(Received for publication, October 4, 1995; and in revised form, January 23, 1996)
From the
Recent studies have shown that substitution of Ala for one or
more Phe residues in calmodulin (CaM) imparts a temperature-sensitive
phenotype to yeast (Ohya, Y., and Botstein, D. (1994) Science 263, 963-966). The Phe residue immediately preceding the
first Ca ligand in site III of CaM (Phe-92) was found
to be of particular importance because the mutation at this position
alone was sufficient to induce this phenotype. In the present work we
have studied the functional and structural consequences of the Phe-92
Ala mutation in human liver calmodulin. We found that the mutant
(CaMF92A) is incapable of activating phosphodiesterase, and the maximal
activation of calcineurin is reduced by 40% as compared with the wild
type CaM. Impaired regulatory properties of CaMF92A are accompanied by
an increase in affinity for Ca
at the C-terminal
domain. To investigate the structural consequences of the F92A
mutation, we constructed four recombinant C-terminal domain fragments
(C-CaM) of calmodulin (residues 78-148): 1) wild type (C-CaMW);
2) Ala substituted for Phe-92 (C-CaMF92A); 3) cysteine residues
introduced at position 85 and 112 to lock the domain with a disulfide
bond in the Ca
-free (closed) conformation
(C-CaM85/112); and 4) mutations 2 and 3 combined (C-CaM85/112F92A). The
Cys-containing mutants readily form intramolecular disulfide bonds
regardless whether Phe or Ala is present at position 92. The F92A
mutation causes a decrease in stability of the domain in the absence of
Ca
as indicated by an 11.8 °C shift in the far UV
circular dichroism thermal unfolding curve. This effect is reversed by
the disulfide bond in the C-CaM85/112F92A mutant. The C-CaMW peptide
shows a characteristic Ca
-dependent increase in
solvent-exposed hydrophobic surface which was monitored by an increase
in the fluorescence of the hydrophobic probe
1,1`-bis(4-anilino)naphthalene-5,5`-disulfonic acid. The fluorescence
increase induced by C-CaMF92A is
45% lower than that induced by
C-CaMW suggesting that the F92A mutation causes a decrease in the
accessibility of several hydrophobic side chains in the C-terminal
domain of CaM in the presence of Ca
. The
Cys-85-Cys-112 disulfide bond causes a 10- or 5.9-fold decrease
in Ca
affinity depending on whether Phe or Ala is
present at position 92, respectively, suggesting that coupling between
Ca
binding and the conformational transition is
weaker in the absence of the phenyl ring at position 92. Our results
indicate that Phe-92 makes an important contribution to the
Ca
-induced transition in the C-terminal domain of
CaM. This is most likely the reason for the severely impaired
regulatory properties of the CaM mutants having Ala substituted for
Phe-92.
Calmodulin is the primary intracellular Ca receptor responsible for regulation of a large number of enzymes
in all eukaryotic cells(2, 3) . The crystal structure
of CaM (
)from various species (4, 5, 6, 7) reveals a
dumbbell-shaped molecule with two globular domains linked by a long
flexible solvent-exposed helix. Each domain contains two
Ca
-binding sites, the helix-loop-helix motifs (termed
EF-hands). The C-terminal domain binds Ca
with high
affinity (K
= 10
M) and the N-terminal with lower affinity (K
= 10
M)(8) . Ca
binding causes
exposure of two hydrophobic patches, one in each domain (the target
binding sites). This is the key event in the transduction of the
Ca
signal. Recently, the structure of
Ca
-free CaM has been solved by multidimensional
heteronuclear magnetic resonance
techniques(9, 10, 11) . A comparison between
the Ca
-free and Ca
-bound CaM
structures shows that the Ca
-induced conformational
transition requires a change in the interhelical angle in each of the
two globular domains of CaM. In the Ca
-free state the
two helices in each EF-hand are almost antiparallel, they are
perpendicular to each other, in the Ca
-bound
conformation. The transition between the two conformations has been
first modeled by Herzberg et al. (12) for the
N-terminal domain of TnC, and it is referred to as the HMJ model. We
have shown previously that this transition can be blocked with a
disulfide bond between Cys residues introduced at specific sites by
site-directed mutations leading to a loss of regulatory properties and
a decrease in affinity for Ca
in both TnC (13) and CaM(14) .
Although the HMJ model provides a
phenomenological description of the Ca-induced
conformational change, the molecular nature of this process is unclear.
In particular, it is not known what are the structural elements in the
Ca
-dependent regulatory proteins TnC and CaM that are
responsible for a tight coupling between Ca
binding
at the Ca
-chelating loops and the subsequent opening
of the structure. It is perplexing that some seemingly very similar
proteins, members of the EF-hand protein family, do not exhibit this
mechanism. For example in calbindin D
, the smallest member
of the calmodulin superfamily consisting of 75 amino acids,
Ca
binding causes a slight rotation of the helical
segments without a change in the interhelical angles and does not lead
to the exposure of hydrophobic sites(15) . A comparison of the
structures of Ca
-free and Ca
-filled
domains in TnC and CaM suggests that the highly conserved Phe residue
immediately preceding the first ligand in the two EF-hand domain may
have some specific contribution to the conformational change. This
residue (Phe-19 and Phe-92 in CaM) undergoes a transition from a
position in which half of the phenyl ring is exposed to solvent in the
Ca
-free form (9, 10, 11) to
a position in which the phenyl ring is deeply embedded in the
hydrophobic core of the protein, with partial exposure of only the
C
and C
atoms (4) which
contribute to the target binding hydrophobic site in the
Ca
-occupied form(16, 17) . This
transition is accompanied by a change in polypeptide backbone folding
at the last two residues in helix A and E in CaM from a 3
helix to an
-helix in the Ca
-free and
Ca
-bound conformation,
respectively(9, 10, 11) . Recent work by Ohya
and Botstein on yeast has shown that one of these residues, the Phe-92
in the C-terminal domain of CaM, is critical for the functional
properties of this protein(18, 19) . Ohya and Botstein
examined 14 temperature-sensitive yeast mutants bearing one or more Phe
to Ala substitutions in the single essential calmodulin gene of yeast.
They found four groups of mutations each showing different
characteristic functional defects in actin organization, calmodulin
localization, nuclear division, or bud emergence. One of the
temperature-sensitive mutants contained a single Phe to Ala mutation at
position 92.
The aim of our work was to evaluate the contribution of
Phe-92 to the functional and structural properties of CaM. We have
substituted Ala for Phe-92 in human liver CaM and found that the mutant
(CaMF92A) is incapable of activating phosphodiesterase, and its ability
to activate calcineurin is decreased by 40%. We used the C-terminal
domain fragment of CaM corresponding to the TRC peptide
(residues 78-148) to analyze the structural consequences of the
F92A mutation alone and in combination with another mutation designed
to lock the domain in the Ca
-free conformation. We
found that the F92A mutation causes a decrease in stability of the
domain in the absence of Ca
, an increase in affinity
for Ca
, and a decrease in the ability to interact
with a hydrophobic probe bisANS and with a synthetic target peptide.
Our results indicate that Phe-92 makes an important contribution to the
Ca
-induced conformational transition in the
C-terminal domain of CaM. This is most likely the reason for the
severely impaired regulatory properties of the mutants having Ala
substituted for Phe-92 in yeast and human CaM.
The C-terminal half-molecule fragment of calmodulin
(C-CaMW) has been produced by introducing into the CaM cDNA an NdeI restriction site (CATATG) at residues 76-77. The
cDNA fragment corresponding to C-CaMW was amplified using polymerase
chain reaction (23) , then purified, digested with NdeI and PstI, and ligated into a T7 expression
vector pEAD4. The same primers and procedures were used to obtain
C-CaMF92A and C-CaM85/112 using the CaMF92A cDNA (this work) and
CaM85/112 cDNA(14) , respectively, as templates. The triple
mutant C-CaM85/112F92A was obtained from C-CaM85/112 cDNA by
substitution of Ala (GCA) for Phe-92 (TTT). All mutations were achieved
using polymerase chain reaction with appropriate primers (21, 22) and then ligated into pAED4 vector. The
nucleotide sequence of the coding region for each construct was
confirmed by dideoxynucleotide sequencing (24) using the
Sequenase kit (U. S. Biochemical Corp.). To overexpress the cloned
cDNA, transformed BL21 cells were inoculated into 4 1 liter of
Luria-Bertani broth (LB) medium and grown at 37 °C until mid-log
phase. Isopropyl-1-thio-
-D-galactopyranoside was then
added at 0.5 mM, and the culture was grown at 37 °C for
135 min. The cells were then harvested by low speed centrifugation.
Purification of the protein was achieved by freeze-thaw of the low
speed cell pellets with a solution containing 50 mM Tris-HCl,
pH 7.5, 2 mM EDTA, 2 mM DTT, 0.2 mM phenylmethanesulfonyl fluoride (60 ml/1 liter of bacterial
culture). The extract was centrifuged at 100,000 g for
30 min; the supernatant was then made up to 0.1 M NaCl and
loaded onto a DE52-cellulose (Whatman) column (30
3 cm)
equilibrated with a solution containing 50 mM Tris-HCl, pH
7.5, 0.1 M NaCl, 1 mM CaCl
(also 2 mM DTT in the case of C-CaM85/112 and C-CaM85/112F92A). The column
was washed with the same solution until absorbance of eluent reached
base-line level. Protein was then eluted by a 0.1-0.3 M NaCl linear gradient (2
500 ml) in a solution containing
50 mM Tris-HCl, pH 7.5, and 1 mM CaCl
.
The fractions containing the CaM or C-CaM fragments (assessed by
SDS-PAGE) were pooled. The further purification steps were different
for different mutants.
For purification of C-CaMW and C-CaM85/112
the combined fractions from the DE52-cellulose column were made to 1 M NaCl and loaded onto a phenyl-Sepharose CL-4B (Pharmacia
Biotech) column (9 3 cm) equilibrated with a solution
containing 50 mM Tris-HCl, pH 7.5, 1 M NaCl, 1 mM CaCl
(also 2 mM DTT for C-CaM85/112). The
column was washed with this solution until absorbance of eluent reached
base-line level. The protein was then eluted with a solution containing
50 mM Tris-HCl, pH 7.5, 1 M NaCl, 2 mM EGTA
(also 2 mM DTT for C-CaM85/112). Fractions eluted with EGTA
were pooled, dialyzed into H
O, and lyophilized.
Since
C-CaMF92A and C-CaM85/112F92A show a significantly decreased binding to
phenyl-Sepharose, further purification of these peptides was achieved
by precipitation of their corresponding fractions from the
DE52-cellulose column, with 8.3% trichloroacetic acid. The precipitate
was ultracentrifuged at 100,000 g for 30 min at 5
°C. The pellet was resuspended in 10 ml of a solution containing 50
mM NH
HCO
and 1 mM CaCl
, and the solution was ultracentrifuged again at
100,000
g for 30 min at 5 °C. The supernatant was
then passed through a Sephacryl S-200 gel filtration column (100
1.5 cm) equilibrated with a solution containing 50 mM NH
HCO
and 1 mM CaCl
.
Fractions containing the peptides as assessed by SDS-PAGE were pooled,
dialyzed into H
O, and lyophilized.
To ensure that
C-CaM85/112 and C-CaM85/112F92A were fully oxidized, they were
incubated at 25 °C at a concentration of 1 mg/ml in a solution
containing 4 M urea, 50 mM Tris-HCl, pH 7.5, 0.1 M NaCl, 2 mM EGTA, 30 mM DTT for 4 h,
followed by dialysis against a solution containing 50 mM Tris-HCl, pH 7.5, 0.1 M NaCl, 2 mM EGTA. Then
5,5`-dithiobis(2-nitrobenzoic acid) at half the concentration of the SH
groups was added to the protein solution, and after 1 h incubation at
room temperature, the samples were dialyzed against 4 liters of a
solution containing 50 mM Tris-HCl, pH 7.5, 0.1 M NaCl for 36 h with 3 changes of buffer. The proteins were then
dialyzed into HO and lyophilized.
Protein concentration
was estimated from UV absorbance using A (0.1%,
1 cm) = 0.18 for CaM and A
(0.1%, 1 cm)
= 0.24 for the C-CaM mutants. The purity of the mutant proteins
was examined by SDS-PAGE gels. The C-terminal domain mutants were also
analyzed by amino acid analysis, partial amino acid sequencing, and
mass spectrometry. Molecular masses of C-CaM mutants were determined by
electrospray ionization mass spectrometry (25) performed on a
Finnigan TSQ700 triple quadrupole mass spectrometer at Harvard
Microchem (Cambridge, MA).
The purpose of these studies was to evaluate the functional
and structural role of Phe-92, the highly conserved Phe residue
immediately preceding the first Ca ligand in the
C-terminal domain of CaM. Initially Ala was substituted for Phe-92 in
the full-length CaM, and the effect of the mutation on CaM-dependent
enzyme activation was tested. For more detailed structural studies the
C-terminal domain recombinant fragment of CaM (residues 78-148)
was used rather than the full-length CaM to facilitate analysis of the
data. A wild type peptide (C-CaMW) and a mutant having Ala substituted
for Phe-92 (C-CaMF92A) were constructed. Moreover, we have obtained two
other mutants with Cys residues substituted for Ile-85 and Leu-112
(C-CaM85/112 and C-CaM85/112F92A). Based on our previous experiments (14) these mutants were expected to form reversibly
intramolecular disulfide bonds locking the two EF-hand domain in the
Ca
-free (closed) conformation. These mutants enabled
us to test the contribution of Phe-92 in the coupling between
Ca
binding and the conformational transition.
Figure 1:
Effect of the F92A mutation on the
regulatory properties of CaM. Activity of phosphodiesterase (A) was measured according to Wallace et al. (26) from the amount of hydrolyzed
[H]cAMP. Activity of calcineurin (B) was
measured according to Newton et al.(28) from the
amount of hydrolyzed phosphonitrophenol.
, CaM; and
,
CaMF92A.
Calcium titrations of the C-terminal domain of CaMF92A
monitored with tyrosine fluorescence have shown a shift of the
transition midpoint to lower Ca concentrations
indicating an increase in the affinity for Ca
as
compared with the wild type protein (data not shown). A similar effect
of the F92A mutation was observed for the C-terminal domain fragment
(see below). It is plausible that the impaired regulatory properties of
CaMF92A result from the missing contribution of the phenyl ring at
position 92 to the binding/activation of the target enzymes. However,
the increase in Ca
affinity suggests that some
structural effects may be responsible. The C-terminal half-molecule
mutants were used to test this hypothesis.
Figure 2:
Electrophoretic mobility of C-CaM mutants
on polyacrylamide gel. C-CaM mutants (3 µg) were run on 10%
polyacrylamide gel slabs containing 80 mM Tris-glycine, pH
8.6, and 1 mM EDTA (A, C) or 1 mM CaCl (B, D) with (A, B) and without (C, D) 4 M urea. DTT
indicates that the samples were incubated with 20 mM DTT at 25
°C for 40 min prior to the electrophoresis. Lanes 1 and 5, C-CaMW; lanes 2 and 6, C-CaM85/112; lane 3, C-CaMF92A; lanes 4 and 7,
C-CaM85/112F92A.
We have compared the structural
stability of the mutants in the presence of EGTA by recording
ellipticity at 222 nm as a function of temperature. All peptides
undergo cooperative heat-induced unfolding with a well defined
transition (Fig. 3) characterized by T, the
temperature at which half of the molecules are unfolded, the enthalpy
of unfolding
H
(at the temperature T
) and heat capacity change
C
(31) . Substitution of Ala for
Phe-92 causes a decrease in stability of the peptide in the absence of
Ca
as evidenced by an 11.8 °C decrease in T
( Fig. 3and Table 2). This effect
is reversed by the Cys-85-Cys-112 disulfide bond in
C-CaM85/112F92A. The computer fit of the data for the DTT-reduced
C-CaM85/112F92A resulted in an obviously incorrect T
value and a much larger value of
C
than
those obtained for other peptides. This was apparently due to a poorly
defined initial slope of the unfolding curve. To correct for this error
a fixed value for the initial slope (an average of those obtained for
other mutants) was used in the calculations. This resulted in stable
values of T
,
H
, and
C
(Table 2). It is somewhat surprising
that the disulfide bond between Cys-85 and -112 does not increase the
stability of the C-terminal domain of CaM except when Ala is present at
position 92. This is in contrast to the N-terminal domain of TnC in
which a disulfide bond caused a large increase in
stability(37) . In the presence of Ca
the
structural stability of all the C-CaM mutants was much higher, and no
cooperative unfolding was observed up to 90 °C.
Figure 3:
Effects of mutations on thermal stability
of C-CaM. Ellipticity at 222 nm was monitored over a temperature range
15-90 °C in 0.5 °C intervals. Each sample contained 0.2
mg/ml protein in 20 mM Tris-HCl, pH 7.5, 50 mM NaCl,
and 0.2 mM EGTA. For clarity every sixth data point is shown
except for C-CaM85/112F92A where every eighth data point is shown.
, C-CaMW;
, C-CaM85/112;
, C-CaMF92A;
,
C-CaM85/112F92A;
, C-CaM85/112F92A +
DTT.
Figure 4:
Calcium
titrations of C-CaM mutants. Calcium binding was monitored by Tyr
fluorescence ( = 280 nm;
= 304 nm). Each sample contained 5 µM protein, 50 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1
mM EGTA, and 1 mM nitrilotriacetic acid. Each data
point is an average from three titrations. Error bars represent the
standard deviation, and the solid lines represent the fit
using the Hill equation. Parameters of the fit are given in Table 3.
, C-CaMW;
, C-CaM85/112;
,
C-CaMF92A;
, C-CaM85/112F92A.
Figure 5:
Interaction of bisANS with C-CaM mutants. A, titrations of bisANS (5 µM bisANS in 50 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM EGTA 2 mM
CaCl) with C-CaM mutants. B, calcium-dependent
increase in fluorescence of bisANS induced by C-CaM mutants. 15
µM of each mutant was added to 5 µM bisANS in
a solution containing 50 mM Tris-HCl, pH 7.5, 50 mM NaCl, and 1 mM EGTA. An emission scan was taken from 400
to 600 nm (1-nm intervals) at an excitation wavelength of 394 nm. Then
2 mM CaCl
was added, and the scan was repeated.
The difference between the two scans is shown. For clarity data points
at 4-nm intervals are shown only.
, C-CaMW;
, C-CaMF92A;
, C-CaM85/112F92A;
,
C-CaM85/112.
C-CaMW induces a characteristic
large Ca-dependent increase in bisANS fluorescence (Fig. 5B). In contrast, the fluorescence increase
induced by the disulfide cross-linked mutants C-CaM85/112 and
C-CaM85/112F92A is very small (Fig. 5B) but becomes
similar to that induced by CaMW upon reduction with DTT (data not
shown). It is interesting that the Ca
-induced
increase in the solvent-exposed hydrophobic surface in C-CaM (513
Å
) represents only 18% of the total hydrophobic
surface in this domain (Table 4). However, the residues exposed
upon Ca
binding form a continuous hydrophobic cluster
having a characteristic concave shape which is apparently necessary for
the binding of bisANS or the target peptide/enzyme (for review see (38) and (39) ). It is clear that this characteristic
property is reversibly abolished by the Cys-85-Cys-112 disulfide
bond. Most remarkably the Ca
-dependent increase in
bisANS fluorescence induced by C-CaMF92A is 45% lower than that induced
by C-CaMW. Calculations of the solvent-exposed hydrophobic surface in
the C-terminal domain of CaM in the presence of Ca
show an increase from 2812 Å
in the wild type
to 2837 Å
in the F92A mutant (Table 4). Thus,
if the structure of C-CaMF92A was the same as that of C-CaMW one might
expect an increase rather than a decrease in bisANS fluorescence. A 45%
decrease in fluorescence observed for C-CaMF92A indicates that the
mutation must have affected many other hydrophobic side chains in this
protein.
Figure 6:
Binding of smM13N to C-CaM mutants.
Tryptophan fluorescence intensity ( = 295 nm;
= 320 nm) of smM13N (5 µM in a
solution containing 50 mM NaCl, 50 mM Tris-HCl, pH
7.5, 0.2 mM CaCl
) was monitored upon addition of
C-CaM mutants. Titration data for C-CaMW and C-CaMF92A were fitted with
a binding isotherm as described under ``Materials and
Methods.''
, C-CaMW;
, C-CaMF92A;
,
C-CaM85/112;
, C-CaM85/112F92A
Our current work on human liver CaM shows the vital
importance of the Phe-92 residue for the regulatory properties of CaM
consistent with the observations of Ohya and Botstein (18, 19) on yeast CaM. It is remarkable that a single
point mutation has such a dramatic effect on the regulatory properties
of CaM. The analysis of the three-dimensional structure of CaM
indicates that Phe-92 is involved in numerous interactions in the
hydrophobic core of the C-terminal
domain(4, 5, 6, 7) . Phe-92 also
contributes to the target interactions in the CaM-smM13
complex(16, 17, 40) . Both these properties
are shared by several other Phe residues in CaM, but a systematic
analysis of F A mutations at all positions in yeast CaM has
shown that the F92A mutation was the only one which alone could impart
a temperature-sensitive phenotype (18, 19) , clearly
indicating a unique role for this residue. An explanation for this
phenomenon stemming from our data is that Phe-92 has an important
structural function and the mutation affects many other residues
interfering with their optimal contribution to the CaM target
interaction/activation.
The Ca-dependent
interaction of CaM with hydrophobic probes such as bisANS is
interpreted to represent the exposure of hydrophobic residues, the
target binding sites. The bisANS was first introduced by Rosen and
Weber (41) and became a popular probe to study hydrophobic
properties of proteins. We have utilized the increase in fluorescence
intensity of bisANS upon binding to C-CaM and its mutants to provide a
relative measure of hydrophobic surface exposure. C-CaM85/112 and
C-CaM85/112F92A in the oxidized form cause little increase in the
bisANS fluorescence due to the disulfide bridge blocking the access of
the probe to the hydrophobic residues regardless of the presence of
Ca
. Most importantly, the fluorescence increase
caused by the C-CaMF92A mutant is approximately 45% lower than that
induced by C-CaMW. This indicates a significant decrease in available
hydrophobic sites for the binding of bisANS which is opposite to what
one would expect based on theoretical calculations. It could be argued
that having one less hydrophobic residue (Ala replaced Phe) could
result in a decrease in the hydrophobicity of the site causing a
reduction in the bisANS fluorescence in the absence of any alterations
of the structure. However, the phenyl ring of Phe-92 accounts for only
a small fraction of the hydrophobic surface in C-CaMW. In fact,
calculations of the solvent-exposed surface area (in the presence of
Ca
) show a slightly larger exposure of hydrophobic
surface in C-CaMF92A (2837 Å
) than in the wild type
protein (2812 Å
). Thus, the Phe-92
Ala
mutation must cause a decrease in the exposure of other hydrophobic
residues. Such an effect would be consistent with an incomplete opening
of the domain upon Ca
binding. Similarly, the large
decrease in the affinity of smM13N peptide for C-CaMF92A, as compared
with the wild type protein, can be interpreted as resulting from
incorrect conformation of the binding site rather than from the lack of
an important contact site (the phenyl ring of Phe-92).
The two types
of mutations used in this study have opposite effects on Ca binding. The large decrease in Ca
affinity
caused by the disulfide bond results from the coupling between the
binding of Ca
to the Ca
-chelating
loop and the subsequent conformational transition which is the hallmark
of the Ca
-dependent regulation. When the opening of
the structure is blocked with a disulfide bond then the coupling
mechanism prevents the Ca
-binding loop from attaining
the correct geometry, which results in a decrease in Ca
affinity. It is less clear why the removal of the phenyl ring at
position 92 should cause an increase in Ca
affinity.
It appears that two different mechanisms could explain such an effect,
each ascribing a different role to Phe-92.
One possibility is that
the hydrophobic phenyl ring of Phe-92 contributes to the stability of
the Ca-free conformation of C-CaM, in particular to
the interhelical interactions that keep the protein in the closed
conformation. The free energy of Ca
interaction with
its ligands, viz. carboxyl and carbonyl groups of the loop, is
used in part for breaking the interhelical interactions. Thus, a
protein modification that decreases such interactions would result in
an increase in apparent Ca
binding constant. The
lower unfolding temperature of C-CaMF92A (in the absence of
Ca
) appears to support this hypothesis. However, in
such a case no difference in Ca
affinity between
C-CaM85/112 and C-CaM85/112F92A should be observed. Since there is no
opening of the structure upon Ca
binding in these
mutants, differences in interhelical interactions in the
Ca
-free conformation should not play any role. This
is clearly not the case. In fact the F92A mutation causes a larger
increase in Ca
affinity in the presence of the
disulfide bond (3.3-fold increase) than in its absence (2.0-fold
increase).
The effect of the F92A mutation may also be interpreted
on the assumption that Phe-92 contributes to the coupling between
Ca binding at the loop and the conformational
transition. This would mean that in the absence of Phe-92 the ligands
at the Ca
binding loop can interact effectively with
Ca
even in the absence of or only after partial
opening of the structure. In such a case a smaller fraction of the
Ca
-binding free energy would be used for the
conformational transition since fewer hydrophobic interhelical
interactions would have to be broken. Moreover, in the
Ca
-bound conformation a smaller hydrophobic surface
would be exposed to solvent. Both factors would cause an increase in
the apparent Ca
binding constant. However, the
accessibility of the target binding hydrophobic surface would be
decreased. Our observations that F92A mutation causes an increase in
Ca
affinity and a decrease in bisANS binding and in
affinity for smM13N agree very well with these predictions. If the
conformational coupling in the F92A mutant is indeed weaker than the
coupling in the wild type protein, then locking this mutant with a
disulfide bond in the Ca
-free conformation should
have a smaller effect on the Ca
affinity. Again, our
finding that there is a 10- and a 5.9-fold decrease in apparent binding
constant depending whether Phe or Ala is present at position 92 is in
good agreement with this prediction. Clearly the hypothesis that Phe-92
makes an important contribution to the conformational coupling is
consistent with all our data. In an extreme case if Phe-92 was solely
responsible for the conformational coupling then C-CaMF92A and
C-CaM85/112F92A should have identical affinities for
Ca
. This is not the case; thus some other yet
unidentified structural elements are also involved.
In conclusion
our results indicate that Phe-92, the highly conserved phenylalanine
residue at the position immediately preceding the first Ca ligand in the C-terminal domain of CaM, makes an important
contribution to the Ca
-induced conformational
transition. This residue appears to be involved in the coupling between
Ca
binding at the loop and the opening of the
structure.