Affinity of S100A1 protein for calcium increases
dramatically upon glutathionylation
Graz_ yna Goch, Sergiusz Vdovenko, Hanna Kozłowska and Andrzej Bierzyn
˜
ski
Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Poland
Calcium ions are one of the most important messen-
gers and regulate numerous vital biological processes.
A crucial role in calcium signal transduction is played
by EF-hand proteins, which upon incorporating cal-
cium change their conformation, exposing hydrophobic
patches to which target proteins bind.
S100 is a subfamily of EF-hand proteins regulating
an amazingly wide variety of biological processes in
either a calcium-dependent or calcium-independent
manner [1–3]. A typical S100 protein is composed of
two subunits, very strongly associated with each other,
and each containing two calcium-binding loops [4–9].
The glutamate residue at the C-terminal position in
both loops plays a crucial role in calcium binding.
To elucidate the calcium-dependent biological activit-
ies of S100 proteins it is of the utmost importance that
their microscopic calcium-binding constants be deter-
mined at physiological conditions. The results of this
study clearly illustrate this point. Only for calbindin D
9k
have such measurements been made [10–12]. These
results, although important, are of limited value in
understanding the calcium-binding mechanism typical
of S100 proteins because of the unique structural fea-
tures of calbindin D

3
m
)1
(C-loops) and K
2
 10
2
m
)1
(N-loops). Only when both
loops are saturated with calcium does the protein change its global confor-
mation, exposing to the solvent hydrophobic patches, which can be detec-
ted by 2-p -toluidinylnaphthalene-6-sulfonic acid – a fluorescent probe of
protein-surface hydrophobicity. S-Glutathionylation of the single cysteine
residue (85) of the a subunits leads to a 10-fold increase in the affinity of
the protein C-loops for calcium and an enormous – four orders of magni-
tude – increase in the calcium-binding constants of its N-loops, owing to a
cooperativity effect corresponding to DDG ¼ )6 ± 1 kcalÆmol
)1
. A similar
effect is observed upon formation of the mixed disulfide with cysteine and
2-mercaptoethanol. The glutathionylated protein binds TRTK-12 peptide
in a calcium-dependent manner. S100A1 protein can act, therefore, as a
linker between the calcium and redox signalling pathways.
Abbreviations
Br
2
-BAPTA, 5,5¢-dibromo-1,2-bis(o-aminophenoxy)-ethane-N,N,N¢,N¢-tetraacetic acid; 5-NBAPTA, 5-nitro-1,2-bis(o-aminophenoxy)-ethane-
N,N,N¢,N¢-tetraacetic acid; TNS, 2-p-toluidinylnaphthalene-6-sulfonic acid.
FEBS Journal 272 (2005) 2557–2565 ª 2005 FEBS 2557

Results
Fluorescence properties of S100A1, its mutants
and derivatives
The fluorescence spectrum of S100A1 is dominated by
the fluorescence of the single tryptophan residues in its
subunits (Trp90) with the maximum at 346 nm. For all
oxidized forms of the protein a 4 nm batochromic shift
in fluorescence is observed. Fluorescence quantum
yields of all apo species are listed in Table 1. They are
affected in various ways by coordination with metals
(Fig. 1). Neither the E32 fi Q (Table 1) mutation nor
E73 fi Q (data not shown) has a measurable effect on
the fluorescence signal intensities of either apo S100A1
protein or its mixed disulfides with 2-mercaptoethanol
and glutathione.
Calcium binding to the reduced and oxidized
forms of the E32Q mutant
The fluorescence signal of E32Q (S100A1 mutant with
a nonactive N-binding loop) increases in the presence
of Ca
2+
ions. Its titration curve (Fig. 2) can be des-
cribed [17] using a simple model assuming that quite
independently, i.e. without any cooperativity effects,
each a subunit of the mutated protein binds only
one calcium ion with the binding constant K
1
¼
Table 1. Fluorescence efficiency U, relative changes in fluorescence signals after binding of the first (f
1

3
1.17 ± 0.03 60 ± 40 1.51 ± 0.04
S100A1–2-mercaptoethanol 0.032 7.6 ± 1.4 · 10
4
1.03 ± 0.03 3 ± 1 · 10
4
1.55 ± 0.02
S100A1–glutathione 0.065 1.1 ± 0.2 · 10
5
0.88 ± 0.03 7 ± 3 · 10
5
0.60 ± 0.03
S100A1–cysteine 0.052 7 ± 2 · 10
4
0.91 ± 0.02 1.2 ± 0.2 · 10
6
0.63 ± 0.02
Fig. 1. Calcium titration curves for fluorescence signals of S100A1
(black) and its disulfides: S100A1–2-mercaptoethanol (red),
S100A1–glutathione (green) and S100A1–cysteine (yellow). In all
cases the protein concentration was 8 l
M. Interpolation curves
have been calculated as described in the text, using the parameters
listed in Table 1.
S100A1 affinity for calcium G. Goch et al.
2558 FEBS Journal 272 (2005) 2557–2565 ª 2005 FEBS
4±2· 10
3
m
)1

glutathione and glutathionylated native S100A1
(Table 1). Evidently, the second calcium ion is still
coordinated by the N-binding loop of the glutathionyl-
ated a subunit of E32Q, despite the Glu32 fi Gln
mutation, although the binding capacity is reduced by
a few orders of magnitude.
Calcium binding to the E73Q mutant and
its oxidized forms
The fluorescence signal of the E73Q mutant changes
only at very high CaCl
2
concentrations (data not
shown). Experiments with 5-nitro-1,2-bis(o-aminophen-
oxy)-ethane-N,N,N¢,N¢-tetraacetic acid (5-NBAPTA) as
a calcium chelator also show that the affinity of the
N-loop for calcium is very low. Both E73Q and
E73Q)2-mercaptoethanol bind calcium with binding
constants not exceeding 10
2
m
)1
, too low to be deter-
mined more precisely using 5-NBAPTA chelator with
aCa
2+
binding constant of the order of 10
4
m
)1
.

subunits of the S100A1 protein. Nevertheless, because
formation of mixed disulfide between the subunits of
E73Q and 2-mercaptoethanol does not affect the affin-
ity of the protein N-loops for calcium, it can be safely
assumed that calcium binding to the N-loops of the
glutathionylated protein can be described by micro-
scopic constants of the order of 10
2
m
)1
as determined
for the reduced protein.
Calcium binding to native S100A1 protein
and its derivatives
The microscopic binding constants to the C-loop of
the S100A1 protein and its oxidized forms, as deter-
mined from studies of the E32Q mutant and its deri-
vatives (K
1
values listed in Table 1), are at least two
orders of magnitude greater than the values for the
N-loops (K
N
) of S100A1 and its derivatives, as evalu-
ated from studies of the E73Q mutant and its mixed
disulfides ( 10
2
m
)1
). This means that the subunits

glutathione and S100A1–cysteine only one inflexion is
observed, although the data analysis clearly shows that
each of these molecules coordinates two calcium ions.
Evidently, the values of K
1
and K
2
are quite similar,
so that determination of all four parameters K
1
, f
1
, K
2
and f
2
using a curve-fitting procedure is not possible.
Therefore, we used K
1
and f
1
parameters found previ-
ously from the studies of E32Q mutant and its deriva-
tives and allowed them to change only within the error
limits of their determination (Table 1) during the fit-
ting procedure of K
2
and f
2
. The best-fit parameters

acid (TNS) fluorescence increases dramatically upon
binding to hydrophobic patches exposed on protein
surfaces [18]. Therefore, TNS is frequently used to
monitor protein conformational transitions accompan-
ied by changes in the hydrophobic area exposed to
water.
Using a TNS probe such a conformational transition
induced by calcium was observed in the S100A1 pro-
tein by Leung et al. [19]. We obtained similar results,
although a strict, quantitative comparison is not pos-
sible because our experiments were carried out at
somewhat different pH and ionic strength.
In calcium-free solutions, TNS fluorescence in the
presence of S100A1 protein and its Cys85–glutathione
derivative is very weak, with the maximum at 420 nm
(Fig. 3). At 200 mm calcium concentration, when the
S100A1 protein is almost completely saturated with
metal ions (Fig. 3B) the maximum of TNS fluorescence
shifts to 440 nm and its intensity increases about four
times (Fig. 3A). Similar effects on TNS fluorescence
are observed when S100A1–glutathione is fully satur-
ated with calcium at a Ca
2+
concentration of 60 lm,
although the increase in the fluorescence signal is smal-
ler (Fig. 3C).
Because calcium binding to C- and N-loops of the
a subunits of S100A1 is not cooperative and the
AB
CD

calcium-dependent binding to S100A1 [1,20,21]. There-
fore, it is commonly used as a convenient probe for
calcium-induced biological activity in this protein.
In the absence of calcium, the fluorescence signal of
an equimolar mixture of 2 lm S100A1–glutathione and
TRTK-12 is equal (see Experimental procedures),
within the limits of error (2%), to the sum of the sig-
nals of each component. In the presence of 200 lm
Ca
2+
, the fluorescence of the protein–peptide mixture
is reduced by 20% relative to the sum of the fluores-
cence signals for TRTK-12 and the protein as meas-
ured separately in the presence of calcium. This proves
that the molecules interact with each other.
Discussion
Under physiological conditions (pH ¼ 7.2, 100 mm
NaCl) unmodified S100A1 protein coordinates calcium
via the C-loops of its subunits, with a binding constant
of K
1
¼ 4±2· 10
3
M
)1
. The N-loops of the protein
bind calcium very weakly, with K
2
values close to the
microscopic binding constant determined from studies

cofactor(s) must be involved in the induction of cal-
cium-dependent intracellular activity of S100A1. Such
a cofactor would need to fulfil the following require-
ments: (a) It should increase the affinity of S100A1
for calcium. (b) Its interaction with S100A1 must not
lead to similar conformational changes as those
induced by calcium coordination. Otherwise, it would
replace, and therefore eliminate, calcium from the sig-
nal pathway because it would keep the protein in the
active conformation even in the absence of calcium.
(c) Calcium-saturated S100A1 protein modified by a
cofactor must preserve its ability to bind target
proteins.
Our results indicate that glutathionylation conforms
to all these requirements. The affinity of S100A1 pro-
tein for calcium is dramatically enhanced when the SH
groups of the cysteine residues of its subunits (Cys85)
are linked covalently to glutathione: the Ca
2+
-binding
constant for C-loops increases  10-fold and that for
N-loops increases by as much as four orders of magni-
tude. The glutathionylated protein binds TRTK-12
peptide in a calcium-dependent manner.
A regulatory role of S-glutathionylation has been
demonstrated for a number of proteins. It is postulated
[26,27] that this reversible protein modification, con-
trolled by the intracellular redox potential and enzy-
matic cleavage of S-S bonds, as well as by reactive
oxygen and nitrogen species, plays a crucial role in the

all, of these proteins for calcium may also be regulated
by post-translational modification of this residue.
The mechanism by which mixed disulfide formation
by Cys85 leads to an increase in the affinity of S100A1
for calcium does not seem to be related to the intro-
duction of some functional groups arranged in space
in any specific manner. Despite the different structure
and number of its carboxyl groups (one, instead of
two) cysteine appears to be an excellent substitute for
glutathione. Even the 2-mercaptoethanol molecule,
devoid of any charged groups, has a similar although
somewhat smaller effect, probably because of its small
size. A large increase in macroscopic Ca
2+
-binding
constants to S100A1 was observed by Baudier et al.as
a result of protein labelling with monobromo(trimethyl-
ammonio)bimane [30].
Remarkably, experiments with E73Q–glutathione
indicate that the microscopic binding constant K
N
does
not change, within the margins of error, and remains
low. Therefore, the tremendous increase in the affinity
of the N-loops for calcium upon protein glutathionyla-
tion is due to the appearance of a large cooperativity
effect, corresponding to Gibbs’ free energy determined
by the ratio of microscopic (K
N
) to macroscopic (K

ently, in a noncooperative way. All our data conform
to this model. It seems, therefore, that the protein
subunits do not exchange any signals regarding their
conformational status. This observation is substan-
tiated by comparative NMR studies of the met and
apo forms of S100B protein [8]. The structure of the
interface between the protein subunits has been
shown to be unaffected by metal binding. Apparently,
it provides a barrier for propagation of calcium-
induced conformational changes from one subunit to
its neighbour.
Experimental procedures
Expression and purification of proteins and
TRTK-12 peptide
S100A1 protein and its mutants were expressed as described
previously [31]. The synthetic gene coding for the bovine
S100a subunit was constructed and cloned into a derivative
of pAED4 plasmid. Genes coding for Glu32 fi Gln and
Glu73 fi Gln mutants of S100a were obtained by site-
directed mutagenesis. The genes were expressed in Escheri-
chia coli utilizing the T7 expression system. The expression
products were isolated using a phenyl–Sepharose column,
purified by reverse-phase HPLC on a semi-preparative
Vydac C
18
column, and identified by the ESI-MS using a
Macromass Q-Tof spectrometer (supplementary Table S1).
Two forms of the proteins: (a) with sequences strictly
corresponding to the respective gene sequences, and (b)
containing the additional initiator methionine at the N-ter-

measured molecular masses is given (supplementary
Table S1). It was checked, using HPLC and MS, that each
derivative could be reduced to the respective original pro-
tein by short incubation with 1 mm dithiothreitol at pH 8.
S100A1 affinity for calcium G. Goch et al.
2562 FEBS Journal 272 (2005) 2557–2565 ª 2005 FEBS
Protein samples
Tris buffer (20 M M), pH 7.2, containing 100 mm NaCl in
MQ water filtered through a Chelex column was used as
the solvent in all experiments. All protein solutions used in
the fluorescence titration experiments were checked for
possible calcium contamination by comparing the fluores-
cence signals of samples measured in the presence and
absence of EDTA. If the difference exceeded 1% the solu-
tion was not used.
Protein stock solutions of  80 lm a subunits were centri-
fuged and stored for no longer than 2 weeks before experi-
ments. Concentrations of the native a subunit, its mutants
and their derivatives were determined from UV absorp-
tion at 280 nm using a molar extinction coefficient of
9300 m
)1
cm
)1
[33]. The absorption spectra were measured
on a Cary 3E spectrophotometer (Varian International AG,
Zug, Switzerland) in thermostated cells of 10 mm path
length. All measurements were made at 25 °C.
Fluorescence measurements
For fluorescence titration experiments we used an appar-

bromo-1,2-bis(o-aminophenoxy)-ethane-N,N,N¢,N¢-tetrraacetic
acid (Br
2
-BAPTA). Both chelators were purchased from
Molecular Probes (Leiden, the Netherlands).
The chelator concentrations were determined by the
absorbance in the presence of excess calcium using the
following molar extinction coefficients: e
340
¼ 6.0 ·
10
3
m
)1
Æcm
)1
and e
239.5
¼ 1.6 · 10
4
m
)1
Æcm
)1
for 5-NBAPTA
[36] and Br
2
-BAPTA [37], respectively. Two-millilitre sam-
ples of 80 lm a subunits and 20 lm of 5-NBAPTA or of
equimolar concentrations ( 25 l m) of protein subunits and

´
rski for his contribution to the discussion
of our results. This study was supported by the Polish
Committee for Scientific Research Grant 6 P04 009 16.
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