In this study, to elucidate the mechanism of Ca21-binding to each EF-hand named EF-A, -B, -C and -D from the N-terminus of p26olf, we examined Ca21-binding in wild-type p26olf and also i
Trang 1Calcium-binding by p26olf, an S100-like protein in the frog olfactory epithelium
Naofumi Miwa, Yukiko Shinmyo and Satoru Kawamura
From the Department of Biology, Graduate School of Science, Osaka University, Japan
Frog p26olf is a novel S100-like Ca21-binding protein found
in olfactory cilia It consists of two S100-like domains
aligned sequentially, and has a total of four Ca21-binding
sites (known as EF-hands) In this study, to elucidate the
mechanism of Ca21-binding to each EF-hand (named EF-A,
-B, -C and -D from the N-terminus of p26olf), we examined
Ca21-binding in wild-type p26olf and also in its mutants
in which a glutamate at the – z coordinate position within
each Ca21-binding loop was substituted for a glutamine
Flow dialysis experiments showed that the wild-type binds
nearly four Ca21 per molecule maximally, while all
the mutants bind approximately three Ca21 Although
EF-B and -D are p26olf-specific EF-hands and their role in
Ca21-binding is not known, the result unequivocally showed
that they actually bind Ca21 The overall Ca21-binding
affinity decreased in the three mutants The decrease was
very large in the mutants of EF-A and -B, which suggested that the Ca21-affinities are high in EF-A and -B in the wild-type Assuming the presence of four steps of Ca21-binding,
we determined the dissociation constant of each step in wild-type p26olf To assign which step takes place at which EF-hand, we measured the antagonistic effect of K1on each step, as the effect of K1is thought to be a function of the number of the carboxyl groups in an EF-hand Although the actual Ca21-binding mechanism may not be so simple, this study together with the mutation study suggested a tentative
Ca21-binding model of p26olf: the order of Ca21-binding to p26olf is EF-B, EF-A, EF-C and EF-D Based on these results, we speculate that similar Ca21-binding takes place
in an S100 dimer
Keywords: calcium-binding; p26olf; S100
We have previously isolated a novel Ca21-binding protein,
p26olf, from the frog olfactory epithelium [1] This protein
localizes in the cilia of olfactory epithelium and interacts
with a frog b-adrenergic receptor kinase (bARK)-like
protein in a Ca21-dependent manner [2] Through the
bARK-dependent phosphorylation, p26olf has been
suggested to have some role(s) in olfactory signal
transduction
The amino-acid sequence of p26olf is most similar to a
pair of S100 proteins aligned in tandem [1,3] The S100
protein family, one of the subgroups of proteins that contain
EF-hands, is known to be involved in many types of
biological function such as cell-cycle progression [4,5],
differentiation [6,7] and regulation of enzyme activity [8]
Abnormal expression of S100 proteins is thought to be a
cause of a number of diseases including cancer [9]
(reviewed in [10,11]) There are two Ca21-binding sites,
known as EF-hands, in S100 [12]: one in the N-terminal half
is S100-specific showing low affinity for Ca21(dissociation
constant, Kd¼ 200–500 mM) and the other in the C-terminal
half is a typical EF-hand showing high affinity (Kd¼ 20 –
50 mM) [13] In p26olf, however, this typical EF-hand is
somewhat modified The amino-acid sequence of this site
fulfills the requirement of the typical EF-hand but there is a
four-amino-acid-insertion between the E and F a helix, which characterizes this EF-hand as p26olf-specific [3] In p26olf therefore there are four EF-hands (named as EF-A, -B, -C and -D from the N-terminus of p26olf), and two of them (EF-A and -C) are S100-specific and the other two (EF-B and -D) are p26olf-specific
S100 proteins form a homo- or a heterodimer, which is the functional form [14,15] In the previous studies, Ca21 -binding was examined in S100 dimers and it was found that the apparent dissociation constants in four steps of the binding in a dimer are 20 – 100 mM [16,17] As the S100-specific site shows low affinity for Ca21, this result suggests the presence of a cooperative interaction in and/or between S100 monomers The mechanism of this cooperative interaction, however, is not known In a homodimer of S100, it is difficult to determine which site is responsible for the nth binding, because there are two identical sites in a homodimer and one cannot be certain which subunit is under consideration As for a heterodimer, the isolation of a heterodimer itself is difficult, because one cannot be sure whether only a heterodimer is present It may be the case that homodimers are formed after dissociation of a hetero-dimer As p26olf is a protein composed of a single peptide, there is no uncertainty about a dimer state
In the present study, we measured Ca21-binding in wild-type p26olf, and determined the dissociation constants of the first to fourth binding of Ca21by assuming the presence of four steps of sequential and ordered binding To identify the site of the EF-hand corresponding to the nth binding of
Ca21, we measured the affinity for K1in the nth binding As the affinity for K1 is suggested to be dependent on the number of the carboxyl groups in an EF-hand in calmodulin [18] and actually the number is different in each EF-hand in
Correspondence to S Kawamura, Department of Biology, Graduate
School of Science, Osaka University, Machikane-yama 1-1, Toyonaka,
Osaka 560 0043, Japan Fax: 1 81 6 6850 5444,
Tel.: 1 81 6 6850 5436, E-mail: kawamura@bio.sci.osaka-u.ac.jp
(Received 15 June 2001, revised 7 September 2001, accepted
12 September 2001)
Abbreviations: bARK, b-adrenergic receptor kinase.
Trang 2laser densitometer (Molecular Dynamics) To measure the
stoichiometry of Ca21-binding to p26olf, we performed flow
dialysis experiments as previously described [2] Briefly, we
incubated p26olf (final concentration; 10 mM) in a 20-mM
Tris/HCl buffer (pH 7.5) at 20 8C with various
concen-trations of45CaCl2(6.2 102Ci:mmol21) at KCl
concen-trations that depended on the type of the experiment Unless
otherwise stated, the Tris buffer always contained 100 mM
KCl in the present study The reaction mixture was placed in
a prewashed microconcentrator (Microcon, Amicon) and
then it was centrifuged briefly We counted the activities of
45Ca in 4 mL portions of both the reaction mixture and the
filtrate using a scintillation cocktail (Clearzol; Nacalai,
Kyoto, Japan) By comparing the activities of45Ca in the
reaction mixture and the filtrate, the amount of Ca21bound
to p26olf was calculated Blank experiments without p26olf
were performed to correct for nonspecific binding of Ca21
to the membrane of microconcentrators The Ca21
concentration in each reaction mixture was calibrated with
a Ca21electrode using diluted solutions of a 20-mMCaCl2
solution (Sigma)
The raw data were analyzed with the Hill equation [19],
the Scatchard plot [20], and then, the Adair equation [21]
The Adair equation is represented as follows
K1
x 1 2 1
K1
1
K2
x21 ::: 1 n 1
K1
1
K2
::: 1
Kn
xn
=
1 1 1
K1
x 1 1
K1
1
K2
x21 ::: 1 1
K1
1
K2
::: 1
Kn
xn
Where R is the molar ratio of bound Ca21to p26olf at a free
Ca21 concentration x, and K1, K2, and Kn are the
macro-scopic dissociation constants for the binding of one and two,
and n Ca21to p26olf in a reaction of
CD measurement
CD spectra of wild-type p26olf and mutants (final
concentration; 10 mM each) were measured with a Jasco
J-720 W spectrophotometer with 1-mm light path in Tris
buffer containing 100 m KCl The Ca21concentration was
directed mutagenesis according to the methods of Tachi-banaki et al [22] We used the following oligonucleotides and the complementary oligonucleotides as PCR primers: AACTTCAAACAGTTTGAGCAG for EF-A-mutation, GACTTTCAACAGTTTCTCAAC for EF-B-mutation, GAT TACACACAGTTCGAGGCA for EF-C-mutation, AATTT CCAGCAGTTCATGAAC for EF-D-mutation The under-lined codons show the sites of mutations, replacing glutamate (E) with glutamine (Q) After PCR reactions, the mutated DNA fragments were digested with Nde I and Bam HI and inserted into pET3a (Novagen), and these recombinant plasmids were introduced into Escherichia coli BL21 pLysS (Novagen) After induction of the expression of p26olf by isopropyl thio-b-D-galactoside, p26olf-mutants were purified according to the method of purification of native p26olf [1]
R E S U L T S
Ca21-binding to p26olf
In p26olf, two S100 homolog domains are located sequen-tially in the molecule [1,3] S100 proteins are generally known to form a homo- or a heterodimer in solution [14,15], but in some experiments the recombinant S100 protein formed a trimer [17] Therefore, it could be the case that our recombinant p26olf prepared here might form a dimer If it
is present, interpretation of the results of Ca21-binding may
be complicated Therefore, to exclude this possibility
we performed gel filtration column chromatography and confirmed that our p26olf exists as a monomer; purified p26olf eluted through the column as a single peak with a molecular mass of < 28 kDa in the absence of Ca21, and
< 16 kDa in the presence of Ca21
(data not shown) Because the calculated molecular mass of p26olf is
< 24 kDa [1], this result indicated that p26olf exists as a monomer in our solution
In order to determine the affinity for Ca21 and the cooperativity of Ca21-binding to p26olf, we next analyzed the stoichiometry of Ca21-binding to p26olf by flow dialysis
in the Tris buffer Ca21-binding to p26olf as a function of
Trang 3free Ca21concentration is shown in Fig 1 Because p26olf
tends to aggregate at high concentrations, we used 10 mM
p26olf in a Ca21-binding experiment Due to this limitation
of the concentration of p26olf, we considered Ca21-binding
to p26olf below < 200 mMCa21 Above this concentration
of Ca21, the Ca21-binding signal (at most 40 mM, see
below) was within a noise level of the total Ca21
concen-tration and therefore, the result would not be reliable
The binding data showed that the maximum number of
Ca21-binding to p26olf is approximately four per molecule
(Fig 1), and therefore we fitted the data with the Hill
equation by assuming that p26olf has four Ca21-binding
sites The fitted curve (solid line in Fig 1) showed a
reasonable fit to the experimental binding data with the KCad
value of 22.3 mMand the Hill coefficient of 2.0 [in order to
distinguish the Kd value determined by the Ca21-binding
studies and that determined by the CD measurement (see
below), we added the superscript Ca or CD to the term of
Kd] The convex curve observed in the Scatchard plot of the
binding data clearly indicates the presence of a positive
cooperativity (see inset in Fig 1)
To estimate the dissociation constants in the Adair
equation (see Experimental procedures), we performed the
curve fitting by assuming the presence of four steps of
Ca21-binding The theoretical curve (dashed line in Fig 1)
fitted well to the binding data with K1¼ 83.3 mM,
K2¼ 6.3 mM, K3¼ 22.2 mMand K4¼ 50 mM, for example,
but due to the experimental errors of the measurement, we
could not determine the four dissociation constants
uniquely However, after many trials, we reasonably
con-cluded that the dissociation constants had a tendency of
K2, K3ø K4, K1 The Adair dissociation constants
obtained were slightly larger than those obtained in S100
dimers [16] This is because in the study in Fig 1,
Ca21-binding was measured in the presence of 100 mMK1 that is known to reduce the affinity for Ca21(see below) Effect of K1on Ca21-binding to p26olf
The antagonistic effect of K1on Ca21-binding was observed
in many of Ca21-binding proteins such as S100 and calmodulin [16,18] Interestingly, Haiech et al assumed that the affinity for K1 is a function of the number of the carboxyl groups in an EF-hand [18], and their rationale is in good agreement with the actual sequence of Ca21-binding [23] Although their assumption should be tested in other
Ca21-binding proteins, we here assumed that a similar analysis can be applied to p26olf Thus, we determined the affinity for K1 in each step of the Adair equation by changing the K1 concentration in order to identify which Adair dissociation constant (K1– K4) corresponds to which EF-hand
As shown in Fig 2, the maximum number of Ca21-binding was nearly four per molecule at all the K1concentrations tested Assuming the presence of four Ca21-binding sites, the data were first fitted by the Hill equation As a result, we obtained KdCavalues of 22, 15.0 and 12.1 mM, and the Hill coefficients of 2.0, 1.9 and 1.7 at 100 (Fig 1), 20 and 0 mM
K1, respectively (inset in Fig 2) Because K1increased the
KCa
d values in the Hill equation, K1competed with Ca21for the EF-hands of p26olf similarly as in the cases of S100 and calmodulin [16,18]
The data were then fitted by the Adair equation and the determined dissociation constants (K1– K4) are summarized
in Fig 3 To calculate the dissociation constant for K1in each step, step 1 for example, we fitted the data of K1 at different K1concentrations with the equation of Kiapp¼ Ki (1 1 [K1]/ki) [18] In the equation, Kiappis the calculated Adair dissociation constant at step i in the presence of K1,
Kiis the Adair dissociation constant at 0 mMK1, and kiis the intrinsic dissociation constant for K1 at step i The calculated result showed that the affinity for K1is high in steps 1 and 4, and low in steps 2 and 3 (see the k values in
Fig 1 Ca21-binding to p26olf The amount of Ca21bound to p26olf
in the Tris buffer containing 100 m M KCl is shown as a function of free
Ca 21 concentration The experimental points represent the average of
16 different experiments using three different preparations of p26olf.
Each bar represents the standard deviation The data were fitted to the
Hill equation (solid line) and the Adair equation (dashed line) The
index of the fit represented as the coefficient of correlation (r2) is 1.0 in
both the fitting to the Hill equation and to the Adair equation.
Fig 2 Effect of K1 on Ca21-binding to p26olf Ca 21 -binding to p26olf was examined at 100 m M KCl (circles), 20 m M KCl (triangles) and 0 m M KCl (crosses) The data points of 20 m M KCl represent the average ^ SD (n ¼ 12) of three different preparations of p26olf, and those of 0 m M KCl represent the average ^ SD (n ¼ 5) of two different preparations The data were fitted to the Hill equation (solid line, see text) The r2values are 0.99 (100 m M KCl), 1.0 (20 m M KCl) and 1.0 (0 m M KCl).
Trang 4Fig 3) Due to the experimental errors, we could not
determine the k-values uniquely, but our best estimate was
k1ø k4,, k2ø k3 These results suggested that the steps
1 and 4 take place in EF-hands which have many carboxyl
groups in the EF-hand motif (see Discussion)
Effect of K1on Ca21-induced conformational change of
p26olf
Our previous CD measurement in the absence of K1showed
that Ca21-binding to p26olf increases the negative signal at
both 210 nm and 222 nm [2] As K1 reduces the affinity
for Ca21, we measured CD spectra in the presence of
100 mM K1 as a function of Ca21 concentration At all
Ca21 concentrations, negative CD signals increased in a
Ca21-dependent manner (inset in Fig 4) Figure 4 shows
the percent change of CD at 222 nm (signal of a helix)
plotted as a function of the free Ca21concentration (lower
axis) and the number of the bound Ca21per p26olf molecule
(upper axis, nCa21) calculated from the binding data in
Fig 1 (circles, data at 100 mMK1; triangles, data at 0 mM
K1 taken from Fig 2 in [2]) The percent changes were
fitted using the Hill equation (solid lines) This fitting for the
data at 100 mMK1indicated the KCD
d value of 8.5 mMand the Hill coefficient of 2.3 When these values were compared
with those in the absence of K1(KCD
d ¼ 2.5 mM, Hill coef-ficient ¼ 1.5 from [2]), it is revealed that the conformational
change of p26olf takes place at higher Ca21concentrations in
the presence of 100 mM K1 than in the absence of K1
Irrespective of the K1 concentration, however, the first
Ca21-binding induced < 80% CD change and the second
Ca21binding < 90% change (Fig 4)
Ca21-binding to mutant p26olf
In order to know how each EF-hand contributes to
Ca21-binding or Ca21-induced conformational changes in
p26olf, we prepared four mutant proteins that lacked the activity of one of the four EF-hands in p26olf To obtain such mutants, we mutated the C-terminal glutamate (E) in each calcium binding loop (Fig 5) This residue is well-conserved among many EF-hand type Ca21-binding proteins, and is believed to be important in providing two oxygen ligands to Ca21[24] In order to minimize the effect
of mutation on the structure of p26olf, we substituted this glutamate (E) for glutamine (Q) As this substitution leaves one ligand intact within the residue, it is possible that the
Ca21-binding activity of the EF-hand is partially retained However, this substitution is shown to be sufficient to suppress Ca21-binding to the EF-hand at 200 mM Ca21 [25,26], which was the highest Ca21 concentration used in the present study The mutant proteins generated were named as DEF-A [glutamate at position 40 was replaced by glutamine (E40Q) in EF-A], DEF-B (E86Q in EF-B), DEF-C (E149Q in EF-C) and DEF-D (E194Q in EF-D)
Ca21-binding in these mutants was measured at various
Ca21concentrations in the Tris buffer The maximum Ca21 -binding in each mutant was close to three per molecule
Fig 3 Dissociation constants of Ca21-binding to p26olf The
dissociation constant of each Ca21-binding step (K 1 – K 4 ) was
determined by the Adair equation for the data obtained in the presence
of 0 m M , 20 m M , and 100 m M KCl The intrinsic dissociation constants
for K1(k ) for each Ca21-binding step is also shown.
Fig 5 Four site-directed mutants of p26olf In each EF-hand motif, the glutamate residue (black boxes) at the C-terminus of each EF-hand motif was replaced by glutamine, and the mutants thus prepared were named as DEF-A, DEF-B, DEF-C and DEF-D The p26olf-specific insertions of four residues are shown by white bars in the upper figure and also double-underlines in the amino-acid sequence.
Fig 4 CD changes at 222 nm as a function of Ca21concentration.
CD spectra of p26olf were measured at various Ca 21 concentrations in the presence of 100 m M KCl (inset, sample records at 12 n M (curve 1), 7.6 m M (2), 109 m M Ca21(3)) Data at 100 m M KCl (circles) and at
0 m M KCl (triangles; taken from Fig 2 in [1]) were fitted to the Hill equation (solid line) (r2¼ 0.99 for 100 m M KCl and 0.99 for 0 m M
KCl) The lower axis of the figure gives the free Ca21concentration, and the upper axis gives the number of Ca 21 bound per molecule of p26olf (nCa 21 ) calculated from the binding data of Figs 1 and 2.
Trang 5(circles, Fig 6); this indicated the lack of one of the four
Ca21-binding sites in the mutated EF-hand Therefore, the
binding data were fitted to the Hill equation assuming the
presence of three Ca21-binding sites (solid lines in Fig 6)
In wild-type p26olf (dashed lines), the KCa
d value was 22.3 mM, and the Hill coefficient was 2.0 (Fig 1) In the
mutants, the KCa
d values varied from 14 to 117 mM
depending on the mutation (insets in Fig 6) The result
indicated that the overall Ca21affinity decreased in some of
the mutants: the decrease was large in DEF-A and DEF-B,
and small in DEF-C, and there was almost no decrease in
DEF-D One of our expectations was that the Ca21-binding
cooperativity might be lost when the responsible EF-hand(s)
was disrupted However, this was not the case because the
Hill coefficient (nH in insets) was always similar to that of
wild-type p26olf (n H ¼ 2.0) (see Discussion)
In the mutation studies, all of the mutants bind
approxi-mately three Ca21 This number might be greater at a higher
concentration of Ca21 However, as stated already, we
measured Ca21-binding at less than 200 mM Ca21 due to
aggregation of our sample (see Experimental procedures)
Ca21-induced conformational changes in mutant p26olf
In Fig 4, we measured the CD spectrum changes by
varying the Ca21concentration and found that the apparent
KCDd value was 8.5 mM and the Hill coefficient was 2.3 In
order to examine how the Ca21-induced conformational
changes in p26olf are affected in the mutants, we measured
the CD spectra of the mutants at various Ca21
concentra-tions To avoid crowding, only the results at a high (200 mM)
and a low (< 10 nM) concentration are shown in Fig 7A
The overall shape of the signal of a mutant was similar to
that of the wild type: the signal at both 210 nm and 222 nm
increased by increasing the Ca21concentration The result
indicated that both the a helix and b sheet content increase
by binding of Ca21 even in the mutants However, the content of these structures decreased somewhat in some of the mutants The signal of DEF-A was similar to that of the wild-type, but those of other mutants were approximately 90% (DEF-C and DEF-D) and 77% (DEF-B) of the wild-type The small signal in DEF-B was surprising, but it has been reported that a single mutation induces a signifi-cant conformational change in a calmodulin mutant [25] Although the mechanism of this effect has not been known, similar effects of the mutation might have affected the p26olf conformation in the DEF-B mutant
The CD signal change at 222 nm was expressed as a function of the Ca21concentration in each mutant, and the data were fitted by the Hill equation to determine the KCDd value and the Hill coefficient (solid lines in Fig 7B) When compared with the result in the wild-type (dashed line), the
KCDd values increased greatly in mutants DEF-A and DEF-B, but the mutation effect was negligible in DEF-C and DEF-D
It should be noted that the increase in the content of a helix (222 nm signal) observed in this study was caused by binding of Ca21 Therefore, the shift of the KCD
d value in a mutant (Fig 7B) would coincide with the shift of the KCa
d value shown in Fig 6 It was actually the case that both the
KCa
d shift and KCD
d shift were large in DEF-A and DEF-B, small in DEF-C and there were almost no shifts in DEF-D (compare the results in Figs 6 and 7B)
In Fig 4, we showed that irrespective of the K1 con-centration, 80 – 90% of the change in the a helix content was attained by binding of two Ca21 Interestingly, in the mutants which have only three Ca21-binding sites, the binding of two Ca21was sufficient to induce more than 70%
of the change (Fig 7B)
Fig 6 Ca21-binding to mutant p26olf We measured Ca21-binding
to four p26olf-mutants (DEF-A, DEF-B, DEF-C and DEF-D) by the
flow dialysis method The raw data (n ¼ 3) were analyzed using the
Hill equation (solid lines) Dashed line represents the fitted curve of
the data of wild-type p26olf obtained in Fig 1B The r 2 values are 0.99
(DEF-A), 0.99 (DEF-B), 0.98 (DEF-C) and 0.99 (DEF-D).
Fig 7 CD spectra of mutant p26olf (A) CD spectra of p26olf mutants (final concentrations; 10 m M each) either in the presence of
Ca21 (< 200 m M ; 1Ca21), or in the absence of Ca21 (< 10 n M ; 2Ca 21
) (B) Ca21-dependent changes in CD signals at 222 nm in p26olf-mutants Data were fitted to the Hill equation (solid line) Dashed line represents the fitted data of the CD change in wild-type p26olf The r2values are 0.99 (DEF-A), 0.99 (DEF-B),0.99 (DEF-C) and 0.99 (DEF-D) The lower axis of the figure gives the free
Ca21concentration, and the upper axis gives the number of Ca21bound per molecule of p26olf (nCa21) calculated from the binding data of Fig 6.
Trang 6each mutant was almost similar to the corresponding KCad
value of the mutant (Fig 7)
EF-hand motifs in p26olf
Our previous Ca21-binding study revealed that p26olf
binds nearly four Ca21per molecule at 200 mMCa21, which
suggests that the four EF-hand motifs in p26olf are
functional [2] However, the presence of an EF-hand motif
does not always mean the presence of the functional
Ca21-binding site Furthermore, in the primary structure of
p26olf, the p26olf-specific EF-hand (EF-B and -D) has a
four-amino-acid residue-insertion between E and F a helix
and therefore, the actual Ca21-binding should be tested In
the present study, in addition to the binding of
approxi-mately four Ca21 in the wild-type p26olf, we observed
nearly three Ca21-binding to each of the mutant that lacked
one of the EF-hands, which unequivocally showed that all of
the four EF-hands in p26olf bind Ca21
In the mutant studies, the Ca21-binding affinity decreased
(Fig 6) The decrease is large in DEF-A and DEF-B, and
small in DEF-C and DEF-D; this indicated that the affinities
of EF-A and EF-B in the N-terminal half of wild-type p26olf
are comparatively high, and those of EF-C and EF-D in the
C-terminal half are low (Fig 8A) It is of interest to test
whether this result is attributed to the intrinsic character of
the EF-hands in the N- and C-terminal halves of p26olf or
number (n ) of the ligand bound to a receptor Therefore, for example, when the first Ca21-binding takes place at a certain EF-hand and then the second binding takes place at two other EF-hands simultaneously, the constant K2 represents
an overall dissociation constant of this multiple second bindings However, it is highly possible that one of the dissociation constants in this multiple binding is lower than the other In this case, K2represents mainly the binding to this rather specific site (second site) Similar situation can be assumed for K3and K4 Alternatively, if Ca21-binding takes place sequentially in an ordered manner, the nth Adair dissociation constant represents the nth binding of Ca21to a specific EF-hand With these sorts of mechanisms in mind,
we assumed that the nth Adair constant represents the nth binding to a specific EF-hand in p26olf
In the presence and absence of K1, we measured
Ca21-binding to p26olf (Fig 2) and calculated the affinity for K1in each of the four binding steps (Fig 3) Haiech
et al [18] have suggested that the affinity for K1 (k in Fig 3) is dependent on the number of the carboxyl groups within an EF-hand of calmodulin Their idea was that the more carboxyl groups are present, the higher the affinity for
K1is As a result, the antagonistic effect of K1is expected
to be higher at the EF-hand loop having more carboxyl groups
The numbers of the carboxyl groups in the EF-hands in p26olf are two (in EF-A), six (EF-B), three (EF-C) and four (EF-D) and therefore the order of the affinity for K1is (from high to low) probably EF-B, -D, -C and -A As our best estimate of the relation among dissociation constants for
K1was k1ø k4,, k2ø k3(see Results), the order of the affinity for K1is (from high to low) steps 1 and 4 (there were no clear differences between these two steps), and steps 2 and 3 (again, no clear differences were observed between these two steps) Therefore, most probably EF-B and -D are responsible for the first and the fourth binding of
Ca21, and EF-A and EF-C for the second and the third binding As the affinity for Ca21is suggested to be higher in EF-B than EF-D (Fig 8A), the first step probably takes place in EF-B and the fourth step in EF-D Similar consideration led us to suggest that the second binding takes place in EF-A (the site showing high affinity for Ca21) and that the third binding in EF-C (low affinity for Ca21) Mechanism of cooperative Ca21-binding to p26olf
Of the four EF-hands in p26olf, EF-A and -C are S100-specific and potentially show low affinity for Ca21 (200 – 500 m [13]) In our present study, however, none of
Fig 8 Possible model of Ca21-binding in p26olf (A) Affinity of
each EF-hand in Ca 21 -binding deduced from the mutation study (B) A
possible simplified scheme of Ca21-binding to p26olf.
Trang 7the dissociation constants measured showed this low affinity,
which suggests the presence of cooperativity in Ca21-binding
to p26olf We will try to explain the mechanism of the
cooperative Ca21-binding, based on the presumed affinities
of S100- and p26olf-specific EF-hands Although the
Ca21-binding affinity to the p26olf-specific EF-hand is not
known yet, based on the similarity of the amino-acid
sequence, we assumed that this EF-hand shows high affinity
for Ca21(see below) As many previous studies reported by
others were performed in the absence of K1, we will use our
result measured in the absence of K1for direct comparison
As discussed above, we already suggested that the order
of Ca21-binding to p26olf is EF-B, EF-A, EF-C and EF-D
In these EF-hands, EF-B and -D are the p26olf-specific
EF-hands and probably show high affinity for Ca21-binding,
while EF-A and EF-C are S100-specific and are expected to
show low affinity From our consideration, the first binding
of Ca21takes place at EF-B with high affinity (K1¼ 21 mM;
Fig 3) The dissociation constants of the following steps
were 5.7 mM(second step dissociation constant K2at EF-A),
17 mM(K3at EF-C) and 18 mM (K4at EF-D) From these
dissociation constants, we speculate that the mechanism of
Ca21-binding to p26olf is as follows (see Fig 8B)
(a) The first Ca21-binding to a high affinity site, EF-B,
induces a conformational change of p26olf, which probably
increases the affinity for Ca21of EF-A to result in the second
Ca21-binding to EF-A It remained uncertain whether the
binding of Ca21to EF-B contributes to this increase in the
Ca21-affinity in EF-A directly or through the interaction
with the C-terminal half of p26olf The major
confor-mational changes (increase in the a helix content measured
with CD) complete at this stage (Fig 4)
(b) The major conformational changes induced by the
second Ca21-binding increase the affinity of EF-C to induce
the third Ca21-binding
(c) Finally, the fourth Ca21-binding takes place at EF-D
with high affinity
Although we assumed the presence of sequential binding
of Ca21in the above, the mechanism may not be so simple
The reason for this is that, in all of the mutants generated, we
observed three Ca21-binding with a positive cooperativity
(Fig 6), which cannot be explained by a simple ordered
sequential binding mechanism It is possible therefore that,
in the wild-type p26olf, most of Ca21binds to EF-B first to
induce a second cooperative binding to EF-A, but that this
process is not exclusive If this is the case, in DEF-A mutant
for example, Ca21firstly binds to EF-B and then also EF-C
(third binding site) with a positive cooperativity In this case,
our suggestion above shows the order of Ca21-binding of the
major population in wild-type p26olf Alternatively, another
explanation for the mutant study is possible It may be the
case that the mutation from glutamate to glutamine in the
mutants induced a similar EF-hand conformational change
that occurs on the binding of Ca21in the wild-type If this is
the case, Ca21-binding in the wild-type can be sequential
and cooperative throughout the course of the binding as
suggested above, and similar behavior can be observed in
the mutant Apparently, further studies are required to
understand the actual mechanism
S100 proteins are known to be functional in the form of a
homodimer or a heterodimer Although S100-specific low
affinity sites are present in an S100 dimer, the Ca21-binding
experiment showed that the calculated Adair dissociation
constants are in a range of 10 – 100 mM[16,17] The loss of the low affinity site (i.e 200 – 500 mM) in the Ca21-binding experiment in S100 dimers would arise from the cooperative mechanism that was found in p26olf and suggested above
A C K N O W L E G E M E N T S
This work was supported by a Grant-in-Aid (13780636) from the Ministry of Education, Culture, Sports, Science and Technology of Japan to N M., and Research for the Future Program of Japan Society for the Promotion of Science under the Project ‘Cell Signaling (JSPS-RFTF97L00301)’ to S K
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