(Received for publication, March 31, 1995; and in revised form, June 26, 1995)
From the
The calcium-binding protein S100A3 is an unusual member of the
S100 family, characterized by its very high content of Cys. In order to
study the biochemical, cation-binding, and conformational properties,
we produced and purified the recombinant human protein in Escherichia coli. The recombinant protein forms noncovalent
homodimers, tetramers, and polymers in vitro with a subunit
molecular weight of 11,712. The Zn-binding parameters
of S100A3 were studied by equilibrium gel filtration and yielded a
stoichiometry of four Zn
per monomer with a
[Zn
]
of 11 µM and a Hill coefficient of 1.4 at physiological ionic strength. The
affinity for Ca
is too low to be determined by direct
methods (K
> 10 mM).
Ca
- and Zn
-binding can be followed
by optical methods: the Trp-45 fluorescence is high in the metal-free
form and addition of Zn
and Ca
, but
not of Mg
, leads to a 4-fold quenching.
Ca
and Zn
promote also quite
similar conformational changes in the Tyr and Trp environment as
monitored by difference spectrophotometry. Fluorescence titrations with
Zn
confirmed that there is one set of high affinity
binding sites with a [Zn
]
of
8 µM and a Hill coefficient of 1.3. Binding of
Zn
to a second set of low affinity sites induces
protein precipitation. Fluorescence titrations with Ca
confirmed the very low affinity of S100A3 for this ion with a
[Ca
]
of 30 mM and
slight negative cooperativity. Mg
has no effect on
this binding curve. Of the 10 Cys residues in S100A3, 5 only are free
thiols, and accessible to 5,5`-dithiobis(2-nitrobenzoic acid); they
display a high reactivity in the metal-free and Ca
form, but a 20-fold lowered reactivity in the Zn
form of S100A3. Ca
-binding promotes the
formation of a solvent-accessible hydrophobic surface as monitored by
the 60-fold fluorescence increase of
2-p-toluidinylnaphthalene-6-sulfonate, whereas Zn
has no noticeable influence. Our data indicate that
Ca
and Zn
do not bind to the same
sites and that under physiological conditions S100A3 is a
Zn
-binding rather than a Ca
-binding
protein; nevertheless, very specific conformational changes are
introduced by either Ca
or Zn
.
Since no Zn
-binding motif of known structure was
identified in the primary sequence of S100A3, the results are
suggestive for a novel Zn
-binding motif.
The S100 protein family constitutes a subgroup of
Ca-binding proteins of the EF-hand type displaying
30% or more sequence identity (Kligman and Hilt, 1988; Hilt and
Kligman, 1991). Under physiological conditions their affinity for
Ca
is rather low but can be increased once S100
proteins are associated with their targets. Different S100 proteins
were also found to bind Zn
with a fairly high
affinity (Baudier et al., 1986; Leung et al., 1987;
Filipek et al., 1990; Dell'Angelica et al.,
1994). Both intracellular roles, such as activation of enzymes,
regulation of motility, and smooth muscle contraction, and
extracellular roles, such as neuronal differentiation, glial
proliferation, and prolactin secretion (for review, see Donato(1991),
Zimmer and Dubuisson(1993), and Heizmann and Braun(1995)), have been
proposed. Intriguingly, in different cases where calmodulin was thought
to be the regulatory CaBP, (
)S100 proteins were finally the
real activators (Bianchi et al., 1993). Most S100 proteins
interact in vitro with hydrophobic matrices, with membranes,
enzymes, cytoskeletal and contractile proteins, and even cell surface
receptors (for review, see Donato(1991)). All of these data point to a
multifunctional role of the S100 family with a particular function for
each of its members. This functional specificity is supported by the
fact that their expression is differentially deregulated in different
types of cancer cells (Hilt and Kligman, 1991; Weterman et
al., 1992; Davis et al., 1993; Pedrocchi et al.,
1994a, 1994b), suggesting participation in tumor progression. However,
for none of these putative functions have the molecular details been
elucidated.
The protein S100A3, ()formerly called S100E,
was recognized for the first time as the product of one of the tightest
gene clusters discovered in the human genome located on chromosome 1q21
(Engelkamp et al., 1993). The S100A3 gene shows a low
but general transcription level in diaphragm, heart, skeletal muscle,
stomach, lung, liver, fat tissue, and placenta. A YAC clone from human
chromosome 1q21 has been recently isolated on which nine different
genes coding for S100 proteins were localized. The clustered
organization of S100 genes in the 1q21 region allowed to introduce a
new logical nomenclature for these genes (Schäfer et al., 1995). The S100A3 gene product is 101 residues long
and possesses one S100-type noncanonical Ca
-binding
loop of 14 residues expanding from Ala-20 to Glu-33, and one canonical
EF-hand loop of 12 residues from Asp-63 to Glu-74, both flanked by two
-helices. In calbindin D-9k, the prototype of this S100 protein
family with a resolved three-dimensional structure (Szebenyi and
Moffat, 1986; Carlström and Chazin, 1993), the
-helices are oriented in an antiparallel fashion, thus forming a
4-helix barrel. Within the S100 subfamily S100A3 is unique for the
exceptionally high number of Cys residues. Despite the Cys frequency,
S100A3 does not display the classical zinc-binding motifs seen in
metallothioneins (Vallee and Auld, 1990), DNA-binding proteins
(Pérez-Alvarado et al., 1994), or
protein kinase C (Hommel et al., 1994).
In order to begin
to understand the role of S100A3 and the molecular mechanisms by which
it exerts its function, we characterized in this study the
Ca- and Zn
-binding properties of
recombinant human S100A3 under physiological conditions. We monitored
the cation-dependent changes in the environment of the Trp and Tyr
residues, probed the thiol/disulfide state and the cation-dependent
reactivity of the thiols, and finally monitored the solvent-exposed
hydrophobic surface. The results suggest that under physiological
conditions S100A3 is a Zn
-binding rather than a
Ca
-binding protein.
Oligonucleotides were synthesized on a Gene Assembler DNA synthesizer (Pharmacia Biotech Inc.). The primers used to amplify S100A3 cDNA for cloning into pMal-c2 were as follows: S100A3-M, 5`-ATGGCCAGGCCTCTGGAGCAGG-3`; S100A3-B, 5`-GGCAAGTCCAGATTGAAAGGGG-3`.
Figure 1: Expression and purification of recombinant human S100A3. Coomassie Blue-stained 15% SDS-Tricine-PAGE under reducing conditions, showing induction and purification steps of MBP-S100A3 fusion protein and of S100A3. Lane 1, 40 µg of crude extract of E. coli; lane 2, 40 µg of flow-through following loading onto amylose-resin column; lane 3, 5 µg of eluate from amylose-resin column: MBP-S100A3 fusion protein; lane 4, 25 µg of fusion protein after factor Xa cleavage; lane 5, 3 µg of MBP; lane 6, 2.5 µg of finally purified S100A3 (monomer).
The native apparent molecular weight of the metal-free,
Ca and Zn
forms of S100A3 was
determined by gel filtration on a 1
70-cm column of Sephadex
G-75 in 50 mM Tris buffer, pH 7.5, 150 mM KCl, 1
mM dithiothreitol (buffer A) containing either no divalent
cations, 100 mM CaCl
, or 100 µM ZnCl
. The column was standardized with the calibration
mixture of Bio-Rad.
Zn binding was measured at room temperature by the
equilibrium gel filtration method of Hummel and Dryer(1962). A Sephadex
G-25 column (0.7
50 cm) was equilibrated in buffer A containing
variable concentrations of Zn
. 0.5-1 ml of
50-200 µM metal-free protein was applied to the
column. In the eluant Zn
concentrations were
determined by atomic absorption with a Perkin-Elmer 2380 atomic
absorption spectrophotometer. For the atomic absorption measurements
EDTA up to 1 mM was added to all solutions, including the
standards (Titrisol, Merck). Protein concentrations were measured by
ultraviolet absorption.
UV absorption spectra and
difference spectra were measured with a Perkin-Elmer Corporation 5
UV/VIS spectrophotometer. Difference spectra were taken at room
temperature on solutions with an optical density at 280 nm close to 1.
To verify recombinant S100A3 for correct synthesis in bacteria we determined its exact mass by electrospray ionization mass spectrometry. Before desalting with butyl-300 microbore reversed-phase HPLC it was again necessary to mix the protein probe with 50 mM DTT to prevent precipitation on the column and to obtain any mass signal. In acidic solvent a molecular weight of 11,712 ± 1.7 was obtained, which is in good agreement with the calculated molecular weight, including the amino-terminal methionine, of 11,713.3 (Fig. 2). This result shows the correct expression of S100A3 in E. coli TB1, including an unprocessed amino-terminal methionine.
Figure 2: ESI-MS of human recombinant S100A3. ESI-MS data for S100A3 including 50 mM DTT.
SDS-PAGE after reduction of S100A3 with
10 mM DTT for 30 min at 37 °C also yielded a band with a
molecular mass of 11 kDa. Determination of the apparent molecular mass
by gel filtration on Sephadex G-75 after thorough reduction yielded
different values depending on the presence of divalent cations: 22.4
kDa in the absence of divalent cations, 24.9 kDa in the presence of 100
µM Zn, and 39.0 kDa in the presence of
100 mM Ca
. Thus, as in other members of the
S100 family, S100A3 forms a noncovalent homodimer. Moreover, 100 mM Ca
promotes formation of a higher order oligomer
(likely tetramers). It is not clear if the latter phenomenon is to be
attributed to the specific binding of Ca
or to an
ionic strength effect, which is known to stabilize hydrophobic
interactions.
We assessed the isoelectric point of S100A3 by two-dimensional gel electrophoresis under reducing conditions using an immobilized pH gradient ranging from pH 3.5 to 10. In contrast to the calculated pI of 4.53, the determined pI of the denatured protein was found to be 5.5 (data not shown). This divergence may be caused by the experimental conditions and not by any modifications of the protein as the measured mass of recombinant S100A3 was found to be identical to the calculated value.
Figure 3:
Zn binding to S100A3 as
determined by the Hummel-Dryer method. The solid lines are the
theoretical isotherms calculated with the Hill equation with
[Zn
]
= 11 µM and n
= 1.4. Inset, Hill plot
of the data assuming four binding sites per
monomer.
Figure 4:
Tryptophan fluorescence characteristics of
S100A3 after excitation at 280 nm. Emission spectrum in the absence of
metals (), in the presence of 30 mM MgCl
(- - - - -), of 150 mM CaCl
(--), of 200 µM ZnCl
(-
-
-
-
), and of 4 M guanidine-HCl (thin dotted line). The protein concentration was 1
µM. The spectra were corrected for the buffer
contribution.
Figure 5:
A, conformational titration of 2
µM S100A3 with Ca in the presence
(
) and absence (
) of 30 mM MgCl
and
with Zn
in the absence of other divalent cations
(
) as followed by changes in the Trp fluorescence. The solid
line is the Hill equation with
[Ca
]
= 35 mM and n
= 0.76 and assuming a total
signal change of 1.18. Panels B and C represent the
Scatchard plot of Ca
binding and of the high affinity
component of Zn
binding,
respectively.
Figure 6:
Difference spectra of S100A3 (66
µM) in buffer A at room temperature after addition of 100
mM Ca (--) or 190 µM Zn
(- - - - -) to the metal-free protein. The
difference in optical density was expressed for a protein solution with
an optical density of 1.0 at 280 nm.
Figure 7:
Thiol reactivity in S100A3 as monitored by
the absorbance at 412 nm after addition of DTNB. Metal-free S100A3
(); S100A3 in the presence of 200 mM Ca
(--), 100 µM Zn
(- - - - -), and 200 mM Ca
+ 100 µM Zn
(-
-
-
-
). Protein concentration was 6.7
µM. After 15 min of reaction time 31 µM thiols were titrated. The reactions do not follow pseudo
first-order kinetics.
Figure 8:
Hydrophobic exposure in S100A3 as
monitored by the fluorescence of TNS after excitation at 328 nm.
Protein and TNS concentrations were 5 and 0.5 µM,
respectively. TNS alone (thin solid line); metal-free S100A3
(); S100A3 + 180 mM
Ca
(--); S100A3 + 190
µM Zn
(- - - - -); S100A3 + 180
mM Ca
+ 86.5 µM Zn
(-
-
-
-
); S100A3 +
190 µM Zn
+ 90 mM Ca
(thin dotted
line).
In this study we report the biochemical characterization and
cation-binding properties of S100A3, a new member of the S100 family
with an unusually high content of Cys residues. The protein is a dimer
and contains two EF-hand motifs per monomer. But, whereas most other
S100 proteins display Ca dissociation constants of
0.1-1 mM, S100A3 is able to bind Ca
only in the 10-100 mM free Ca
range, i.e. very far from the cytosolic Ca
levels. Nevertheless, this binding seems specific since it is
accompanied by Tyr and Trp conformational changes very similar to those
caused by Zn
binding, by a well defined exposure of
hydrophobicity and an oligomerization. The reason for this low affinity
for Ca
is not clear, since its primary structure is
quite classical for a S100 member. However, our data indicate that each
dimer contains five disulfide bridges. This may stabilize the protein
but can impose strong constraints for the efficient binding of
Ca
. Reduction of all the disulfide bridges of S100A3
under denaturing conditions and alkylation of the thiols yields a
protein product which binds Ca
with a dissociation
constant of 0.8 mM, (
)i.e. an affinity
close to that of most other S100 proteins. It is still possible that
S100A3 displays a real Ca
-dependent function when
associated with its target or when secreted in the
Ca
-rich extracellular fluid. In contrast to
calgranulin C (Dell'Angelica et al., 1994) and S100B
(Baudier et al., 1986), there are no indications that
Zn
increases the affinity of S100A3 for
Ca
. But an interesting dynamic regulation may occur
through zinc binding, since the affinity is rather high (K
10 µM). It is
estimated that 99% of the 36 mg of Zn
per kg human
wet weight is intracellular and 25% of this amount is not, or loosely
bound (Vallee and Falchuk, 1993). This represents intracellular free
Zn
concentrations of 40-400 µM,
depending on the tissue, with a maximum of 2 mM in the retina.
It is thus very likely that in vivo S100A3 is mostly in
Zn
-bound form. Its precise role in the direct
activation of response systems and/or in promotion of exchange with
other important Zn
-regulated proteins must still be
evaluated.
Where are the four Zn sites per
monomer, or rather the eight Zn
sites per native
dimer located and what kind of sequence motifs in S100A3 could be
responsible for Zn
binding? Since the
three-dimensional structure of most S100 proteins has not been
elucidated, one can only compare with Zn
-binding
motifs in proteins where the ligands responsible for binding have been
identified. Zn
sites are either of the tetradentate
or tridentate type (Vallee and Auld, 1990). Tetradentate sites are
found in small Zn
-binding domains (zinc fingers) in
which the cation is strongly held by four ligands composed of
Cys and/or His (Schabe and Klug, 1994). These motifs have affinities of
the order of 10
M
or more
(Zeng et al., 1991). The tridentate type of site, found in
different extracellular (Vallee and Auld, 1990) and intracellular
enzymes (Perlman and Rosner, 1994), bind Zn
with an
affinity constant of 2
10
M
(Francis et al., 1994). In these enzymes Zn
is held by three ligands: two His residues in a typical
H-E-x
-H or H-x
-H sequence implanted on a
-helical segment and a third coordinating Glu residue,
located at a variable distance, up- or downstream, of the His motif
(Vallee and Auld, 1990). S100A8, S100A9, and calgranulin C display such
a motif and bind Zn
with an affinity of at least
10
M
. The single Zn
site is clearly distinct from the two
Ca
-binding sites. However, other S100 proteins do not
possess one of the motifs frequently encountered in proteins where
Zn
binding has been proven to be functionally
important. Strong binding of 8 Zn
ions per dimer was
also reported for S100B (Baudier et al., 1986). S100A1 (Leung et al., 1987) binds Zn
with a rather low
affinity. S100A6 (calcyclin) binds Zn
with a
[Zn
]
of about 2 mM
(Filipek et al., 1990; Pedrocchi et al., 1994b). But
S100A3 is the only S100 protein with 10 Cys residues, most of which are
clustered at the opposite site of the two Ca
-binding
loops. This abundance of sulfur atoms and the fact that the fully
reduced and alkylated protein does not bind Zn
at all
anymore
suggest that the Zn
ions are
bound in thiolate clusters of the Kagi and Kojima type (reviewed in
Vallee and Auld(1990)). Direct binding of Zn
to the
Cys residues also would explain the strong reduction of the thiol
reactivity in the presence of Zn
, but not of
Ca
. The metallothioneins bind 7 Zn
per mol (8 for the S100A3 dimer) to 20 cysteinyl residues in
clusters of the type Zn
S
and
Zn
S
. The Zn
-thiolate cluster
has recently also been observed in DNA-binding proteins (Pan and
Coleman(1990). However, since Co
-binding does not
induce the characteristic absorption bands at 700 nm as it does in
metallothioneins, and since half of the Cys residues in S100A3 are not
in the free thiol form, it is tempting to postulate that a new type of
cluster is present in the latter protein. Structural work is in
progress to provide a more detailed description of this novel
Zn
-binding motif.