-terminal primer copz-p1 and in the C-terminal primer copz-ap6
Fig 1 3D NMR structure of apo-EhCopZ (PDB ID:1CPZ) and Cu(I)-loaded BsCopZ (PDB 10:1K0V) apo-EhCopZ (right) BsCopZ (left) Hydrogens have been omitted for clarity, and only one of the multiple structures is represented for both proteins Selected bond (A˚) and ang les ():
S C13 –Cu 2.16, S C16 –Cu 2.17, S C13 –Cu–S C16 , 115.31.
Table 1 Conditions used and observed stoichiometries for different metal–chaperone complexes DTT, dithiothreitol, ICP-AES, inductively coupled plasma-atomic emission spectrometry.
Protein
[Protein]
(l M ) Metal Buffer used Reducingagent
Stoichiometry (metal:protein) Analytical method Reference EhCopZ 5, 10 Cu, Cd Mops No 0.5 Fluorescence, CD, UV This work EhCopZ 5, 10 Cu, Cd Mops TCEP 1 Fluorescence, CD, UV This work EhCopz 0.5 Cd, Cu, Co, HgMops No 1 Fluorescence This work
BsCopZ 2000 Cu Phosphate DTT 0.77 ICP-AES 11 BsCopZ 300 Cu Mops No 0.5 Gel filtration 15 BsCopz 300 Cu Mops DTT 1 Gel filtration 15
yAtx1 2400 Cu, HgPhosphate, Mes DTT removed 0.6–0.8 ICP-AES 25 yAtx1 100–400 Cu Tris/Mes
phosphate
No, DTT, GSH 1 ICP-AES 26 HAH1 500–1300 Cu, Cd, HgMes No 0.2–0.5 ICP-AES, X-Ray 17
Trang 3Construction of the synthetic geneEhcopz and
expression vector PQE-cop
The six oligonucleotides were assembled in a first PCR step
The reaction was carried out in 25 lL, using3 pmol of each
oligonucleotide, 0.2 mM each dNTP, 1ở Pfu buffer and
1.75 U Pfu turbo (Stratagene) The assembling PCR was
performed in an MWG-Biotech Primus thermocycler with
the followingprogram: 94C for 60 s; 94 C for 45 s, 47 C
for 30 s, 72C for 15 s (55 times); and 72 C for 5 min The
assembled fragment was amplified by a second PCR step
The reaction was performed in 25 lL, using7 pmol of
the N-terminal and C-terminal oligonucleotides (1 and 6),
0.2 mM each dNTP, 1ở Pfu buffer, 1.75 U Pfu turbo
(Stratagene) and 1 lL of the previous PCR product The
PCR program was: 94C for 60 s; 94 C for 45 s, 47 C for
30 s, 72C for 35 s (30 times); and 72 C for 5 min After
analysis of the PCR product on a 1.6% (w/v) agarose gel in
a TAE buffer (40 mM Tris, 20 mMsodium acetate, 1 mM
EDTA, pH 8.3), the 220-bp fragment of interest was
digested with NcoI and BglII, purified usinga Nucleospin
extraction kit (Macherey Nagel) and ligated into PQE60
(Qiagen) digested with NcoI and BglII The final construct
PQE-cop was verified by DNA sequencing E coli XL1 blue
(Stratagene) was used as the host strain for plasmid
propagation The expected molecular mass calculated by
MassLynx from this sequence is thus 7592.9 Da It is in
good agreement with the experimental molecular mass
found of 7592.3 ổ 0.6 Da It should be noted that the
calculated mass of Eh-CopZ in the NCBI database
(acces-sion No 1361370) is 7521.8 Da The 71-Da difference
between this value and the experimental mass is due to the
insertion of an N-terminal alanine duringthe primer design
for the plasmid construction
Expression and purification of recombinant EhCopZ
E coliM15 (Qiagen) was used as the host strain for the
expression of EhCopZ The cells were grown at 37C in
LuriaỜBertani medium containing200 mgẳL)1 ampicillin
and 25 mgẳL)1 kanamycin to an absorbance of 0.6 at
600 nm Protein expression was induced by the addition of
isopropyl b-D-thiogalactoside to a final concentration of
200 lM, and the cells were further incubated for 4 h The
cells were harvested, resuspended in 50 mMsodium
phos-phate, pH 7.2, containing5% glycerol, 2 mMEDTA, 5 mM
dithiothreitol, and lysed by Eaton pressure cell The cell
extract was incubated for 1 h at 4C with 1 mM
phenyl-methanesulfonyl fluoride, DNase I and RNase After
filtration on a 0.45-lM nitrocellulose membrane, it was
loaded on to a 3ở 10 cm S15 Sepharose fast flow column
(Pharmacia) equilibrated in buffer A (50 mM sodium
phosphate, pH 7.2) EhCopZ was eluted with a linear
gradient (0Ờ1M) of NaCl in buffer A The EhCopZ
fractions were pooled and concentrated to less than 5 mL
with an Amicon YM3 membrane, and stored at)20 C
after the addition of 2 mM dithiothreitol The purity was
checked by SDS/PAGE on 20% (w/v) polyacrylamide
gels after silver staining Gel filtration was performed to
exchange the buffer before functional characterization of
the protein
Protein and metal quantification for titration experiments
A mean e280of 2000M )1ẳcm)1was determined by amino-acid quantification for EhCopZ in buffer C (20 mM
Mops, 150 mM NaCl, pH 7.2) used for fluorescence experiments Protein concentration was then measured usingthe UV absorbance at 280 nm for all titration experiments All metal solutions were prepared in water except for Cu(I)Cl which was prepared as a 4 mM
solution in acetonitrile or 0.1MHCl/1M NaCl [22] Tris(2-carboxyethyl)phosphine (TCEP) was prepared in the buffer used for the titration
Metal titration by fluorescence Fluorescence measurements were performed with a Cary Eclipse spectrofluorimeter (Varian) in a thermostatically controlled cell holder, usinga 1-cm-path-length quartz cell All the experiments were carried out under argon The spectra were recorded with a bandwidth of 5 nm for both excitation and emission beams at a scan rate of 250 nmẳ-min)1 Intrinsic protein fluorescence measurements were recorded at 22C between 260 and 400 nm usingan excitation wavelength of 278 nm The protein was reduced
in 5 mM dithiothreitol and desalted by gel filtration on Superdex 75 (Pharmacia) in buffer C (20 mM Mops,
150 mM NaCl, pH 7.2) or in the appropriate buffer for further functional characterization (NaHCO3) The fluor-escence emission spectrum of EhCopZ exhibited a maxi-mum at 305 nm, which is consistent with its single tyrosine Tyr63 Typically 500 lL of 5 lM, 0.5 lMor 50 nMEhCopZ
in buffer C was titrated with additions of 0.5Ờ1 lL CdCl2at the appropriate concentration In some experiments, TCEP was added as a reducingagent As no effect of CuCl, HgCl2
or CoCl2 was detected with direct fluorescence measure-ments, the titration of these metals was performed by competition in the presence of Cd Equilibrium was established within 2 min of the addition of the metal The data correspondingto the titration of EhCopZ with cadmium were fitted usingeither the bindingisotherm or a Scatchard plot In the first case, the isotherm corresponds to the equilibrium: CopZ + Cdề CopZỜCd At equilib-rium, the law of mass action gives:
KdỬ đơCopZ ơCdỡ=ơCopZCd đ1ỡ The fluorescence intensity of the protein can be written as:
IỬ ImaxơCopZCd=ơCopZ0 đ2ỡ where [CopZ]0is the initial protein concentration and Imaxis maximum intensity correspondingto 100% of the complex
in solution At equilibrium, the concentrations in solution can be expressed as:
ơCopZ Ử ơCopZ0ơCopZCd;
ơCd Ử ơCdiơCopZCd đ3ỡ where [Cd]i is the concentration of added cadmium in solution InsertingEqn (3) into Eqn (1) leads to the equation:
Trang 4ơCopZCd2đơCopZ0ợ ơCdiợ KdỡơCopZCd
ợ ơCopZ0ơCdiỬ 0
which can be combined with Eqn (2) to give:
IỬ đ1=2ơCopZ0ỡImax đđơCopZ0ợ ơCdiợ Kdỡ
fđơCopZ0ợ ơCdiợ Kdỡ2 4ơCopZ0ơCdig1=2ỡ
A Scatchard plot is obtained by calculatingthe free and
bound cadmium concentration at equilibrium with the
followingexpressions:
ơCdfreeỬ ơCdI ơCopZ0 đI I0ỡ=đImax I0ỡ;
ơCdboundỬ ơCopZ0 đI I0ỡ=đImax I0ỡ
where I0and Imaxare, respectively, the initial and maximal
fluorescence intensities
CD spectroscopy measurements
CD measurements of EhCopZ were recorded on a JS 810
spectropolarimeter (Jasco) The scans were recorded usinga
bandwidth of 2 nm and an integration time of 1 s at a scan
rate of 100 nmẳmin)1 For near-UV measurements between
250 nm and 310 nm, a 1-cm-path-length quartz cell
con-taining2 mL protein sample was used A total of 20 scans
were recorded and averaged for each sample All resultant
spectra were baseline subtracted Aliquots of volume
200 lL of these protein sample solutions were used for
far-UV measurements between 190 nm and 250 nm in a
1-mm-path-length quartz cell A total of 10 scans were
recorded and averaged for each sample
Protein samples of concentration 20 lMwere prepared in
an anaerobic atmosphere in 40 mM Mops/10 mM NaCl,
pH 7.0 CD spectra were recorded after each addition of
1 lL metal aliquots The total volume added to the 2
mL-buffered protein solution was less than 20 lL of the metal
stock solution The titration experiment was performed
under argon The pH and ionic strength of the reaction
mixture remained unchanged throughout the titration
UV-vis absorption spectra
UV-vis absorption spectra were recorded on a Lambda 35
spectrophotometer (PerkinỜElmer) usinga
1-cm-path-length quartz cell Protein samples of 10 lM or 20 lM
apo-EhCopZ were prepared in buffer C Optical spectra
were recorded from 190 to 700 nm after each TCEP, GSH
or metal addition Corrected spectra were obtained after
baseline subtraction
Sample preparation for electrospray ionization (ESI)-MS
analysis
For functional ESI-MS analysis under nondenaturing
conditions, the protein samples were thawed on ice, reduced
with 5 mMdithiothreitol and desalted by g el filtration on a
Superdex 75 column (Pharmacia) equilibrated in freshly
prepared 20 mMNH4HCO3, pH 8.0 The fractions
collec-ted were freeze-dried and stored under argon at 4C before
use The protein was suspended in MS buffer (4 mM
NHHCO, pH 8.0), centrifuged (7200 g) and quantified
For the functional ESI-MS study, the samples were prepared as 50 lL aliquots of 15 lM EhCopZ in 4 mM
NH4HCO3 (pH 8.0)/15% methanol with the appropriate metal or GSH concentrations
ESI-MS measurements ESI mass spectra were acquired usinga Micromass Q-TOFII instrument under control of the MassLynx 3.5 data acquisition and analysis software (Micromass Ltd, Manchester, UK) The MS buffer was used as the electrospray carrier solvent Samples were introduced into the ion source at a flow rate of 10 lLẳmin)1, and mass spectra were acquired from m/z 400Ờ2200 in positive ionization mode with a scan time of 5 s External calibration
of the mass scale was performed with horse heart myoglobin (Sigma) The spectra were analyzed withMASSLYNX3.5 Light-scattering measurements
Dynamic light-scattering data were obtained with the DynaPro-801 instrument (Protein Solutions Inc, High Wycombe, Bucks, UK) usinga 30 mW, 833 nm wave-length argon laser at 22C and equipped with a solid-state avalanche photodiode Duringillumination, the photons scattered by proteins were collected at 90C on a 10 s acquisition time and were fitted with the analysis software,
DYNAMICS Intensity fluctuations of the scattered light resultingfrom Brownian motion of particles were analyzed with an autocorrelator to fit an exponential decay function and then measuringa translational diffusion coefficient D For polydisperse particles, the autocorrelation function was fitted as the sum of contributions from the various size particles usingthe regularization analysis algorithm D is converted into a hydrodynamic radius R through the StokesỜEinstein equation (RỬ kBT/6pgD where g repre-sents the solvent viscosity, kBthe Boltzmann constant, and
T the temperature) R is defined as the radius of a hypothetical hard sphere that diffuses with the same speed
as the particle under examination However, the particle may be nonspherical and solvated Therefore, the molecular mass M of a macromolecule is estimated using M vs R calibration curves developed from standards of known molecular mass and size Thus, the estimated mass of a given particle is subjected to error if it deviates from the shape and solvation of the molecules used as standards (globular proteins) The molecular mass for a protein is estimated from the curve that fits the equation
MỬ (1.68 ở R)2.3398 as implemented in the DYNAPRO
software
Results
The analysis of the 3D structure of EhCopZ shows that Cys11 and Cys14 were at 5.73 A˚ CaỜCa distance This value is within the range of the average CaỜCa distance of bridged cysteine residues generally found in proteins [23] The spatial proximity between the two cysteine residues could make the protein very sensitive to oxidation While these two residues are involved in the metal-bindingsite, it is crucial that the protein remains reduced throughout the experiment A control experiment was performed under
Trang 5conditions favorable to oxidation: an apoprotein sample
was left under aerobic conditions for 2 h and the free-thiol
quantification usingEllman’s reagent showed that less than
8% of the protein was oxidized Consequently, all the
experiments described hereafter were performed within 2 h
under argon to further minimize CopZ oxidation
Interaction between copper and EhCopZ
In a first set of experiments the bindingof copper to the
chaperone was studied usingCD and UV-vis spectroscopy
To a 20 lM solution of apo-EhCopZ in 40 mM Mops/
10 mM NaCl, pH 7, were added aliquots of a solution of
Cu(I), stabilized using0.1M HCl, under anaerobic
condi-tions [22] The far-UV region of the CD spectrum displayed
no significant modification on the addition of the metal,
indicatingthat the global fold of the protein was preserved
throughout the titration The dichroic signal at 265 nm
increased with the concentration of copper, as could be
expected with a change in the hydrophobicity of the local
environment of Tyr63 and/or a contribution of the binding
of the copper to the thiolates of the protein A plot of the
intensity of the signal at 265 nm against the concentration
of added copper showed that a plateau was reached when
0.5 equivalents of copper had been added, compatible with
a 2 : 1 EhCopZ–Cu complex (Fig 2) The UV-vis spectrum
of the reaction mixture in the presence of the metal ion
exhibited strongabsorption at 260 nm compatible with a
metal to ligand charge transfer band (data not shown) The
intensity of this band increased with the concentration of
added copper in solution, and the plot of A260vs
concen-tration of copper indicated in this case also a 2 : 1 EhCopZ/
Cu ratio
This result is in contrast with the 1 : 1 stoichiometry
reported for a similar UV-vis experiment described
previ-ously [9] Several hypotheses were explored to explain this
difference As mentioned above, experiments were
per-formed under conditions in which the oxidation of EhCopZ
is not significant Partial oxidation of the protein can
therefore be excluded to explain the 2 : 1 protein to metal stoichiometry
Although precautions were taken to avoid any oxidation
of the metal, a change in the oxidation state of the metal may be responsible for this unexpected stoichiometry The
CD experiment described above with Cu(I) was therefore repeated with Cu(II) in order to study the influence of the oxidation state of copper on the complex stoichiometry The spectra showed an increase in the signal of the tyrosine at
265 nm with increasingconcentrations of copper up to a plateau reached for 0.5 molar equivalents of Cu(II) added per protein A similar experiment was described by Kihlken
et al [15] with BsCopZ It was shown that Cu(II) was reduced to Cu(I) on coordination to the protein A similar process cannot be excluded in our case However, no difference in stoichiometry in the complex was detected usingeither Cu(I) or Cu(II) SDS/PAGE analysis of EhCopZ was performed under nondenaturingconditions for the protein in the presence of increasingcopper equivalents A band correspondingto the molecular mass expected for a dimer appeared in the presence of the metal, confirmingthe dimeric nature of the EhCopZ–Cu (Fig 3) Lastly, in previous studies [11,15,24–26], reducingmole-cules such as dithiothreitol or TCEP were added in the solution of copper chaperone to prevent the formation
of the disulfide bridge The interaction of such a small organic molecule present in the solution with the metal center could greatly influence the stoichiometry by changing the form of the complex As these compounds can compete with the protein to bind the metal ion, their influence on the stoichiometry of the complex EhCopZ–metal was studied Cadmium was substituted for copper to avoid any redox reaction involvingthe metal ion Although Cd(II) is not a usual substitute for Cu(I), the available 3D structures of a homologous protein, HAH1, show that the copper-loaded and cadmium-loaded structures of the chaperone are very similar (PDB ID: 1FEE and 1FE0, respectively [17]) Moreover, the single tyrosine Tyr63 located on loop 5
at the beginning of the last strand b4 is close to the
Fig 2 CD titration of EhCopZ against Cu(I) CD spectra of EhCopZ
(20 l M ; 40 m M Mops/10 m M NaCl, pH 7) in the presence of Cu(I)
(from top to bottom) at 0, 2, 4, 6, 8, 10, 12, 16, 20 l M The insert shows
the plot of the intensity of the dichroic signal at 265 nm vs the
con-centration of introduced copper.
Fig 3 SDS/PAGE analysis of the protein EhCopZ in the presence of various concentrations of copper Left lane, MultiMark Multi-Colored standard (Invitrogen); lane 1, apo-EhCopZ; lane 2, in the presence of 1 equivalents CuCl 2 ; lane 3, in the presence of 4 equiva-lents CuCl 2 Experiments were performed with a solution of 25 l M
EhCopZ in 20 m M Mops/150 m M NaCl, pH 7.2 The 6· sample buffer was: g lycerol 50%, Bromophenol Blue 0.5%, Mops pH 7 Samples containing3 lgprotein were loaded on a 4–12% NuPAGE Bis/Tris gel (Invitrogen) The electrophoresis was performed with a Mes run-ningbuffer (Invitrogen).
Trang 6metal-bindingcysteine Cys14 (distance OHTyr63–SCys14c ¼
3.6 A˚) Preliminary experiments showed that excitation of a
solution of EhCopZ at kexcit¼ 278 nm led to a maximum
in the fluorescence emission at 305 nm, which increased on
addition of Cd(II) whereas no change was detected on
addition of copper Therefore, the fluorescence of Tyr63
was used as a probe to monitor the bindingof metal ions to
the protein
Study of the binding of Cd(II) to EhCopZ by fluorescence
spectroscopy, dynamic light scattering and ESI-MS
spectroscopy
In the first experiment, a spectrofluorimetric titration of
EhCopZ (5 lM solution in 20 mM Mops/150 mM NaCl,
pH 7.2) against an aqueous acidic solution of Cd(II) was
performed at room temperature With a 278 nm excitation
wavelength, the emitted fluorescence of the tyrosine in
position 63 was observed Its intensity was found to increase
on addition of the metal up to a limit correspondingto
0.5 ± 0.1 equivalents cadmium added (Fig 4) The change
in the intensity of the tyrosine fluorescence may be
attributed to the formation of a dimer in solution, as was
the case with copper To test the hypothesis of potentially
coordinating exogenous ligands, a similar experiment was
carried out in the presence of 5 molar equivalents TCEP,
and the plateau was reached when 0.9 ± 0.1 equivalents
Cd were added (Fig 4) TCEP is often used as a reducing
agent instead of dithiothreitol, and bears three carboxylate
functions instead of thiols If the change in the stoichiometry
is due to the coordination of a molecule of TCEP to the
metal ion via the phosphorous atom [26] or a carboxylate
function [27], the use of a potentially coordinatingbuffer
should provide the same result Therefore, the Mops buffer
was replaced by a sodium hydrogenocarbonate buffer The
carbonate function is well known as a coordinatinggroup
for which a great number of binding modes have been
described [28] In contrast, the sulfonate function of Mops is
known as a poor ligand for transition metals and has
recently been described as noncoordinatingfor copper [29]
The correspondingfluorimetric titration of EhCopZ (5 lM
in 20 mMNaHCO3/150 mMNaCl, pH 7.2) was achieved in the absence of TCEP, and a 1 : 1 stoichiometry was also found in this case (Fig 4)
To confirm the nature of the oligomeric EhCopZ–Cd complex, dynamic light-scattering experiments were carried out on EhCopZ/cadmium solutions in the absence and presence of TCEP Apparent molecular masses were found
to be 13.6 ± 2 kDa (hydrodynamic radius R¼ 18.5 ± 0.9 A˚) in the case of the apoprotein in the absence and presence of 5 molar equivalents TCEP On addition of cadmium to apo-EhCopZ, the apparent molecular mass increased to 23.7 ± 2.5 kDa (R¼ 23.4 ± 1.1 A˚) This increase is in the range that could be expected from dimerization and consistent with the results obtained by Wimmer et al [10] for the complex EhCopZ–Cu When cadmium was added to apo-EhCopZ in the presence of TCEP, the apparent molecular mass was 16.5 ± 2 kDa (R¼ 20.0 ± 1.1 A˚) close to the value obtained for the apoprotein and hence consistent with the formation of a monomeric complex in these conditions
Complementary studies of the interaction between EhCopZ and metal ions were performed usingMS High-quality ESI mass spectra of proteins can be obtained in a
4 mMammonium carbonate buffer, pH 8.0, in the presence
of 15% (v/v) methanol [30] In these conditions, EhCopZ retains its structure (CD data not shown) and is desorbed
in the gas phase as multiply charged ions corresponding predominantly to the charge states +5 and +6 This charge distribution indicates a folded protein with fewer basic residues available for protonation [31] The protein was incubated in the presence of increasingconcentrations of cadmium, copper, mercury and cobalt, and a set of spectra were recorded for each metal ion In each case, the spectrum displayed peaks with charge states of +5 and +6, only compatible with a 1 : 1 monomeric EhCopZ–metal com-plex (Fig 5) [32] Taken together, these results are
compat-Fig 4 Fluorimetric titration of EhCopZ against Cd(II) in the presence
and the absence of TCEP Normalized fluorescence intensities at
305 nm of EhCopZ (5 l M ) with increasingconcentrations of Cd(II) in
20 m M Mops/150 m M NaCl, pH 7, in the absence of TCEP (d), in
20 m M Mops/150 m M NaCl, pH 7, in the presence of 25 l M TCEP
(m) and in 20 m M NaHCO 3 /150 m M NaCl, pH 7, in the absence of
TCEP (j).
Fig 5 MS of the EhCopZ–metal complexes ESI-MS spectra of 15 l M
EhCopZ in 4 m M NH 4 HCO 3 (pH 8.0)/15% methanol in the pre-sence of 0.75 equivalents (11.25 l M ) metal ions (A) Apo-EhCopZ; (B) Cu(I)Cl; (C) Hg(II)Cl ; (D) Cd(II)Cl ; (E) Co(II)Cl
Trang 7ible with the formation of ternary complexes corresponding
to the formula EhCopZỜCdỜL where L is an exogenous
coordinatingmolecule (dithiothreitol, TCEP, buffer anion)
On dilution of the reaction mixture in the absence of
TCEP, the monomer/dimer equilibrium is expected to be
displaced in favor of the monomeric species The titration of
a 0.5 lM solution of EhCopZ in 20 mM Mops/150 mM
NaCl, pH 7.2, by a solution of Cd(II) led indeed to the
detection of a 1 : 1 EhCopZ to Cd stoichiometry A fit of
the data by the bindingisotherm led to a dissociation
constant of 65 nM To ensure that the monomer/dimer
equilibrium is displaced to close to 100% monomer in
solution, the concentration of the protein was decreased by
another order of magnitude Therefore, a new titration was
carried out usinga 50 nM protein solution A Hill plot
yielded a straight line with a slope nỬ 1.05 confirmingthe
formation of an adduct with a 1 : 1 stoichiometry The
correspondingScatchard plot led to a dissociation constant
of KdỬ 30 ổ 5 nM(Fig 6)
Interaction of EhCopZ with Co, Hg, Cu and determination
of the apparent dissociation constants
Of the metal ions tested, a change in fluorescence intensity
of Tyr63 was only detected with Cd(II) Therefore
compe-tition experiments were run to determine the dissociation
constants of the metalỜprotein complexes with cobalt,
mercury and copper In a typical experiment, the
mono-meric EhCopZỜCd complex was formed (0.5 lMprotein in
20 mMMops/150 mMNaCl, pH 7.2) before the addition of
the competingmetal M The concentration range of the
protein was increased to 500 nM in order to have nearly
100% of the EhCopZỜCd complex with the minimum
amount of cadmium (Ậ 1.5 equivalents vs protein) The
decrease in the fluorescence intensity of the cadmium
complex was followed, and the dissociation constants were
determined at half fluorescence intensity
In each case, the followingreaction takes place in the solution:
Cop ZCd ợ M $ CopZM ợ Cd(II) The correspondingreaction constant is:
KRỬ KdđCdỡ=KdđMỡ
Ử đơEhCopZMơCd(II)ỡ=đơEhCopZCdơMỡ Startingfrom an initial intensity I0and an initial protein concentration C0, the concentrations of the compounds present in solution can be determined at I0) [(I0) Imin)/2], where Iminis the intensity at high [M] At this point, the concentrations of free species in solution are:
ơEhCopZCd Ử C0=2; ơM Ử C0đNM 0:5ỡ;
ơCd Ử C0đNCd 0:5ỡ; ơEhCopZCd Ử C0=
A new form of the reaction constant can be written as follows:
KdơM Ử KdđCdỡđNM 0:5ỡ
đNCd 0:5ỡ
in which NMis the number of equivalents of the competing metal M introduced at IỬ I0) [(I0) Imin)/2], and NCd
is the number of equivalents of cadmium in solution This concentration of cadmium in solution is chosen as a function of the affinity of the competingmetal for EhCopZ
to obtain a value for NM different from 0.5 equivalents (Table 2) The competition curves obtained for Hgare shown in Fig 7 The dissociation constant for mercury was
Kd(Hg)Ử 2 ổ 0.5 nM In the case of cobalt, no significant change in the intensity of the tyrosine was detected up to
500 molar equiv cobalt added As EhCopZ precipitates at higher cobalt concentrations, the dissociation constant could therefore be estimated to be greater than 15 lM This
is in good agreement with the value of KdỬ 20 lM
obtained from ESI-MS titration previously described [32]
In the case of copper, the affinity appeared to be so high that the value found for NCu (0.52) in the presence of 1000 equivalents of cadmium was still very close to 0.5 equiv and could only lead to an estimation of a maximum value of the dissociation constant for copper of 10)12M A hig her initial concentration of cadmium led to precipitation of the protein
The fact that copper binds to the protein with higher affinity than cobalt or cadmium could be predicted from thermodynamic data However, it was more surprisingthat mercury had a weaker affinity than copper A confirmation
Fig 6 Fluorimetric titration of EhCopZ with Cd(II) at low
concentra-tion Fluorescence spectra of EhCopZ (50 n M in 20 m M Mops/150 m M
NaCl, pH 7.2; k excit Ử 278 nm) in the presence of
increasingconcen-trations of Cd(II) ions (from bottom to top: 0, 10, 20, 30, 40, 50, 75,
100, 150, 200, 400 n M ) The insert shows the correspondingScatchard
plot.
Table 2 Dissociation constants between metal ions and CopZ The K d values were calculated at half-intensity usingcompetition fluorescence experiments between cadmium and other metal ions For each experiment, the concentration of the protein CopZ was 0.5 l M N M is the amount of competingmetal at half-intensity.
Competing metal ion
[Cd(II]
(l M ) N M Estimated K d Hg(II) 2.5 0.8 2 ổ 0.5 n M
Co(II) 0.75 >500 >15 l M
Cu(I) 500 0.52 ặ 10)12M
Trang 8of this result was obtained usingESI-MS competition
experiments As it reported previously [32] cadmium can
easily be displaced by copper and mercury but not by
cobalt Moreover, mercury can be displaced by copper
leadingto the order of affinity Cu > Hg> Cd > Co
which is the same as we obtained in the fluorescence
experiments
The coordination of an exogenous thiol to the EhCopZ–
Cu complex was also studied by ESI-MS Given the high
concentration of glutathione in cells [33] and its ability to
bind Cu(I) [34], it is a good candidate to act as the third ligand
for the Cu(I) ion in the complex The ESI-MS experiments
were carried out in the ammonium carbonate buffer (15 lM
EhCopZ, pH¼ 8) The spectrum of apo-EhCopZ exhibits
a peak at m/z¼ 1519.2 correspondingto the +5 charged
state of EhCopZ which shifts to m/z¼ 1531.9 on addition of
a stoichiometric amount of Cu(I) Subsequent addition of
aliquots of glutathione to the reaction mixture led to the
appearance of a new peak at m/z¼ 1593.7 compatible with
the formation of a ternary adduct of formula EhCopZ–Cu–
GSH Moreover, a mixture of Cu(I) with 2 molar equiv
glutathione exhibits a spectrum with peaks at m/z¼ 613.1,
674.0 and 676.0 which correspond, respectively, to the
oxidized glutathione dimer (GS)2and to both isotopes of
the oxidized copper complex Cu(I)(GS)2 On addition of
1 equivalents EhCopZ in this solution, the peak at 674.0
disappears and new peaks at m/z¼ 308.4, 1531.8 and 1592.9
are detected, corresponding, respectively, to free glutathione,
EhCopZ–Cu and EhCopZ–Cu–GSH
Discussion
Our understandingof copper traffickingwithin the cell
took a great step forward with the discovery of
metallochaperones In the presence of an extremely low
free copper concentration in cells under normal growth
concentration ([Cu]free< 10)17M), copper chaperones have been shown to be key partners in the delivery of the metal ion to their target proteins [35] These proteins have been studied extensively over the past few years However, several characteristics remain to be determined Among these is the metal-loaded form of the chaperone in vivo which is a key element to further understandingthe mechanism of the metal transfer from a metallochaperone
to its target protein Another point of interest is the selectivity of the chaperone for one type of metal ion We here present results from in vitro experiments in these two fields of interest usingthe protein EhCopZ
Our experiments show that, of the metals tested, EhCopZ has a high preference for copper; the following order of affinity was found: Cu(I) Hg(II) > Cd(II) Co(II) These results can be compared with those reported for the homologous protein MerP from CD analysis [36] MerP shares the same consensus bindingmotif and a similar 3D structure The higher affinity was found for Hg with a dissociation constant of 2.8 lM, and similar affinities were reported for Cu and Cd (respectively 5 and 20 lM) The strikingdifference between these values and our results on EhCopZ may be due to the experimental conditions used by Veglia et al [36] All the measurements were made in the presence of 100 lMdithiothreitol, which can compete for the metal ions [37] Surprisingly, whereas the metal-binding affinities of MerP follow the order found for inorganic thiolates (Hg> Cu > Cd > Co), this is not true for EhCopZ As these two proteins share the same structural bindingmotif C-X-X-C (first co-ordination sphere), our results suggest that the molecular determinants for the preference for copper may lie in the second co-ordination sphere of the metal The presence of different amino-acid side chains near the metal-bindingsite may play a major role
in the discrimination between metal ions, as it would be a source of different local electrostatic properties In parti-cular, several basic residues lie close to the metal-bindingsite
of CopZ whereas there are none in this area in the 3D structure of MerP As Hg(II) and Cu(I) differ by one charge,
a small difference in the electrostatic potential generated by the nearby residues could generate a significant change in the affinity for the two ions The molecular mechanism by which these different residues discriminate amongmetals requires further experiments, which are in progress in our laboratory
So far, metallochaperones have been reported to bind Cu(I) in different types of complex: monomers (protein– Cu), in which the copper ion is either two or three coordinated [11–14]; dimers, in which the metal ion is coordinated by the four cysteine residues of two protein molecules [15,17] In this study we show that two distinct types of complex can be stabilized in solution, and that they are highly sensitive to the experimental conditions used
In the experiments performed in the absence of any coordinatingmolecule and at high concentration of protein (> 10)6M), the titration curves showed a saturation at 0.5 equiv metal added per protein monomer Alongwith the change in intensity of the fluorescence of Tyr63 in the presence of cadmium and dynamic light-scattering analysis, these observations are consistent with the formation in this case of a homodimer Such a sandwich complex has been reported in the crystal structure of the homologous protein
Fig 7 Binding competition experiment between Cd(II) and Hg(II) to
EhCopZ Fluorescence spectra of a mixture of EhCopZ (0.5 l M in
20 m M Mops/150 m M NaCl, pH 7.2; k excit ¼ 278 nm) and 2.5 l M
CdCl 2 in the presence of increasingconcentrations of Hg(II) ions (from
top to bottom: 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 1, 1.2, 1.5, 2,
2.5 l M ) The insert shows the plot of the emission of fluorescence at
305 nm vs the concentration of added Hg(II).
Trang 9HAH1 loaded with Cd, Cu or Hg[17] In contrast, a recent
study has reported the formation of a monomeric complex
between BsCopZ and its metal cargo in the presence of the
reducingagent dithiothreitol [15] To avoid any
competi-tion between the thiols of the protein and the thiol of
dithiothreitol, we chose to use TCEP as the reducingagent
In its presence, the titration curves showed saturation at
1 equiv metal per protein correspondingto a monomeric
protein complex, as shown by dynamic light-scattering
experiments These data suggest a probable interaction of
such an exogenous molecule with the metal center which
would therefore be coordinated by the thiols of Cys11 and
Cys14 and by the phosphine of TCEP, as recently shown in
the case of the homologous protein HAH1 [26] The
presence of a third ligand on the metal ion is also in
agree-ment with the X-ray absorption fine structure (EXAFS)
experiments recently reported by Cobine et al [9], who
showed that the copper ion is coordinated in a trigonal
geometry by three atoms with an average distance of
2.241 A˚ Moreover, the substitution of Mops buffer by a
more coordinatingbuffer molecule such as carbonate also
led to the formation of a monomeric species Banci et al
[38] have recently described an effect of the buffer on the
coordinatinggeometry of a copper ion in a copper
chaperone complex Our results are in agreement with a
complex in which the buffer molecule acts as a weak and
labile ligand for the metallic center When the protein
concentration is lowered to 500 nM or less, the protein to
metal stoichiometry is always 1 : 1 In this case, the copper
ion may be bis-coordinated to the two sulfur atoms of the
cysteine residues in a linear geometry It may also be
three-coordinated in a trigonal planar geometry, a water molecule
completingthe coordination sphere as a weak and labile
ligand This hypothesis is in good agreement with the 3D
structures obtained for the copper complex of BsCopZ in
the presence of dithiothreitol [11] and for the copper
complex of the homologous protein Atx1 [13] In these
complexes, EXAFS studies have shown that the Cu(I) lies in
a trigonal geometry [24,38] In the 3D structure of Atx1-Cu,
the Cu(I) is coordinated to the two thiols of the cysteine
residues with an SCys15c –Cu–SCys18c of 120 This angle is what
would be expected for a perfect plane trigonal geometry of a
three-coordinated copper with a buffer or solvent molecule
as the third ligand
In yeast cells, the concentration of free copper is very low
(<10)17M), and the concentration of the chaperone is
thought to range from 0.1 to 1 lM[2] Assumingthat this
copper concentration is similar in other cells and that there
is also a high concentration of free thiol, a EhCopZ–Cu–SG
ternary complex could be the species present in vivo Indeed,
the ESI-MS results show the ability of the copper chaperone
to extract copper from a Cu(GS)2complex which could be
formed in cells and demonstrate the formation of such a
ternary complex with glutathione The origin of the metal
supply to a chaperone is a question of great interest A
recent study on the copper chaperone Atx1 from
Synecho-cystisPCC 6803 has shown that Atx1 acquires copper from
another protein CtaA, but can also scavenge the metal from
other sources [39] Such a Cu(GS)2complex could be one of
these sources
Taken together, these results suggest a mechanism for the
transfer of copper to the protein CopY In the presence of
glutathione, EhCopZ may form a ternary complex with copper of formula EhCopZ–Cu–GSH Transfer of the metal ion to CopY would then probably be achieved via a multiple ligand exchange with the cysteine residues of CopY helped by the geometry of the CopY active site and its higher affinity for copper, as proposed recently [9,40] The very high affinities of EhCopZ, and consequently CopY, for copper are consistent with the recent studies of cadmium-regulatory and zinc-regulatory proteins (CadC [41], SmtB [42] and ZntR [43]) Indeed, in the presence of a cellular overcapacity for bindingof transition metals, high affinities for such metal-regulatory proteins appear to be critical for specific traffickingpathways in vivo [35]
Conclusion
We have here described a study of the bindingof several metal ions to the metallochaperone EhCopZ In the presence of a metal ion, EhCopZ is able to form monomeric or dimeric compounds, and we have shown that the experimental conditions can be controlled to obtain either one form or the other Under physiological conditions, the presence of potentially coordinatingmole-cules probably leads to the formation of a monomeric ternary copper complex, EhCopZ–Cu–L L is an exogen-ous molecule that may be glutathione or a phosphate or carbonate ion The dissociation constant found for the protein–copper complex shows that the protein has a very high affinity for copper and may take up its copper ion from a potential intracellular Cu(GS)2 species These results suggest the possibility that, in vivo, the transfer of copper from EhCopZ to the target protein CopY could be achieved through a multiple ligand exchange mechanism between the glutathione molecule and the cysteine residues
of the two proteins involved A comparison between EhCopZ and MerP suggests that the determinants for the metal-bindingselectivity do not reside only in the struc-tural bindingmotif, but the environment surroundingthe metal-bindingsite has to be taken into account We are currently extendingthis study to other metallochaperones
to identify further molecular determinants of this metal-bindingselectivity
Acknowledgements
This work was supported by the Commissariat a` l’Energie Atomique (CEA), which provided a Ph.D fellowship to A U We would like to thank Dr V Forge for giving us access to the spectropolarimeter and for helpful discussions, Dr S Bressane for giving us access to light-scatteringfacilities, Dr B Amekraz and Dr C Moulin for givingus access to the Q-TOF mass spectrometer, Dr F Rollin-Genetet for invaluable advice, and Professor A Me´nez, Dr R Sto¨cklin and Dr
E Que´me´neur for continuous support.
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... copper of formula EhCopZ–Cu–GSH Transfer of the metal ion to CopY would then probably be achieved via a multiple ligand exchange with the cysteine residues of CopY helped by the geometry of the. .. of free thiol, a EhCopZ–Cu–SGternary complex could be the species present in vivo Indeed,
the ESI-MS results show the ability of the copper chaperone
to extract copper from. .. be one of
these sources
Taken together, these results suggest a mechanism for the
transfer of copper to the protein CopY In the presence of
glutathione, EhCopZ may