Energetics of Target Peptide Binding by Calmodulin Reveals Different Modes of Binding*

Richard D. BrokxDagger §, Maria M. Lopez§, Hans J. VogelDagger , and George I. Makhatadze||

From the Dagger  Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada and  Department of Biochemistry and Molecular Biology, Penn State College of Medicine, Hershey, Pennsylvania 17033

Received for publication, December 6, 2000, and in revised form, January 24, 2001




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ABSTRACT
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Thermodynamic parameters of interactions of calcium-saturated calmodulin (Ca2+-CaM) with melittin, C-terminal fragment of melittin, or peptides derived from the CaM binding regions of constitutive (cerebellar) nitric-oxide synthase, cyclic nucleotide phosphodiesterase, calmodulin-dependent protein kinase I, and caldesmon (CaD-A, CaD-A*) have been measured using isothermal titration calorimetry. The peptides could be separated into two groups according to the change in heat capacity upon complex formation, Delta Cp. The calmodulin-dependent protein kinase I, constitutive (cerebellar) nitric-oxide synthase, and melittin peptides have Delta Cp values clustered around -3.2 kJ·mol-1·K-1, consistent with the formation of a globular CaM-peptide complex in the canonical fashion. In contrast, phosphodiesterase, the C-terminal fragment of melittin, CaD-A, and CaD-A* have Delta Cp values clustered around -1.6 kJ·mol-1·K-1, indicative of interactions between the peptide and mostly one lobe of CaM, probably the C-terminal lobe. It is also shown that the interactions for different peptides with Ca2+-CaM can be either enthalpically or entropically driven. The difference in the energetics of peptide/Ca2+-CaM complex formation appears to be due to the coupling of peptide/Ca2+-CaM complex formation to the coil-helix transition of the peptide. The binding of a helical peptide to Ca2+-CaM is dominated by favorable entropic effects, which are probably mostly due to hydrophobic interactions between nonpolar groups of the peptide and Ca2+-CaM. Applications of these findings to the design of potential CaM inhibitors are discussed.




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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 alpha -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 alpha -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 alpha -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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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.

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% beta -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, Delta Hcal, and the other thermodynamic parameters as follows (40).
Q=<FR><NU>n · [<UP>CaM</UP>]<SUB><UP>t</UP></SUB><UP> · &Dgr;</UP>H<SUB><UP>cal</UP></SUB> · V<SUB><UP>a</UP></SUB></NU><DE>2</DE></FR> · <FENCE>A−<RAD><RCD>A<SUP>2</SUP>−<FR><NU>4 · [<UP>pep</UP>]<SUB><UP>t</UP></SUB></NU><DE>n · [<UP>CaM</UP>]<SUB><UP>t</UP></SUB></DE></FR></RCD></RAD></FENCE> (Eq. 1)
where
A=1+<FR><NU>[<UP>pep</UP>]<SUB><UP>t</UP></SUB></NU><DE>n · [<UP>CaM</UP>]<SUB><UP>t</UP></SUB></DE></FR>+<FR><NU>[<UP>pep</UP>]<SUB><UP>t</UP></SUB></NU><DE>n · K<SUB>a</SUB> · [<UP>CaM</UP>]<SUB><UP>t</UP></SUB></DE></FR>, (Eq. 2)
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, Delta ASA, were calculated as follows.
&Dgr;ASA=ASA<SUP><UP>i</UP></SUP><SUB><UP>CaM</UP>−<UP>pept</UP></SUB>−ASA<SUP><UP>i</UP></SUP><SUB><UP>CaM</UP></SUB>−ASA<SUP><UP>i</UP></SUP><SUB><UP>pep</UP></SUB> (Eq. 3)
where ASA<UP><SUB>CaM−pept</SUB><SUP>i</SUP></UP> is the water-accessible surface of CaM-peptide complex, ASA<UP><SUB>CaM</SUB><SUP>i</SUP></UP> is the water-accessible surface area of free CaM, and ASA<UP><SUB>pep</SUB><SUP>i</SUP></UP> 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, Delta 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, Delta 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.
ASA<SUB><UP>CaM</UP></SUB>=ASA<SUP>1–76</SUP><SUB><UP>CaM</UP></SUB>+ASA<SUP>76–148</SUP><SUB><UP>CaM</UP></SUB> (Eq. 4)
where ASA<UP><SUB>CaM</SUB><SUP>1–76</SUP></UP> and ASA<UP><SUB>CaM</SUB><SUP>76–148</SUP></UP> 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<UP><SUB>CaM</SUB><SUP>1–76</SUP></UP> and ASA<UP><SUB>CaM</SUB><SUP>76–148</SUP></UP> 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 Delta 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.
&Dgr;ASA<SUB><UP>TII</UP><SUB><UP>N</UP></SUB></SUB>=ASA<SUB><UP>CaM</UP><SUB><UP>N</UP></SUB>−<UP>pept</UP></SUB>−ASA<SUB><UP>CaM</UP><SUB><UP>N</UP></SUB></SUB>−ASA<SUB><UP>pep</UP></SUB> (Eq. 5)

&Dgr;ASA<SUB><UP>TII</UP><SUB><UP>C</UP></SUB></SUB>=ASA<SUB><UP>CaM</UP><SUB><UP>C</UP></SUB>−<UP>pept</UP></SUB>−ASA<SUB><UP>CaM</UP><SUB><UP>C</UP></SUB></SUB>−ASA<SUB><UP>pep</UP></SUB> (Eq. 6)
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.
&Dgr;ASA<SUB><UP>TII</UP></SUB>=ASA<SUB>1<UP>CFF</UP></SUB>−ASA<SUB><UP>CaM</UP><SUB><UP>1CFF</UP></SUB></SUB>−ASA<SUB><UP>pep</UP></SUB> (Eq. 7)
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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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, Delta Hcal. The experimentally measured enthalpy, Delta 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. open circle , CaMKI, 25 °C; triangle , PDE, 20 °C; black-square, CaD-A, 15 °C. Solid lines show the fit to Equation 1 using parameters listed in Table I.


&Dgr;H<SUB><UP>cal</UP></SUB>=&Dgr;H<SUB><UP>b</UP></SUB>+&Dgr;n<SUB><UP>+</UP></SUB> · &Dgr;H<SUB><UP>ion</UP></SUB> (Eq. 8)
where Delta Hb is the enthalpy of binding per se and Delta n+·Delta 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, Delta 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, Delta Hion; a plot of Delta Hcal versus Delta Hion will allow the estimate of Delta n+. Fig. 2B compares the experimental enthalpies, Delta Hcal, of the CaMKI-Ca2+-CaM complex formation in four different buffers with different enthalpies of ionizations (51): sodium cacodylate (Delta Hion = -4 kJ/mol), PIPES (Delta Hion = 12 kJ/mol), MOPS (Delta Hion = 23 kJ/mol), and imidazole (Delta Hion = 36 kJ/mol). The absence of the dependence of Delta Hcal on the Delta Hion of the buffers shown in Fig. 2B indicates that the interactions of Ca2+-CaM with CaMKI are devoid of LPE (Delta 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. Delta Hcal = Delta 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.


                              
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Table I
Thermodynamics of CaM-peptide interactions
ND, not determined.

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, Delta 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 Delta Hcal versus temperature represents the heat capacity change upon complex formation, Delta Cp. The values of Delta 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 Delta 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.

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).
&Dgr;C<SUB><UP>P</UP></SUB>=2.14 · &Dgr;ASA<SUB><UP>alp</UP></SUB>+1.55 · &Dgr;ASA<SUB><UP>arm</UP></SUB>−1.81 · &Dgr;ASA<SUB><UP>bb</UP></SUB>− 0.88 · &Dgr;ASA<SUB><UP>pol</UP></SUB> (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 Delta Cp.


                              
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Table II
Experimental and calculated values of the heat capacity changes upon peptide/Ca2+-CaM complex formation



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Fig. 4.   Comparison of the experimental (blue bars) heat capacities changes upon peptide/Ca2+-CaM complex formation with the Delta Cp calculated according to Equation 9 using structures modeled as Type I (red bars) or Type II (green bars) structures. The Delta Cp, calc values for Type II binding are average of three values shown in Table II.

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 Delta Cp for PDE/Ca2+-CaM complex is -2.0 kJ·mol-1·K-1. This value is almost 40% lower than the Delta 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 Delta 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 Delta 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 Delta 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 (Delta H) and entropic (-Delta 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 (Delta Hcal, blue bars) and entropic (-T·Delta S, red bars) contributions to the Gibbs energy (Delta G, green bars) of peptide/Ca2+-CaM complex formation at 25 °C. The Gibbs energy values were calculated as Delta G = -R·T·ln(Ka), where Ka values were taken from the last column of Table I. The entropy changes were calculated as Delta S = (Delta Hcal - Delta G)/T. Data for smMLCK peptide are from Ref. 19.

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 left-right-arrow 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 alpha -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 alpha -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 alpha -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 alpha -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.


    FOOTNOTES

* This work was supported in part by National Institutes of Health Grant GM54537 (to G. I. M.) and Medical Research Council of Canada Grant MT15237 (to H. J. V).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ These authors contributed equally to this work.

|| To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology/H171, Penn State University College of Medicine, 500 University Dr., Hershey, PA 17111. Tel.: 717-531-0712; Fax: 717-531-7072; E-mail: makhatadze@psu.edu.

Published, JBC Papers in Press, January 29, 2001, DOI 10.1074/jbc.M011026200

2 T. Yuan, H. Ouyang, M. E. Huque, K. Siivari, and H. J. Vogel, manuscript in preparation.

3 H. Yoshino, personal communication.

4 J. M. Richardson and G. I. Makhatadze, unpublished observations.


    ABBREVIATIONS

The abbreviations used are: CaM, calmodulin; CaD, chicken gizzard caldesmon; CaD-A, peptide of residues 651-666 of CaD; CaD-A*, CaD-A with an extra C-terminal cysteine residue; CaD-B1, peptide of residues 674-696 of CaD; CaMKI, CaM-dependent protein kinase I; cNOS, constitutive (cerebellar) nitric-oxide synthase; ITC, isothermal titration calorimetry; MEL, melittin; MLC, C-terminal 13-residue fragment of MEL; MLCK, skeletal muscle myosin light chain kinase; MOPS, 3-(N-morpholino)propanesulfonic acid; PDE, 3':5'-cyclic nucleotide phosphodiesterase; PIPES, 1,1-piperazinediethanesulfonic acid; smMLCK, smooth muscle myosin light chain kinase; ASA, accessible surface area; LPE, linked protonation effects.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSIONS
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