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INTRODUCTION |
Understanding detailed molecular mechanisms that govern
macromolecular interactions represents one of the major goals of
structural biology. One of the important prerequisites for success in
this line of research depends on the choice of an appropriate
biological system. Calcium-dependent target recognition by
calmodulin might be an ideal model system, because of its well
characterized nature and its central role in cellular metabolism.
Calmodulin (CaM)1 is a small,
acidic, eukaryotic Ca2+-binding protein of 148 amino acid
residues that is arranged into two lobes of similar size and structure
(1, 2). Each lobe consists of two EF hand helix-loop-helix
Ca2+-binding motifs, and thus CaM is capable of binding two
Ca2+ ions per lobe, four in total. NMR solution structures
of apo-CaM reveal that Ca2+ binding leads to significant
structural rearrangements of the CaM molecule (3, 4). The alignment of
the helices within each lobe changes upon Ca2+ binding,
resulting in the exposure of two methionine-rich hydrophobic "patches," one in each lobe (5, 6). These hydrophobic patches enable CaM in the Ca2+-loaded form (Ca2+-CaM)
to interact with a number of intracellular proteins and enzymes that
are involved in a wide variety of different biochemical processes
(7-9). CaM-binding sequences are generally 15-25 amino acids long
with little amino acid sequence homology, but the majority do have the
propensity to form an amphipathic
-helix with one or two aromatic or
bulky hydrophobic "anchor" residues (often tryptophans) located on
the hydrophobic face (10-12). Binding of a target peptide to
Ca2+-CaM occurs via distinct conformational changes upon
which the Ca2+-CaM molecule wraps around the peptide,
forming a globular complex (13-15). This is enabled by the unwinding
of the central linker region of CaM, which is actually quite flexible
in solution (16, 17). The peptide, which is unstructured in the unbound
state, forms an
-helix upon binding to CaM, and residues on the
hydrophobic face of the peptide, especially the anchor residues,
interact extensively with the hydrophobic patches of CaM. All
interactions between CaM and the peptide are through amino acid side
chains, which is unique for a protein-protein complex.
It is curious how so many different target sequences with relatively
little sequence homology can be bound by CaM with such high affinity. A
primary reason no doubt has to be hydrophobic effects, because of the
well known importance of the hydrophobic patches of CaM and their
interaction with the hydrophobic face of target sequences.
"Classical" hydrophobic interactions involve the favorable burial
of nonpolar groups from the contact with water and are considered to be
entropic in nature (18). However, in a recent calorimetric study, it
was shown that binding of a CaM target peptide from smooth muscle
myosin light chain kinase (smMLCK) is driven by enthalpic not entropic
factors (19). It could be that the increase in solvent entropy is
offset by other phenomena, such as the loss of mobility of some amino
acid side chains (20, 21) or the loss of backbone entropy in the target peptide as it forms an
-helix in the complex (22). Enthalpic effects
must also play a key role, and these could include van der Waal's
interactions among the hydrophobic residues in the complex, as well as
salt bridges and hydrogen bonds between acidic residues on CaM and
basic residues on the target peptide. Because of the promiscuous nature
of CaM in its ability to bind such a wide range of target sequences, it
is quite possible that the relative contribution of the various factors
involved in binding could be different for different target peptides.
Thus, a detailed calorimetric investigation of the binding of many
different peptides to CaM is warranted. Because of the vast amount of
knowledge and the pivotal importance of CaM/target recognition in
eukaryotic cells, a more complete understanding of the detailed
mechanism of Ca2+-dependent CaM/peptide
interactions can lay down the foundation for design of specific targets
and inhibitors for this and other related systems.
In this paper we report the results of the direct calorimetric
measurements of thermodynamics of complex formation of nine different
peptides with Ca2+-CaM. These peptides were derived from
the CaM-binding sequences of CaM-dependent protein kinase I
(23), cyclic nucleotide phosphodiesterase (22), caldesmon (24-26),
constitutive cerebellar nitric-oxide synthase (27, 28), and bee venom
melittin.2 The peptides
studied are illustrated in Fig. 1. Analysis of the thermodynamic data
for CaM-peptide complex formation allowed us to shed more light on the
nature of physical forces underlying Ca2+-CaM/target interactions.
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MATERIALS AND METHODS |
Calmodulin and Peptides--
The CaM expression plasmid pCaM,
which contains a synthetic mammalian CaM gene, was a gift from
Dr. T. Grundström (University of Umeå, Sweden) and has
been described elsewhere (27, 30). CaM was purified from
Escherichia coli cells containing pCaM by published methods
(31-33). Melittin (MEL) was purchased from Sigma. The peptides
corresponding to the CaM binding sequence of cerebellar nitric-oxide
synthase (cNOS), cyclic nucleotide phosphodiesterase (PDE),
calmodulin-dependent protein kinase I (CaMKI), caldesmon (CaD-A, CaD-A*, CaD-B1, and CaD-B2), and the C terminus of melittin (MLC) were commercially synthesized at the Core Facility for
Protein/DNA Chemistry at Queens University (Kingston, Canada). The
sequences of these are shown in Fig. 1.
All peptides were additionally purified on Sephasyl-Peptide C18 12 µ (Amersham Pharmacia Biotech) reverse-phase column attached to the
AKTA system using 0-100% acetonitrile gradient in the presence
of 0.1% trifluoroacetic acid (34). The purity of peptides was >95%
as judged by analytical high pressure liquid chromatography.

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Fig. 1.
Alignment of the CaM-binding peptides used in
this study, as well as smMLCKp (19). The peptides are aligned
according to the position of their important hydrophobic anchor
residues (shaded), which bind to the C-terminal lobe of CaM.
The exceptions to this are MEL and MLC, which are shown in the reverse
(C to N termini) orientation; MEL binds to Ca2+-CaM with
80% being in a parallel orientation, i.e. C-terminal
hydrophobic anchor residue binds to C-terminal lobe of
CaM,2 in contrast to the other peptides.
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For the titrations of Ca2+-CaM with all but PDE and CaD-A*
peptides, the stock solutions were dialyzed extensively against at least two changes of corresponding PIPES buffer containing 5 mM PIPES, 100 mM NaCl, 2 mM
CaCl2, pH 7.0. In the case of the CaMKI peptide, additional
experiments were performed in buffers that contained 5 mM
imidazole, sodium cacodylate, or MOPS, pH 7.0, supplemented with 100 mM NaCl and 2 mM CaCl2.
The PDE and CaD-A* peptides contain cysteine residues potentially
capable of forming intermolecular disulfide bonds. To keep PDE and
CaD-A* in the reduced form, these peptides were incubated for 2 h
in 50 mM Tris pH 7.5 buffer containing 1%
-mercaptoethanol followed by dialysis against 5 mM
PIPES, 100 mM NaCl, 2 mM CaCl2, pH
7.0, buffer containing the reducing agent 1 mM
tris-(carboxyethyl)-phosphine. For PDE and CaD-A* titration
experiments, CaM was also dialyzed against the
tris-(carboxyethyl)-phosphine-containing buffer.
Spectrapor CE dialysis membranes with a 1000-Da molecular mass
cut-off were used for dialyses. The concentrations of stock solutions
of CaM and peptides after dialysis were measured
spectrophotometrically. The following molar extinction coefficients
have been used: CaM, 2,900 M
1
cm
1 at 276 nm; CaMKI, PDE, MEL, MLC, CaD-A, and CaD-A*,
5690 M
1 cm
1 at 280 nm; and
cNOS, 386 M
1 cm
1 at 258 nm. The
extinction coefficients were calculated according as described (35)
taking into account the corrections for light scattering (36).
Methylation of the cysteine residue of the CaD-A* peptide was performed
using methyl-4-nitrobenzenesulfonate, as described (37). The degree of
modification was checked by the reaction with the Cys-specific reagent
5,5-dithiobis-2-nitrobenzoic acid by monitoring the changes in
absorbance at 412 nm. The reaction mixture was subjected to
chromatography on Sephasyl-Peptide C18 12µ (Amersham Pharmacia
Biotech) reverse-phase column on an AKTA system using 0-100%
acetonitrile gradient in the presence of 0.1% trifluoroacetic acid.
Isothermal Titration Calorimetry--
The overall procedure for
ITC experiments was similar to that described previously (38). Briefly,
5 µl of the peptide solution at concentrations between 0.5 and 1.3 mM were injected into the cell containing
Ca2+-CaM. The concentration of CaM in the cell varied
between 0.01 and 0.06 mM depending on the magnitude of the
observed heat effects. In the case of melittin, the titration was
reversed, i.e. melittin at concentration 0.016 mM was in the cell, and CaM at concentration 0.6 mM was used in the titration syringe. This was done to
avoid the heat of melittin dilution because melittin forms tetramers at
high concentrations (39). The blank injections of titrant into
corresponding buffer were used to account for the heat of mixing and
dilutions. It was found that in all cases the heat effects upon blank
injections were less than 1% of the heat effect of CaM-peptide
interactions. The heat of the reaction, Q, was obtained by
integrating the peak after each injection of peptide ligand using
ORIGIN software provided by the manufacturer. The heat of the reaction
at each injection is related to the calorimetric enthalpy of binding,
Hcal, and the other thermodynamic parameters as follows (40).
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(Eq. 1)
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where
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(Eq. 2)
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n is the stoichiometry of the peptide/CaM complex,
Ka is the association constant, [CaM]t
is the amount of calmodulin in the ITC cell with the volume
Vo, and [pep]t is the total
concentration of a peptide.
Water-accessible Surface Area (ASA) Calculation--
ASA values
were computed using the modeled three-dimensional structures of CaM
complex with the studied peptides as described (41). The changes in the
surface area were divided into four types (41): 1)
ASAbb, the surface area of the backbone atoms C,
O, N; 2) ASAarm, the aromatic surface area that
is defined as all carbon atoms for Phe, Tyr, Trp except CA, CB; 3)
ASAalp, the surface area for aliphatic group
that includes all carbon atoms except those that are defined as
backbone or aromatic; and 4) ASApol, the surface
area for the polar groups that include all side chain oxygen, nitrogen,
and sulfur atoms. Two different models for the calculations of the
changes in ASA upon peptide/Ca2+-CaM complex formation have
been used.
For Type I binding, the changes in the ASA upon complex formation,
ASA, were calculated as follows.
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(Eq. 3)
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where
ASA
is the
water-accessible surface of CaM-peptide complex,
ASA
is the
water-accessible surface area of free CaM, and
ASA
is the
water-accessible surface area of an unstructured peptide; superscript
"i" represents one of the four type of the surface areas: backbone,
polar side chains, aliphatic, and aromatic.
The CaM-peptide complex was modeled according to one of the several
known x-ray and NMR structures, 1CDL, which represents an x-ray
structure of the smMLCK/Ca2+-CaM complex (14). The rest of
the peptides were threaded into this structure using sequence alignment
of Yap et al. (42) performed in the environment of
Swiss/Protein Data Bank viewer as described (43). Several control
calculations have been performed to ensure that the threading procedure
is reasonably reliable in terms of ASA calculations. The most
compelling evidence for the correctness of our threading is comparison
of the surface area of smMLCK/Ca2+-CaM complex calculated
using Protein Data Bank entry 1CDL with the area of the complex after
smMLCK was threaded into the Protein Data Bank entry 1CDM instead of
the original sequence corresponding to calmodulin-dependent
protein kinase II. The difference is very small particularly in terms
of predicted heat capacity change upon complex formation,
Cp (see Table II). This is mostly due to the
fact that the peptides become largely buried upon binding to CaM.
The surface area of the free CaM was calculated as follows. We decided
not to use the reported structural coordinates of CaM based on the fact
that even small perturbations in the conformation because of the
refinement procedure, crystal symmetry, and resolution significantly
affect the absolute values of ASA. This might lead to erroneous results
because we are looking at small difference,
ASA, between
two large numbers, ASACaM
pept and
ASACaM. Thus, free CaM for the purpose of ASA
calculations was modeled as a sum of surfaces.
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(Eq. 4)
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where ASA
and
ASA
are the
water-accessible surfaces of the N-terminal (amino acid residues 1-76)
and the C-terminal (amino acid residues 76-148) domains of CaM.
ASA
and
ASA
were calculated
using the coordinates of the CaM-peptide complex by simply removing the
coordinates of the peptide and using only the coordinates for the
residues 1-76 or 76-148, respectively. The change in the surface area
calculated using the structure of unligated CaM or as a sum of isolated
domains did not have significant effect on the absolute values of the
ASA change (data not shown).
The Type II binding model describes binding of the peptide to only one
of the N- or C-terminal lobes of CaM. For the purpose of surface area
calculations for Type II binding, hypothetical complexes were modeled
as follows. The coordinates of one of the domains in the threaded
structure were removed, and surface area was calculated using
coordinates for the remaining peptide and another domain plus a hinge
region (amino acid residues 72-86). The surface area change upon
complex formation was estimated as follows.
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(Eq. 5)
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(Eq. 6)
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where the subscripts TIIN and TIIC
reflect Type II binding of peptides to N- and C-terminal lobes of CaM,
respectively; ASACaMN
pept and ASACaMC
pept are the surface
areas of the peptide/N-terminal domain and peptide/C-terminal domain
complexes; ASACaMN and
ASACaMC are the surface areas of N-
or C-terminal domains of CaM in the absence of the other domain; and
ASApep is the surface area of the unbound
peptide in the fully extended conformation (see above). As a test of
correctness of this model, we also performed calculations based on the
NMR structure of the complex of Ca2+-CaM with a target
peptide from a Ca2+ pump using Protein Data Bank entry 1CFF
(44). The peptide in this structure binds to the C-terminal domain. The
surface area change upon complex formation was calculated as
follows.
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(Eq. 7)
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where ASA1CFF is the surface area of the
CaM-peptide complex and ASACaM1CFF is the surface area of the CaM only. The results of the calculations using Equations 6 and 7 are very similar (Table II).
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RESULTS AND DISCUSSIONS |
The Enthalpy of Ca2+-CaM/Peptide Complex
Formation--
Fig. 2A shows
representative results of calorimetric titrations of
Ca2+-CaM with two different peptides, CaMKI and PDE. The
interaction of CaMKI with Ca2+-CaM is exothermic. In
contrast, the reaction of PDE with Ca2+-CaM is endothermic.
The sum of the areas of the peaks after each injection normalized per
amount of CaM in the cell represents the enthalpy of binding of a given
peptide to Ca2+-CaM,
Hcal. The
experimentally measured enthalpy,
Hcal,
consists of two parts.

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Fig. 2.
A, calorimetric traces of the titration
profiles. Injection of PDE into the ITC cell containing
Ca2+-CaM (a) or buffer (b) at
20 °C are compared with the injections of CaMKI peptide into the
cell containing Ca2+-CaM (c) or buffer
(d) at 25 °C. The buffer in all these experiments was 5 mM PIPES, 100 mM NaCl, pH 7.0. B,
the dependence of the experimental enthalpy of interactions between
CaMKI and Ca2+-CaM at 25 °C on the enthalpy of
ionization of the different buffers used. The solid line
represents a linear fit of the experimental data. C, the
dependence of the heat of peptide/Ca2+-CaM interactions on
the concentration of the peptide in solution. Symbols indicate
experimental data. , CaMKI, 25 °C; , PDE, 20 °C; ,
CaD-A, 15 °C. Solid lines show the fit to Equation 1
using parameters listed in Table I.
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|
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(Eq. 8)
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where
Hb is the enthalpy of binding
per se and
n+·
Hion is the
enthalpy of linked protonation effects (LPE). The latter arises from
the fact that the pKa value(s) of one or several
groups on the ligand (peptide) or macromolecule (Ca2+-CaM)
can change as a result of complex formation, as has been observed in a
variety of other systems (45-50). Hence, the change in the protonation
upon binding can be characterized as the difference in the number of
protons bound before and after complex formation,
n+. Equation 8 provides an experimental test
for LPE by measuring the binding reaction in the presence of buffers
with very different enthalpies of ionization,
Hion; a plot of
Hcal versus
Hion will allow the estimate of
n+. Fig. 2B compares the
experimental enthalpies,
Hcal, of the
CaMKI-Ca2+-CaM complex formation in four different buffers
with different enthalpies of ionizations (51): sodium cacodylate
(
Hion =
4 kJ/mol), PIPES
(
Hion = 12 kJ/mol), MOPS
(
Hion = 23 kJ/mol), and imidazole
(
Hion = 36 kJ/mol). The absence of the
dependence of
Hcal on the
Hion of the buffers shown in Fig.
2B indicates that the interactions of Ca2+-CaM
with CaMKI are devoid of LPE (
n+ is only
-0.03). This conclusion is in accord with the results obtained for
other peptide/Ca2+-CaM complexes (19, 52). Because peptide
binding to Ca2+-CaM is not accompanied by LPE, most of the
experiments were performed in PIPES buffers, and the measured
enthalpies were considered to be entirely the enthalpies of binding,
i.e.
Hcal =
Hb. The enthalpies of binding of eight
different peptides to Ca2+-CaM measured at different
temperatures are presented in the Table I. It is remarkable that there is a great
variation in the enthalpies of binding ranging from +90 kJ/mol to
66
kJ/mol.
The Stoichiometry of Peptide/Ca2+-CaM
Complex--
Under the conditions of the ITC experiment, the binding
of most peptides to Ca2+-CaM is close to stoichiometric.
This can be seen in Fig. 2A, which shows that the heat
effects during initial injections are comparable and are followed by a
quick disappearance of heat effects in later injections, indicating
that saturation of Ca2+-CaM with the peptide has been
achieved. This means that the molar concentration ratio of peptide to
Ca2+-CaM at which the heat of binding on the titration
curve reaches 50% of total heat corresponds to the stoichiometry of
the binding. For the studied peptides this stoichiometry is essentially
one molecule of peptide per one molecule of Ca2+-CaM (Table
I). This includes the PDE peptide, which in a previous study had been
observed to bind Ca2+-CaM with a stoichiometry of 2 peptides per CaM molecule (53). The results here, as well as those of
another study (22), however, strongly suggest a 1:1 mode of binding for
this PDE peptide to CaM. An alternative way of estimating the
stoichiometry of the peptide/Ca2+-CaM complex formation is
from the fit of the experimental data. In all cases the difference
between the stoichiometry obtained from the saturation point on the
titration curve and the fit of the data to the Equation 1 was
insignificant (less than 5%) compared with the absolute value of 1. In
only one case was significant deviation of stoichiometry from 1:1
observed. It was found that the stoichiometry of
CaMKI/Ca2+-CaM complex formation in sodium cacodylate
buffer is on the order of 0.8:1. Somewhat peculiar effects of
cacodylate on the conformation and stability of calmodulin have also
been previously observed (54).
The Association Constants for Peptide/Ca2+-CaM Complex
Formation--
In some cases the binding of the studied peptides to
Ca2+-CaM was very tight (>108
M
1) and thus precluded reliable determination
of the association constants for the peptide/Ca2+-CaM
complex (Fig. 2C). Nevertheless, one can obtain a rough
estimate of the binding constants by fitting the ITC titration data to a single-site binding model (equation 1). The results are presented in
Table I. Comparisons with the binding constants obtained using spectroscopic methods under equilibrium conditions certainly indicate that our estimates are in general close to the results of these more
precise measurements. For example, according to other studies (55, 56),
the association constant for cNOS/Ca2+-CaM is 5 × 108 M
1, comparable with our
estimate of 1.3 × 108 M
1.
Association constants lower than 108
M
1 can be reliably estimated from ITC
profiles using a nonlinear fit to the Equation 1. The results of the
fits are presented Table I. The quality of the fit of the
representative titration profiles with binding constants on the order
of 108 M
1, 107
M
1, and 106
M
1 are shown in Fig. 2C. The
Ka for CaD-A/Ca2+-CaM was estimated to
be 1.3 × 106 M
1 (24), in
excellent agreement with our estimate of (1.4 ± 0.1) × 106 M
1. In the case of CaMKI the
reported values are somewhat higher than our estimates (Table I). The
largest difference was observed in the case of PDE. The reported
Ka value for this peptide, 4.5 × 106 M
1 (22), is 2 orders of
magnitude lower than our estimate of 1.1 × 108
M
1. This difference might be because the
binding constant of Yuan et al. (22) was measured using a
competition experiment in the presence of high concentrations of
another divalent ion, magnesium, which can decrease the affinity of CaM
for some peptides (57). We note, however, that even this large
difference in the association constant results in only a relatively
small (25%) difference in the Gibbs energy of binding,
G. As a consequence the resulting estimates of the
entropy change upon peptide/Ca2+-CaM complex formation are
qualitatively similar.
Effect of Amino Acid Sequence on the Thermodynamics of
Binding--
The thermodynamics of binding of different peptides to
Ca2+-CaM depends on their sequence. For example MEL at
15 °C has an enthalpy of binding of 77.8 kJ/mol, whereas the MLC
peptide, which represents the C terminus of MEL, has a much lower
enthalpy of 16.7 kJ/mol. Both peptides have the same stoichiometry of
binding, but the shorter peptide MLC has 2 orders of magnitude lower
affinity (Table I). The difference in sequence between CaD-A and CaD-A*
is more subtle; CaD-A* has an additional Cys residue at the C terminus, which was originally included as a potential labeling position for
other studies. This leads to an increase in the enthalpy of binding
from
66 kJ/mol for CaD-A to
49 kJ/mol for CaD-A* but does not
affect the stoichiometry or the binding constant (Table I).
Interestingly, methylation of the Cys in CaD-A* did not notably affect
the thermodynamics of the interaction with Ca2+-CaM, which
suggests that the differences between CaD-A and CaD-A* are due to the
presence of an extra residue rather than the properties of the cysteine
itself. In addition to the peptides listed in Table I, we studied the
peptide CaD-B1, derived from the second CaM-binding region of caldesmon
(26, 58). We did not, however, observe any appreciable heat effect in
ITC experiments. It was thus concluded that this peptide has a
relatively low binding affinity (<104
M
1) in agreement with previous reports (26,
58).
The Effect of Temperature on the Enthalpy of Binding--
To
evaluate the effects of temperature on the thermodynamics of
peptide/Ca2+-CaM complex formation, experiments were
performed at different temperatures. Detailed calorimetric studies have
shown that temperature induced unfolding of CaM starts at ~30 °C
(59). Thus, to avoid complications from the effects of CaM
folding/unfolding reaction, we limited our titration experiments to the
temperature range of 5-25 °C. The enthalpy of binding for all
peptides to Ca2+-CaM has a strong temperature dependence,
as can be seen from Fig. 3. For all
studied peptides, an increase in temperature leads to a decrease in the
enthalpy of binding. The slope of
Hcal
versus temperature represents the heat capacity change upon
complex formation,
Cp. The values of
Cp are in all cases negative and depending on
the peptide vary between
1.3 and
3.7
kJ·mol
1·K
1. It is interesting to note,
however, that although the
Cp values are
different for different peptides, they can be separated into two
groups, clustered around
3.2 ± 0.5 kJ·mol
1·K
1 (smMLCK, CaMKI, MEL, and
cNOS) and
1.6 ± 0.3 kJ·mol
1·K
1
(PDE, MLC, CaD-A, and CaD-A*). This is particularly surprising because
there is no difference in the association constants or the absolute
values of enthalpy between these two groups of peptides (Table I). To
get insight into the source of the differences in the heat capacity
change upon peptide/Ca2+-CaM complex formation, we built
and analyzed model structures of peptide/Ca2+-CaM
complexes.

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Fig. 3.
The temperature dependence of the
experimental enthalpy of peptide/Ca2+-CaM
interactions. Green diamond, MEL; black
circle, cNOS; white inverted triangle, CaMKI; red
square, smMLCK (19); blue square, MLCK (78); blue
circle, PDE; orange triangle, MLC; gray inverted
triangle, CaD-A*; yellow square, CaD-A.
Solid lines (MEL, cNOS, CaMKI, smMLCK, and MLCK) and
dashed and dotted lines (PDE, MLC, CaD-A*, and CaD-A)
represent the linear fit of the experimental data with the slopes
representing the heat capacity change upon peptide/Ca2+-CaM
interactions and given in Table II.
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The Heat Capacity Change upon Binding--
It has been shown in
numerous instances that the heat capacity change upon protein unfolding
and protein-protein interactions can be calculated reasonably well
based on the changes in the water-accessible surface area for a given
process (41, 60-67). Because the structures of Ca2+-CaM in
complex with several peptide targets have been solved by x-ray
crystallography and NMR spectroscopy, we used these structures to
estimate the changes in the water-accessible surface areas upon complex
formation. For all calculations, we used the crystal structure of CaM
in complex with the CaM-binding peptide from smooth muscle myosin light
chain kinase, Protein Data Bank accession code 1CDL (14), with the
different peptide sequences threaded in as described under "Materials
and Methods." However, very similar results were obtained when the
Protein Data Bank entry 1CDM of the structure of the complex of CaM
with a target peptide from CaM-dependent protein kinase II
(15) was used (data not shown). These calculations are considered as
Type I binding for the reasons given below.
The changes in the surface area upon peptide/Ca2+-CaM
complex formation were used to estimate the expected heat capacity
change for this process using the following empirical relationship
(41).
|
(Eq. 9)
|
where the subscripts alp, arm, bb, and pol represent the changes
in the surface areas of aliphatic, aromatic, backbone, and polar atoms
respectively, and all four numerical coefficients are expressed in
J/(K·mol·Å2).
The results of the calculations using the surface area
changes obtained for Type I binding are
presented in the Table II and compared with the experimental values in
Fig. 4. In the case of four peptides
smMLCK, CaMKI, cNOS, and MEL, there is an excellent correspondence
between the experimental and calculated values. Similar calculations
performed by Wintrode and Privalov (19) on smMLCK/Ca2+-CaM
complex also found an excellent correlation between the experimental and calculated values of
Cp.

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Fig. 4.
Comparison of the experimental (blue
bars) heat capacities changes upon
peptide/Ca2+-CaM complex formation with the
Cp calculated according to
Equation 9 using structures modeled as Type I (red
bars) or Type II (green bars)
structures. The Cp, calc values for
Type II binding are average of three values shown in Table II.
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On the other hand these calculations fail to predict the observed heat
capacity changes for the other four peptides PDE, MLC, CaD-A, and
CaD-A*. For example, the experimental
Cp for
PDE/Ca2+-CaM complex is
2.0
kJ·mol
1·K
1. This value is almost 40%
lower than the
Cp calculated using the
three-dimensional structure modeled as a Type I
peptide/Ca2+-CaM complex.
It is important to note that the calculations using the Type I binding
model are able to predict the heat capacity changes for the group of
peptides that have
Cp values clustered around
3.2 kJ·mol
1·K
1 but not those around
1.6 kJ·mol
1·K
1. Most likely, PDE,
MLC, CaD-A, and CaD-A* bind differently to Ca2+-CaM, which
could be expected because these peptides are truncated, or partial,
CaM-binding sequences that do not align completely with the other four
peptides (Fig. 1). A preponderance of evidence supports a mode of
binding where these peptides interact primarily with one lobe of CaM
(22, 58, 68, 69).2 For example, Yuan et al. (70)
have shown that the Trp in the MLC peptide becomes significantly buried
upon interaction with the C-terminal lobe of Ca2+-CaM, but
MLC interacts only weakly with the N-terminal lobe. The PDE peptide
interacts significantly with Ca2+-CaM in what appears to be
a canonical fashion (22), but another study suggested that this peptide
might not represent the entire CaM-binding sequence of PDE (71). There
is another sequence upstream in the protein that can also bind
Ca2+-CaM, and it is thought that Ca2+-CaM binds
the two sequences simultaneously in activating the PDE enzyme (71).
This binding of CaM to a target molecule in two noncontiguous
stretches is also seen with smooth muscle caldesmon, although here the
CaM-binding sequences are considerably closer together such that the
entire CaM-binding region of caldesmon is within a 54-residue stretch
(Refs. 26 and 72 and references therein). It must be noted that in
these cases CaD-A and CaD-A* are the upstream CaM-binding sequence and
CaD-B is the downstream CaM-binding sequence. Small angle x-ray
scattering data show that Ca2+-CaM remains in an extended
conformation when it binds to a full-length peptide containing both
CaM-binding sequences of caldesmon (73). A relatively small structural
collapse upon binding is also seen in small angle x-ray scattering
results with the PDE
peptide.3 In contrast,
binding of an MLCK peptide or MLCK protein leads to a fully collapsed
conformation of Ca2+-CaM (13, 14, 74-77). It is important
to note that the heat capacity change for the MLCK/Ca2+-CaM
interactions is
3.8 kJ/mol (78), in agreement with the formation of
Type I complex.
To explain the difference between the calculated and experimentally
measured values of
Cp for PDE, MLC, CaD-A,
and CaD-A* peptides, we propose that these peptides bind primarily to
one lobe of Ca2+-CaM, most likely the C-terminal lobe,
which is supported by previous spectroscopic studies (79).2
The binding of an incomplete CaM-binding sequence to only the C-terminal lobe of Ca2+-CaM is also seen in the solution
NMR structure of the complex of Ca2+-CaM with a peptide
from a Ca2+ pump (44). We call this mode of binding Type
II. Obviously the interactions with just one lobe of CaM will be
accompanied by a much smaller change in water-accessible surface area
and thus might lead to smaller heat capacity changes. To estimate the
heat capacity change from the interactions of peptide with just one
domain of CaM, we calculated the expected surface area change for such interactions.
Table II presents the surface area changes calculated using
hypothetical Type II models, in which the helical peptide is bound only
to the N- or C-terminal lobe of CaM. These structures were modeled as
described under "Materials and Methods" (Equations 5-7).
Using these values of ASA, we estimated the expected heat capacity
changes for Type II binding. The results of the calculations performed
using Equation 9 are compared with the experimental data (Fig. 4). The
Type II binding model performed well in predicting the heat capacity
changes for the PDE, MLC, CaD-A, and CaD-A* peptides. In contrast, the
Type II model failed to predict the heat capacity changes for smMLCK,
CaMKI, cNOS, and MEL peptides. Thus, the structural calculations
suggest that smMLCK, CaMKI, cNOS, and MEL peptides form similar
structures in complex with Ca2+-CaM, i.e. both
lobes of CaM wrap around a helical peptide. The structural calculations
of
Cp also predict that the peptides PDE,
MLC, CaD-A, and CaD-A* interact primarily with just one lobe of
Ca2+-CaM. In addition to being the final CaM-bound state
for peptides such as the Ca2+ pump peptide (44),
interactions with only the C-terminal lobe of CaM are also thought to
represent an initial step in recognition of complete target sequences
by Ca2+-CaM (79). In the case of caldesmon, this is
supported by this study and other results. We have observed that the
CaD-B sequence binds relatively poorly to CaM, which is in agreement
with other work (24, 58). Intact caldesmon appears to bind CaM in an antiparallel fashion such that the CaD-B sequence interacts with the
N-lobe of CaM (72). Therefore, it seems likely that the CaD-A sequence
binds first to the C-terminal lobe of CaM, which raises the local
concentration of the CaD-B sequence, enabling it to bind to the
N-terminal lobe of CaM. Similar results were found with melittin, which
binds to CaM in a parallel manner; hence, MLC, which binds to the
C-lobe of CaM, binds tightly, whereas the N-terminal peptide of
melittin, MLN, binds poorly to CaM.2 An analogous situation
is also found with CaM-binding peptides from simian immunodeficiency
virus transmembrane glycoprotein (70). In some cases, the tighter
binding peptide can bind to CaM in a 2:1 ratio at higher concentrations
or under different sample conditions. For example, the PDE peptide was
observed by one group to bind CaM in a 2:1 stoichiometry (53), as was
MLC,2 and a shorter version of the CaD-A peptide (25).
What Is the Driving Force for the Peptide/Ca2+-CaM
Complex Formation?--
Based on the analysis of binding of smMLCK
peptide to Ca2+-CaM, it was proposed that van der Waal's
and electrostatic interactions and not hydrophobic effects are
primarily responsible for the peptide target recognition by
Ca2+-CaM. This conclusion is based on the fact that
smMLCK/Ca2+-CaM complex formation is enthalpically driven
(19). The results on an additional eight peptides presented in this
paper indicate, however, that such generalization of thermodynamics of
the peptide/Ca2+-CaM interactions is unjustified. Fig.
5 shows the enthalpic (
H) and entropic
(
T·
S) contributions to the Gibbs energy of the peptide/Ca2+-CaM complex formation. The difference is
striking and importantly is not associated with the mode of binding
(i.e. Type I versus Type II). The smMLCK, CaMKI,
CaD-A, and CaD-A* peptides interact with Ca2+-CaM primarily
through enthalpic interactions. The interactions of the
cNOS, MLC, PDE, and MEL peptides are defined by the entropic factors. Although the variations in the enthalpic and entropic components are enormous (e.g. the enthalpy varies from
68
to 60 kJ/mol) the resulting Gibbs energy change upon complex formation varies only between
33 and
47 kJ/mol. Thus,
peptide/Ca2+-CaM interactions, as well as other systems,
exhibit the well known enthalpy-entropy compensation phenomenon
(80-82).

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Fig. 5.
Comparison of the enthalpic
( Hcal, blue
bars) and entropic ( T· S,
red bars) contributions to the Gibbs energy
( G, green bars) of
peptide/Ca2+-CaM complex formation at 25 °C. The
Gibbs energy values were calculated as G = R·T·ln(Ka), where
Ka values were taken from the last column of
Table I. The entropy changes were calculated as S = ( Hcal G)/T. Data
for smMLCK peptide are from Ref. 19.
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To look deeper into possible mechanisms of peptide/Ca2+-CaM
interactions, we attempted to find a correlation between the observed differences in the enthalpies of binding and certain physico-chemical parameters characterizing these peptides. No correlation of the enthalpy of binding with the length of the peptides, their relative hydrophobicity, number of ionizable groups, relative amount, and type
of surface area buried upon binding. The only notable property for all
peptides is that although they are unfolded to a different degree in
the unbound state, they all form helical structure in the complex with
Ca2+-CaM. Furthermore, helical structure is formed in both
Type I and Type II binding modes. Thus, helix folding is linked to the binding to Ca2+-CaM, and contribution of this linkage to
the observed thermodynamics of the peptide/Ca2+-CaM complex
formation will depend on the helix propensity of a peptide. The
thermodynamics of the helix-coil transition is well studied (83, 84),
and recent direct calorimetric studies (34)4 have shown that the
enthalpy of helix folding (coil
helix transition) is negative and
is on the order of
4 kJ/mol/amino acid residue. Therefore, the
binding enthalpy of a fully helical peptide to Ca2+-CaM
will be relatively small but positive and will be defined by the
positive enthalpy of dehydration of the binding interfaces and the
negative enthalpy of interactions between these interfaces (41,
86).
The entropy change upon binding of an
-helical peptide to
Ca2+-CaM will be defined by several factors. There is a
relatively small negative entropy associated with the loss of
translational and rotational degrees of freedom upon complex formation
(87-90). The immobilization of the side chains (20, 21) and the loss of side chain entropy will also have a small and negative contribution (91, 92). The major contribution to the entropy of binding will,
however, be defined by the large positive entropy of dehydration of the
binding interface (41). Thus, it is expected that the entropy change
upon a peptide
-helix binding to Ca2+-CaM will be large
and positive. Thus, in the absence of linkage to the folding of a
helix, the peptide/Ca2+-CaM interactions are
entropically driven, which is consistent with the dominant
role of hydrophobic interactions for the complex formation (18).
Interestingly, in a CaM-binding peptide from skeletal myosin light
chain kinase, alanine substitutions, which presumably increase the
-helical propensity of the sequence, increased the CaM-binding
affinity of the peptides (93), illustrating the importance of entropy
in the binding process. This is also supported by a large negative heat
capacity change upon complex formation (Fig. 4), by the amphipathic
character of the peptides (11, 94, 95), and by the importance of the
Met-residues of Ca2+-CaM for peptide binding (5, 6, 11) and
is consistent with structural calculations indicating that ~65-70%
of the surface area buried upon complex formation is nonpolar (Table
II).
Conclusions--
There is an important consequence of these
findings: one might expect that the peptide sequences recognized by
Ca2+-CaM are already in a helical conformation when they
are part of an intact protein. A structure of a
Ca2+-CaM-activated protein that illustrates the structural
basis of CaM activation is the x-ray crystal structure of
calcium/calmodulin-dependent protein kinase I (96). The
C-terminal calmodulin recognition sequence in this protein (which
corresponds to the sequence of CaMKI peptide studied here) is partially
in a helical conformation, but Trp303, the important
anchoring tryptophan residue, is in a random coil region of the protein
and strikingly points away from the rest of the protein into the
solvent, ready to be bound by Ca2+-CaM. Trp303,
which binds to the C-terminal lobe of CaM, presumably binds CaM first
in a Type II fashion, after which a structural rearrangement occurs to
the final complex we refer to as Type I (23). As mentioned earlier,
induction of
-helical structure in the CaM-binding sequence of a
target protein is an important step in the activation of target enzymes
(22). If the target sequence is already in a helical conformation, less
of a rearrangement would occur, and there would be no need to spend
energy on coil-helix transition, and thus the affinity of
Ca2+-CaM to the target will be much higher. On a practical
side, an efficient peptide-based calmodulin inhibitor will require, in addition to being amphipathic (97), to have a stable helical conformation. Restricted conformational entropy of the peptide in the
unbound state will have an important favorable contribution to the
peptide/Ca2+-CaM complex formation. Of course such a
prediction will require direct experimental validation.