NMR experiments for structure and dynamics determination, molecular simulations, competition reactions for metal chelation by different metabo-lites c-Glu-Cys-Gly, a-Glu-Cys-Gly and c-Gl
Trang 1insights into heavy metal detoxification
Olivier Delalande1,*, Herve´ Desvaux2, Emmanuel Godat1,3, Alain Valleix4, Christophe Junot3, Jean Labarre1and Yves Boulard1
1 Laboratoire de Biologie Inte´grative ⁄ Service de Biologie Inte´grative et Ge´ne´tique Mole´culaire ⁄ Institut de Biologie et de Technologies de Saclay, CEA-Saclay, Gif-sur-Yvette Cedex, France
2 Laboratoire Structure et Dynamique par Re´sonance Magne´tique ⁄ Service de Chimie Mole´culaire, URA CEA-CNRS 331 ⁄ IRAMIS,
CEA-Saclay, Gif-sur-Yvette Cedex, France
3 Laboratoire d’Etude du Me´tabolisme des Me´dicaments ⁄ Service de Pharmacologie et d’Immuno Analyse Mole´culaire ⁄ Institut de Biologie
et de Technologies de Saclay, CEA-Saclay, Gif-sur-Yvette Cedex, France
4 Service de Chimie Bioorganique et de Marquage ⁄ Institut de Biologie et de Technologies de Saclay, CEA-Saclay, Gif-sur-Yvette Cedex, France
Introduction
Cadmium is a very toxic metal with mutagenic
proper-ties It also causes oxidative stress, but the mechanisms
involved remain unclear [1] In most eukaryotic cells, the
first line of defence against cadmium is thiol-containing
molecules (glutathione, phytochelatin or metallothionein
depending on the cell type) that have the property to chelate and sequester the toxic metal Glutathione is
a thiol-containing tripeptide, c-Glu-Cys-Gly, which
is ubiquitous and one of the most abundant cellular metabolites in many cell types, such as yeast or
Keywords
cadmium chelation; glutathione; heavy metal
toxicity; NMR; yeast
Correspondence
Y Boulard, CEA – Direction des Sciences
du Vivant, Institut de Biologie et de
Technologies de Saclay, Service de Biologie
Inte´grative et Ge´ne´tique Mole´culaire,
Baˆt.144, 91101 Gif-sur-Yvette Cedex,
France
Fax: +33 1 69084712
Tel: +33 1 69083584
E-mail: yves.boulard@cea.fr
*Present address
Centre de Biophysique Mole´culaire, CNRS
UPR 4301, Rue Charles Sadron, 45071
Orle´ans Cedex 2, France
(Received 12 July 2010, revised 6 October
2010, accepted 12 October 2010)
doi:10.1111/j.1742-4658.2010.07913.x
Cadmium is a heavy metal and a pollutant that can be found in large quantities in the environment from industrial waste Its toxicity for living organisms could arise from its ability to alter thiol-containing cellular com-ponents Glutathione is an abundant tripeptide (c-Glu-Cys-Gly) that is described as the first line of defence against cadmium in many cell types NMR experiments for structure and dynamics determination, molecular simulations, competition reactions for metal chelation by different metabo-lites (c-Glu-Cys-Gly, a-Glu-Cys-Gly and c-Glu-Cys) combined with bio-chemical and genetics experiments have been performed to propose a full description of bio-inorganic reactions occurring in the early steps of cad-mium detoxification processes Our results give unambiguous information about the spontaneous formation, under physiological conditions, of the Cd(GS)2 complex, about the nature of ligands involved in cadmium chela-tion by glutathione, and provide insights on the structures of Cd(GS)2 complexes in solution at different pH We also show that c-Glu-Cys, the precursor of glutathione, forms a stable complex with cadmium, but biological studies of the first steps of cadmium detoxification reveal that this complex does not seem to be relevant for this purpose
Abbreviations
GSH, glutathione reduced form; GSSG, glutathione oxidized form.
Trang 2mammalian liver cells, where it is present at millimolar
range concentrations [2,3] In vivo, it has a key role
in protecting cells against reactive oxygen species,
xenobiotics and heavy metals such as cadmium [4]
Glutathione exists in two forms: the antioxidant
reduced form conventionally called glutathione (GSH)
and its oxidized form known as glutathione disulfide
(GSSG) In vivo, the GSH⁄ GSSG ratio is in the range
of 20–100 depending on the cell type and growth
conditions [5,6]
Driven by this biological relevance, numerous
spec-troscopic studies of cadmium(II) complexes, in
particu-lar of simple thiol-containing ligands [7–11], have been
performed, revealing a large diversity of
cadmium–pep-tide interactions varying according to pH and metal
concentration [7,12–14] Nevertheless, the nature of the
metal binding in the case of GSH remains subject to
debate, as cadmium has been proposed to link to
amide [15], carboxylates of both glycine [13,14] and
glutamate residues [13,14,16] or the amine NH2 lone
pair [11,13,16].113Cd NMR [12] was used to
character-ize this interaction, but without success, in contrast to
many 113Cd–protein experiments [17–19] Simulation
of theoretical chemical shifts [20,21] or EXAFS
experi-ments [22] were also performed to analyse the
cad-mium(II) sphere of co-ordination From these studies,
it appears that the Cd(GS)2dimer is the major
biologi-cally active form of the complex [15,23], but it is not
necessarily the main stable form of the complex in
solution at neutral pH [7] Also, despite the high levels
of GSH in cells, the kinetics of the formation of
Cd(GS)2complexes at physiological pH (6.5–7.0 in the
cytosol and 6.0–6.5 in the vacuole) have not been
stud-ied Furthermore, it is not known whether glutathione
S-transferase activities are important for the formation
of the complex in vivo, as previously suggested [24–26]
Finally, cadmium detoxification in yeast cells is based
on export of Cd(GS)2 complexes outside the cell or
into the vacuole compartment These movements are
performed by ABC transporters, respectively Yor1p
[27] and Ycf1p (similar to human MRP1) [15,28]
Genetic data unambiguously indicate that the Ycf1p
vacuolar transporter has a more important role in
vac-uolar sequestration of cadmium compared with the
Yor1p transporter [27] A third efflux recently
described is also present in some yeast strains It
con-sists of a P1B-type ATPase able to directly expulse
Cd2+ions outside the cells [29]
Here we provide further insights into cadmium
com-plexation by metabolites We considered four
glutathi-one-related peptides, GSH, c-Glu-Cys, a-Glu-Cys-Gly
(a-GSH) and the free GSSG oxidized form Despite
the wide range of peptides considered for cadmium
chelation studies, c-Glu-Cys has never been studied, even though it is a precursor used by glutathione syn-thetase for c-GSH production Its study seems biologi-cally relevant as this metabolite is overproduced in yeast under cadmium stress conditions [3] Also, because its cellular concentration is in the range of that of c-GSH, it could compete with glutathione for cadmium chelation in the detoxification process [30] The choice of the synthetic peptide (a-GSH) was moti-vated by its ability to modulate cadmium complexa-tion Finally, because the thiolate group is strongly implicated in metal co-ordination, we also considered the free GSSG glutathione oxidized form as a reliable model to validate the solution structure refinement procedure Indeed, this molecule bearing a disulfide bridge leads to a global structure close to Cd(GS)2 where the cadmium is bridging sulfur atoms Because
of the absence of a definitive structural model of the Cd(GS)2 complex, we combined absolute distance determination using off-resonance ROESY experiments with molecular dynamics simulations and biochemical observations to provide insight into the solution struc-ture of GSH complexes of cadmium at different bio-logically relevant pH We also describe competitive experiments giving indications on the relative affinity
in vivoof these different natural peptides for cadmium The data suggest that the biological importance of Cd(GS)2 for detoxification is more driven by the selec-tivity of the transporter than by the stability of the complex
Results
Co-ordination of cadmium from NMR studies The chelation of cadmium by the thiol-containing pep-tides GSH, a-GSH and c-Glu-Cys (chemical structures are given in Fig 1) in aqueous solution and physiolog-ical pH can visually be observed and characterized by simple 1D NMR experiments Indeed, after the addi-tion of cadmium to the GSH sample in a 1 : 2
Cd⁄ GSH stoichiometry (0.5–5 mm solutions in our experiments), a white precipitate instantaneously formed and the solution became acidic Integration of NMR signals relative to a reference peak (CH2 of
l-glycine) indicated that the precipitate corresponded
to 10% at pH 6.4 to 20% at pH 7.2 of the total amount of GSH Notably, the precipitate was resolubi-lized after restoring the pH to a neutral value and shaking the sample These results were confirmed using radioactive 35S-GSH to quantify both precipitate and soluble forms of complexes (Table S1) Similarly, the addition of cadmium to the a-GSH sample resulted in
Trang 3the formation of a precipitate, but in this case, its
dis-solution was impossible, even after changing the pH,
vigorous shaking or sonication Furthermore, analysis
of NMR spectra indicated the precipitation of a 1 : 1
stoichiometric complex A simple pH-dependent
analy-sis of the electric charges indicated that because the
precipitated complex is necessarily neutral at this pH,
the cysteine residue should be in its thiolate S) form
Indeed, at neutral pH, a-GSH bears two carboxylate
groups (COO)) and the amino group of the glutamate
is protonated (NHþ3)
From these simple observations, information about
the co-ordination modes of a-GSH and GSH can be
deduced The transient precipitate observed in the case
of GSH should have a very similar complexation mode
to that of a-GSH, with a 1 : 1 stoichiometry and a
glo-bal charge of zero Restoring the pH to its initial value
allows this precipitated form to be transformed to the
more stable 1 : 2 complex [13,14,31,32] In both cases,
a-GSH and GSH peptides form a bidentate complex
with cadmium where the sulfur of the cysteine and the
carboxylate group of the glutamate or the glycine
resi-due are implicated These two carboxylate groups are
fully equivalent in terms of metal co-ordination
struc-tures Consequently, the difference observed in cadmium
chelation with a-GSH and GSH is due to the different location of the amino group of the glutamate residue
in both peptides The Cd(a-GSH) complex in 1 : 1 stoichiometry is structurally stable, whereas intermo-lecular interactions between GSH chains are necessary
to stabilize the 2 : 1 complex of GSH with cadmium Analyses of 1H 1D NMR spectra of GSH and c-Glu-Cys in the presence of cadmium are also very informative (Fig 1) and show that both Cd(GS)2 and Cd(c-Glu-Cys)2 complexes have common properties First, the broadening of both cysteine a and b proton resonances after cadmium addition to the sample sug-gests the existence of an exchange process involving the metal ion and the cysteine residue Second, we observed that the two cysteine b protons, which are equivalent in the absence of cadmium (only one chemi-cal shift in NMR spectra), are well differentiated (two chemical shifts) after metal addition [see 1D spectra for GSH (Fig S1) or 2D spectra for c-Glu-Cys (Fig S2)] This observation clearly indicates an asym-metry of the final complex due to metal co-ordination and represents a direct probe to follow cadmium chela-tion Finally, the dependence of NMR spectra on pH values is a way to probe the chemical structure of the complex In acidic conditions (pH = 5.6), the addition
GSH GSH + Cd GSSG
α-GSH
γ-EC + Cd
γ-EC
NH2E
NHG
NHC
β′ C / β′′ C
9.0 8.5 p.p.m 4.5 4.0 3.5 3.0 2.5 p.p.m.
Fig 1 1D1H NMR spectra of the different glutathione species Spectra were recorded in H 2 O at 280K and pH 7.2 From bottom to top, GSH, Cd(GS)2, GSSG, c-Glu-Cys, Cd(c-Glu-Cys)2and a-GSH The chemical structures from bottom to top of GSH, c-Glu-Cys and a-GSH are indicated on the right *Corresponds to impurities present in the aGSH sample # Indicates the resonance of the CH2group of L -glycine, which was used as a reference signal for peak integrations Arrows indicate characteristic resonances of cadmium chelation by GSH or c-Glu-Cys and of the oxidized form of glutathione Spectra were aligned with respect to L -glycine CH 2 resonance.
Trang 4of cadmium to the sample did not affect the amino
group resonance at 7.6 p.p.m of the GSH peptide
(Fig S3), indicating that this potential ligand is not
involved in metal chelation At neutral pH (pH = 6.4
and pH = 7.2), the exchange rate with the solvent of
amide protons (NH) of both cysteine and glycine as a
function of temperature was of the same order
(Fig S4), demonstrating that no amide deprotonation
occurs after cadmium chelation to GSH or c-Glu-Cys
peptides, as has been previously suggested for the
cysteine amide nitrogen [15]
The lability of cadmium bound to the GSH
mole-cules was assessed by 14N versus 15N glutathione
competition reactions (Fig 2) Chelation of cadmium
to the 15N-labelled GSH pool led to the formation of
the Cd(15N-GS)2 complex, which induced (despite a
partial signal overlap with the cysteinyl proton) the
disappearance of the glycine amide cross-peak on the
spectra After the addition of 14N-GSH in the same
proportion to the 15N-enriched GSH (1 : 1
stoichiom-etry), the glycine 15N-1H cross-peak was restored
This indicates the presence of free 15N-GSH peptide
The observed reappearance consequently resulted
from a chemical exchange process, in the 0.1–10 ms
range, between bound and unbound GSH molecules
to the cadmium
The relative affinity of cadmium to GSH and other peptides was explored by competitive complexation experiments, as shown in part of the TOCSY spectrum
in Fig S2 The quantification of NMR data allowed the evaluation of the chelation fraction: 42.5 ± 5.5 and 57.5 ± 5.5% for GSH and c-Glu-Cys, respec-tively These values clearly indicate the similar affinities
of both natural metabolites for cadmium
NMR structural models for Cd(GS)2and comparison with the GSSG model Because of the small molecular mass of GSH (307.5 Da), NOESY experiments are not appropriate to determine internuclear distances and ROESY-type experiments are also known to lead to quantification problems [33] To circumvent this major problem, we decided to use an alternative approach based on the off-resonance ROESY pulse sequence [34] This method allows the determina-tion of absolute internuclear distances and of local corre-lation times These parameters were used to build initial structural models of both GSSG and Cd(GS)2 com-plexes A refinement protocol with an explicit solvent was first performed on GSSG structures and then applied
to the Cd(GS)2complex The best structures (shown in Fig 3) were obtained, in agreement with the co-ordina-tion study, using the protonated N-terminal c-glutamate residue A total of 26 and 25 NMR constraints per monomer (GSH unit) were respectively used for GSSG and Cd(GS)2 structure determinations (Table 1) Sur-prisingly, strong differences were observed for the major-ity of the distances recorded for both molecules, despite their similar topological arrangement Because the exis-tence of a disulfide bond between the two glutathione units in the oxidized form cannot induce those striking variations, this suggests that the tridimensional organiza-tion of the Cd(GS)2 complex is very different from GSSG When the pH was varied between 6.4 and 7.2, sig-nificant differences for several distances were observed for the Cd(GS)2complex (distances marked in Table 1) Some of them were characterized as effects of inter-GSH interactions This interpretation resulted from sampling
of numerous conformations in molecular dynamic trajec-tories, which indicated that these NMR data could not
be due to interproton distances in a unique GSH unit of the dimer (Table S2) This was confirmed by bad refine-ment convergence of simulated annealing calculations parametrized only for intra-GSH distances, in agreement with the fact that we did not observe the 1 : 1 stoichiome-tric complex in the experimental conditions
The pairwise local correlation times (sc) extracted from off-resonance ROESY experiments were longer for Cd(GS)2 complexes ( 1.5 · 10)9s) than for oxidized
p.p.m.
p.p.m.
116
117
118
119
120
121
+ Cd
+ 14 N-GSH
8.1 8.2 8.3
8.4
Fig 2 Expanded contour plot of three superimposed 15 N- 1 H
heteronuclear single quantum coherence spectra: 15 N-GSH without
cadmium (red), after the addition of cadmium and the formation of
the Cd( 15 N-GS)2complex (green), after the addition of 14 N-GSH in a
1 : 1 stoichiometry with respect to 15 N-GSH (purple) Spectra were
recorded in H 2 O at 275 K and pH = 7.0 Because of the overlap of
the cross-peaks, the 2D spectra (green and purple) were shifted
towards the 15 N dimension (vertical axis).
Trang 5glutathione ( 0.4 · 10)9s) A detailed analysis of the
local correlation times revealed that pH changes mainly
affected inter-GSH unit glutamic acid⁄ glycine chain
interactions with an increase in correlation times for
higher pH revealing a greater complex rigidity
Cd(GS)2 structures determined by simulated
anneal-ing calculations were clustered into six families based on
minimal penalties induced from NMR distance
restraints 3D models representative of each
conforma-tional family and calculated for pH = 6.4 or pH = 7.2
are presented in Fig 3 and coordinates are given in
Tables S3–S4 Those of GSSG model are given in Table
S5 Our model clearly indicates that between zero and
two water molecules are present and complete the
cad-mium co-ordination sphere This number depends on
the pH value and the cadmium charge, which varies
from 0 to +2.0 in our calculations Despite these
varia-tions, the final models are very similar The pH value
mainly influences the interactions between the two GSH
units, whereas the presence of a positively charged
cadmium clearly favours the bonding of the glycine
carboxylate group It should be noted that symmetric
structural models are severely penalized because of their
deviation from imposed NMR restraints
Discussion
Cadmium glutathione complexes
When cadmium is complexed by glutathione, different
species exist in solution in equilibrium: a mixture of the
Cd(GS)2 1 : 2 complex and the Cd(GS) 1 : 1 complex
A recent study [7] suggested that the 1 : 1 monochelate
is one of the major complexes formed at low glutathi-one concentration On the other hand, at physiological
pH and higher glutathione concentration, which are the relevant in cellulo conditions, the 1 : 2 complex is pre-dominant, as shown by speciation studies [7,14] As a consequence, in this work, we focused on the Cd(GS)2 complex, as apart from its biological relevance, it is the transport-active complex [15] To this end, we per-formed NMR experiments at the optimal conditions for the formation of the Cd(GS)2complex, considering that the 1 : 1 complex that precipitates is almost totally transformed into the water-soluble Cd(GS)2 1 : 2 com-plex after restoring the pH and shaking Consequently,
as shown by NMR, we observed only the major and the most stable soluble Cd(GS)2 complexes formed under the conditions of the study (pH 6.4 and 7.2, a temperature of 17C, concentration over 1 mm and at
a favourable 1 : 2 stoichiometry) Eventual disturbing effects on spectra arising from additional minor species could not be excluded, but the absence of such NMR spectral signatures substantiates the assumption that these forms are negligible in our experimental condi-tions Finally, the characterization of unambiguous inter-GSH unit cross-peaks was also in accordance with the hypothesis of the predominance of the dimeric form
in solution On the other hand, interproton distance variations observed after pH changes may result from local conformational changes in the dimeric Cd(GS)2 complex
88%
12%
66%
71%
25%
59%
Fig 3 Representation of the best 3D con-formational families obtained for Cd(GS)2 NMR-refined structures at pH = 6.4 and
pH = 7.2 The first two rows present two different rotated views of the major confor-mations (population in %) obtained for the Cd(GS) 2 complex model refinement at both
pH 6.4 and 7.2 and using noncharged cadmium The bottom row presents two views of the major conformation of refine-ments carried out with a +1 charged cadmium In these structures, the sphere of co-ordination of cadmium (green sphere) is completed with two water molecules in the case of the Cd(GS)2models with
noncharged metal For models with charged cadmium there is one water molecule in the case of pH = 6.4 (bottom left) and no water molecule for pH = 7.2 (bottom right).
Trang 6Other cadmium sulfur–metabolite complexes
NMR experiments performed on complexation of
cad-mium with other sulfur metabolites revealed some
characteristics of their chelation with glutathione
First, at biologically relevant concentrations, GSSG
does not chelate cadmium, demonstrating that the
thiol group is essential Second, a-GSH and c-GSH
have different co-ordination modes with cadmium
(1 : 1 stoichiometry for Cd⁄ a-GSH with implication of
glutamate and 1 : 2 stoichiometry for Cd⁄ c-GSH)
showing that c-glutamate is not implicated in the
che-lation of GSH Third, GSH and c-Glu-Cys have the
same co-ordination mode for cadmium with nearly the
same affinity, indicating that the carboxylate group of
glycine is not a key cadmium ligand Finally, NMR
experiments demonstrated unambiguously that the
chelation is a spontaneous and rapid (millisecond time scale) phenomenon
Dynamic co-ordination effects
In the complex formed in aqueous solution with both glutathione and c-Glu-Cys at biologically relevant con-centrations and pH, cadmium is mainly linked by thiols from two distinct GSH or c-Glu-Cys units Cysteine sulfur affinity for cadmium provides a strong anchoring site for glutathione derivatives [8–10,13] Nevertheless,
we observed during our14N-GSH versus15N-GSH com-petition experiments, a significant lability of cadmium, suggesting that we need to reconsider the strength of cadmium chelation by glutathione in a biological con-text Based on the disappearance of the1H-15N cysteine resonance, this exchange rate typically occurs in the millisecond time range This phenomenon could explain our inability to directly observe the 113Cd resonance at high magnetic field (data not shown) An obvious con-clusion of the in vitro part of the present study is that the formation of Cd(GS)2 complexes is spontane-ous and rapid in the tested conditions, which were close
to physiological (ambient temperature, pH tested from 5.6 to 7.2, GSH concentration in the millimolar range) Consequently, it is unlikely that glutathione S-transfer-ases are required to catalyse complex formation [24–26]
In our structural models, cadmium always has a tet-rahedral co-ordination sphere in which two ligands are thiolate groups Precipitation observations and NMR hydrogen exchange data between amide protons of GSH and bulk water show that cadmium ligation by nitrogen (amino group of glutamic acid or amide of cysteine) does not seem to occur, conversely to what has been previously proposed for cadmium [15] as derived from zinc studies [35] This was confirmed by numerical simulations, which never led to a structure where nitrogen was involved in cadmium complexation Moreover, it is in agreement with the pKa measured for the amino group of the N-terminal c-glutamate resi-due (9.42–9.48 from references [14,16]) In most of our structural models, metal completes its co-ordination with two water molecules Furthermore, at neutral pH, for the best calculated NMR structures, no carboxylate group is involved Our results demonstrate that the complexation of cadmium by glutathione primarily involves the deprotonated sulfhydryl groups from cysteine residues and two water molecules
Dimerization effects Although cadmium is only complexed through the thio-late groups, the relevance of glutathione for cadmium
Table 1 NMR interproton distances Distances were calculated
from build-up curves measured at different h angles for oxidized
glutathione GSSG (pH = 7.0) and Cd(c-GS)2complexes at pH = 6.4
and 7.2, respectively.
Glutathione state
GSSG
pH = 7.0
Cd(c-GS)2
pH = 6.4
Cd(c-GS)2
pH = 7.2 Interproton distances r (A ˚ ) ± 0.7 r (A ˚ ) ± 0.2 r (A ˚ ) ± 0.3
observed a
3.2 a
observed
Not observed
observed a
4.0 a,b
a Significant distance differences when pH varied from 6.4 to 7.2
for Cd(GS)2 b Inter-GSH unit distance for the Cd(c-GS)2complexes.
Trang 7detoxification clearly depends on the whole structure
and not just the co-ordination modes Indeed, strong
interactions between two GSH units are directly
observed on the off-resonance ROESY spectra, leading
to clear structural constraints Based on most
struc-tural models of the dimer complex, the driving force
for these peptide interactions seems to involve
gluta-mate side chains of both GSH units, which form a
hydrophobic core and electrostatic interactions
between glycine and glutamate side chains Under
these conditions, the atypical c-configuration of the
N-terminal glutamate seems to lock the structure of
the Cd(GS)2 complex and decrease the accessibility to
the metal on one side This breaks the symmetry of the
complex, explaining 1D NMR spectral modifications
after metal addition
Off-resonance ROESY experiments also provided a
reliable description of flexibility occurring in the
com-plex Pairwise correlation times collected for Cd(GS)2
complexes are much longer (about 1.5 ns) than those
derived from GSSG (0.4 ns) Even if this is partially
expected due to the increase in the relative molecular
mass, the observed difference, almost a factor of 4,
cannot be ascribed to this sole effect This result thus
reveals the appearance of a significant structural
rigidi-fication after metal co-ordination Furthermore, local
correlation times also confirmed that the side chain of
glutamate is more rigid in Cd(GS)2compared with GSSG,
substantiating the previous comment on the importance
of the GSH side chains in dimer stabilization
Cadmium–glutathione complex and the
detoxification process
The protonation state of the cadmium–glutathione
com-plex is strongly dependent on the pH, which can
signifi-cantly differ in the different subcellular compartments
Intracellular pH values are in the range from 6.5 to 7.2
in the cytosol and from 6.0 to 6.5 in the vacuole [36–38]
The complex should thus be stabilized in the cytosol,
favouring specific recognition and efficient transport by
Ycf1p In vacuolar acidic conditions, the equilibria
should be displaced to protonated forms with
enhance-ment of inter-GSH interactions and destabilization of
Cd(GS)2leading to possible ligand substitution In this
schema, thiolate reprotonation could be the first
chemi-cal event in the cadmium releasing process by
glutathi-one and so a key step in the detoxification process
The competition experiment showing similar
effi-ciency in the formation of Cd(GS)2 and
Cd(c-Glu-Cys)2 complexes suggests that the latter complex can
also be formed in vivo, as c-Glu-Cys pools can reach
high concentrations in the range of GSH levels
following cadmium exposure [3] In addition, the het-erologous complex involving the two metabolites Cd(GS)(c-Glu-Cys) is also expected Interestingly the mutant strain Dgsh2, devoid of glutathione synthase activity and unable to produce glutathione, accumu-lates c-Glu-Cys at high intracellular levels (Fig 4) This strain has a high chelating capacity, demon-strated by a global level of free thiols (GSH + c-Glu-Cys) higher than the wild-type (Fig 4A) In addition, although our data indicate that Cd(c-Glu-Cys)2 complexes are efficiently formed, this strain was shown to be hypersensitive to cadmium [39] This phenotype suggests an impaired detoxification of cadmium in this strain due to a decreased rate of transport of Cd(c-Glu-Cys)2 compared with Cd(GS)2 complexes This defect may concern the transport into the vacuole through Ycf1 or the export outside the cell through Yor1 or both Using wild-type cells labelled with 35S-GSH, we observed that the total export of glutathione [including free GSH and Cd(GS)2 complexes] outside the cells is not significant (5.9–8.7% in cells treated for 3 h with 0.1 mm cad-mium compared with 4.4% in untreated cells; Table 2) This very low level of Cd(GS)2 export is consistent with the very slight cadmium-sensitive phe-notype of the yor1D strain [27] Thus, considering the low contribution of Cd(GS)2 complex export to cad-mium resistance, we assume that the gsh2D pheno-type is caused by a low efficiency transport into the vacuole of Cd(c-Glu-Cys)2 compared with Cd(GS)2 complexes Consistent with this interpretation is the observation that, even under standard conditions, c-Glu-Cys is far less efficiently transported than GSH into purified vacuoles overexpressing YCF1 (M Lazard
γ-Glu-Cys
0 5 10 15 20
WT Dgsh2
GSH
0 5 10 15
WT
A
B
Fig 4 c-Glu-Cys concentration is strongly increased in Dgsh2 cells (A) Wild-type and Dgsh2 cells grown in minimum medium supple-mented with 400 l M glutathione were treated with 200 l M cad-mium for 3 h The intracellular metabolites were extracted and analysed by LC ⁄ MS as previously described [3] (B) Representation
of the glutathione biosynthesis pathway Gsh1, c-glutamyl-cysteine synthetase; Gsh2, glutathione synthetase.
Trang 8& P Plateau, personal communication) The absence
of cadmium co-ordination via carboxylate groups of
glycine residues in structural models supports the idea
that detoxification differences observed for GSH and
c-Glu-Cys only occur at the recognition step of
cad-mium–metabolite complexes by the transporter We
thus suggest that the glycine residue may be involved
in metal-complex recognition by the Ycf1p transporter
and not in direct interaction with cadmium
The transporter Ycf1 is a key element in cadmium
detoxification, as shown by the cadmium-sensitive
phenotype of the ycf1D mutant strain and the
cad-mium-resistant phenotype of strains overexpressing
YCF1 [40] Our data suggest that under physiological
conditions, the formation of Cd(GS)2 complexes is
spontaneous and should not constitute a bottleneck in
the detoxification process The next steps, transport
into the vacuole and the metabolism of the complexes
in the vacuole, remain to be fully understood
Experimental procedures
Strains and culture conditions
The Saccharomyces cerevisiae strain used for the production
of15N-GSH was S288C (Mata SUC2 mal mel gal2 CUP1)
The strain used for the production of 15N-c-Glu-Cys was
Dgsh2 previously constructed in the BY4742 genetic
back-ground (MATa, ura3D0, his3D1, lys2D0, leu2D0) [41] by
EUROSCARF (Intitute of Molecular Biosciences, Johann
Wolfgang Goethe-University Frankfurt, Germany) This mutant
has the KanMX4 marker inserted into the GSH2 locus
Cells were grown at 30C in minimal yeast nitrogen base
medium (0.67%) supplemented with 2% glucose as a
carbon source and with auxotrophic requirements (uracil,
histidine, lysine, leucine and 30 lm glutathione) when
nec-essary (strain Dgsh2) The standard yeast nitrogen base
medium contains 30 mm 14N-ammonium sulfate For 15N
labelling, 30 mm 14N-ammonium sulfate was replaced by
10 mm 15N-ammonium sulfate (Eurisotop, Gif-sur-Yvette,
France) as the sole source of nitrogen
Preparation of15N-enriched metabolites
After growth for at least 25 generations in15N-ammonium
sulfate, the cell culture (400 mL corresponding to
1010
cells) was treated with 50 lm Cd2+ to induce an overproduction of15N-GSH [3] After 4 h of treatment, the cells were collected by centrifugation, washed quickly with cold water and resuspended in 3 mL of 0.1% perchloric acid Cells were transferred to boiling water for 5 min, cen-trifuged and the supernatant was collected This extract contained soluble yeast metabolites, including 15N-GSH (S288C strain) and15N- c-Glu-Cys (Dgsh2 strain)
The extracts containing 15N-GSH and 15N-c-Glu-Cys were purified on a carbohydrate analysis column (4.6· 250 mm, 5 lm) from Waters (Saint-Quentin en Yvelines, France) Chromatographic separations were performed using a Surveyor pump and a Surveyor auto-sampler (ThermoFisher Scientifics, Les Ulis, France), under isocratic conditions with a flow rate of 0.8 mLÆmin)1 The mobile phase consisted of water containing acetic acid at 0.4% The effluent from the liquid chromatography was split by a factor of 1⁄ 20 before its introduction into the
MS ESI MS was performed using an LCQ-Duo ion trap
MS fitted with an electrospray source (ThermoFisher Scientifics) operated in the positive mode The mass spec-trometer was operated with the capillary temperature at
250C, sheath gas at 80 (arbitrary units) and the auxiliary gas at 20 (arbitrary units) The target was fixed at 2· 107 ions and the automatic gain control was turned on The electrospray voltage was 4.5 kV, the capillary voltage 10.6 V and the tube lens offset )6 V The injection time was 50 ms MS were recorded at unit mass resolution with-out in-source fragmentation using the single ion recording detection mode The signals for 15N-GSH and 15N- c-Glu-Cys were monitored at m⁄ z 311 and 253, with retention times of 23 and 40 min, respectively The fractions corre-sponding to these retention time ranges were collected and finally lyophilized before NMR experiments
Sample preparation
In the case of samples used for distance extraction, chelation
or competition reaction experiments, GSH or GSSG were dissolved in 500 mL (90% H2O 10% D2O) resulting in 1 mm minimal ionic strength samples, with a final 1 : 2 Cd⁄ GSH stoichiometry To decrease oxidation processes, all samples were sealed after bubbling with dry nitrogen gas for a few minutes Classical peptides were purchased from Sigma-Aldrich (St Louis, MO, USA) and a-GSH was synthesized and purified for NMR quality by Eurogentec (Seraing, Belgium)
NMR spectroscopy All NMR experiments were performed on Bruker Advance DRX spectrometers (Bruker, Ettlingen, Germany) 1D and 2D 1H spectra in H2O were recorded at 500 MHz by using
a Watergate [42] or an excitation sculpting sequence [43] to suppress the water signal Peak assignments were carried out using classical techniques, in particular for proton
Table 2 Total export of 35 S-glutathione by wild-type cells The
values reported in the Table are the ratio S ⁄ T (see Radioactive
experiments section in Experimental procedures).
Trang 9resonances through TOCSY and off-resonance ROESY
experiments Our attribution agreed with those obtained in
other studies [16,44] l-glycine at a final concentration of
2.5 mm was added to the sample before recording 1D
NMR spectra because the CH2resonance was not affected
by the addition of cadmium and this resonance did not
overlap with the other signals (see Fig 1) It was used as
an internal reference for peak integration and species
quantification
Proton–proton distances were extracted from
off-reso-nance ROESY build-up curves using a procedure already
described [45] The off-resonance ROESY pulse sequence
[46] was adapted for the excitation sculpting water
suppres-sion method Seven h angles between the effective and static
magnetic field directions (5, 15, 25, 35, 45 and 54.7) and
six mixing times (25, 50, 75, 100, 150 and 200 ms) were
used For metal-reduced glutathione, the spectra were
recorded at three different pH values: 5.6, 6.4 and 7.2 For
the samples with c-Glu-Cys and metal-free oxidized
gluta-thione, the experiments were performed at pH = 7.0 All
off-resonance ROESY spectra were collected at 500 MHz
at 274.3K with TXI or BBI probes
1
H,15N-heteronuclear single quantum coherence
experi-ments were carried out using gradient coherence selection
and sensitivity enhancement Backbone 15N amide
reso-nances were observed on a 600 MHz spectrometer equipped
with a TCI cryoprobe (Bruker) Natural abundance 13C
NMR experiments were also performed, using
heteronucle-ar multiple bond correlation sequences and a TCI
cryo-probe All chemical shifts for 1H were referenced to an
internal TSP signal
Molecular modelling
Molecular mechanics calculations and molecular dynamic
simulation methods were used for model construction using
the amber 9 suite programs Parameters for the
c-gluta-mate residue were developed from the Gaussian03 DFT
charge calculations method and adapted to the Parm99
force-field [47] using the Resp module and a standard
charge fitting protocol Both protonated and
nonprotonat-ed states for the amino group of the c-glutamate residue
were implemented and simulated Cadmium was considered
as a hard sphere with a modulated charge varying between
0.0 and +2.0 and a Cd-S distance of 2.46 A˚ [8] Other
main parameters for angles and dihedrals were adapted
from Amber force-field data previously depicted for zinc
ion in the four-cysteine tetrahedral environment [8,48] An
explicit solvation model (TIP3P water model and +1.0
dielectric constant) was used in all simulations Structure
refinements were performed using NMR interproton
dis-tances as restraints implemented as harmonic functions into
a simulated annealing protocol with 5000 final structures
collected Free and restraint molecular dynamic simulations
were used to analyse characteristic key distances for
co-ordination-type discrimination Cd-N (2.3 ± 0.1 A˚ [8])
or C-O (2.2 ± 0.1 A˚ [49]) restraint distances were imposed between cadmium and potential ligand atoms during the production period Structures obtained from simulated annealing calculations were sorted and divided into homog-enous conformational groups leading to the best NMR refined models
Radioactive experiments Cells (3 ml at D = 0.4) grown in minimum medium were labelled with 2 lCi of 35S-GSH (PerkinElmer) for 40 min
at 30C Cells were washed and re-suspended in the same medium (with or without 100 lm cadmium) Total
35
S-GSH pools were counted (T) After 3h incubation, the cultures were centrifuged and the amount of radioactivity present in the supernatant was measured (S)
Acknowledgements
This work was supported by the Commissariat a` l’Energie Atomique (grant from the Programme de Toxicologie Nucle´aire Environnementale) We thank
R Genet for kindly providing 15N-ammonium sulfate
We thank Dr Carl Mann for careful reading and helpful comments on the manuscript
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