Peptide binding by calmodulin and its proteolytic fragments and by troponin C

Calmodulin and troponin C exhibit calcium-dependent binding of 1 mol/mol of dynorphin. The dissociation constants of the complexes, determined in 0.20 N KC1-1.0 mM CaCI2, pH 7.3, are 0.6 microM for calmodulin, 2.4 microM for rabbit fast skeletal muscle troponin C, and 9 microM for bovine heart troponin C. Experiments with deletion peptides of dynorphin show that peptide chain length and especially charge affect the binding of the peptides by calmodulin. Dynorphin, but not mastoparan or melittin, inhibits adenosinetriphosphatase activity in a reconstituted rabbit skeletal muscle actomyosin assay. The inhibition is partially reversed by the addition of calmodulin or troponin C in the presence of calcium. Calmodulin also exhibits calcium-dependent binding of a synthetic peptide corresponding to positions 104-115 of rabbit fast skeletal muscle troponin I. Mastoparan is a tetradecapeptide from the vespid wasp having exceptional affinity for calmodulin, with Kd approximately 0.3 nM [Malencik, D.A., & Anderson, S.R. (1983) Biochem. Biophys. Res. Commun. 114, 50]. The addition of 1 mol/mol of mastoparan to the complex of calmodulin with dynorphin results in complete dissociation of dynorphin. Similar titrations of the skeletal muscle troponin C-dynorphin complex produce a gradual dissociation consistent with a dissociation constant of 0.2 microM for the troponin C-mastoparan complex. Fluorescence anisotropy measurements using the intrinsic tryptophan fluorescence of mastoparan X show strongly calcium-dependent binding by proteolytic fragments of calmodulin. binding by proteolytic fragments of calmodulin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conformation and structural transitions in the EF-hands of calmodulin

Calcium plays a key role in cellular signal transduction. Calmodulin, a protein binding four calcium ions, is found in all eukaryotic cells and is believed to activate such processes. The calcium binding loop found in this protein, the canonical EF-hand, is also found in a large number of other proteins such as troponins, parvalbumins, calbindins etc. Earlier analysis of the amino acid sequences of these proteins with a view of understanding evolution of protein families and signaling mechanisms have provided extensive evidence for a characteristic double gene duplication event in this family of proteins. These analyses have been extended here to the three dimensional structures and the biophysical properties of the sequence segments of calmodulin EF-hands. The clear evolutionary history that shows up in sequences is not reflected as clearly in the conformation of individual EF-hands, which may be a consequence of the much higher conservation pressure on the structure. Some evidence for the proposed gene duplication is implicit in the apo-holo structural transitions of the EF-hands. The profile of amino acid properties that might be significant for calcium binding, however, clearly reflects the gene duplication. These profiles might also provide insightful information on the calcium affinity of the EF-hand motifs and the nature of amino acid residues that constitute them.     

Conformational changes upon calcium binding and phosphorylation in a synthetic fragment of calmodulin

We have recently investigated by far-UV circular dichroism (CD) the effects of Ca(2+) binding and the phosphorylation of Ser 81 for the synthetic peptide CaM [54-106] encompassing the Ca(2+)-binding loops II and III and the central alpha helix of calmodulin (CaM) (Arrigoni et al., Biochemistry 2004, 43, 12788-12798). Using computational methods, we studied the changes in the secondary structure implied by these spectra with the aim to investigate the effect of Ca(2+) binding and the functional role of the phosphorylation of Ser 81 in the action of the full-length CaM. Ca(2+) binding induces the nucleation of helical structure by inducing side chain stacking of hydrophobic residues. We further investigated the effect of Ca(2+) binding by using near-UV CD spectroscopy. Molecular dynamics simulations of different fragments containing the central alpha-helix of CaM using various experimentally determined structures of CaM with bound Ca(2+) disclose the structural effects provided by the phosphorylation of Ser 81. This post-translational modification is predicted to alter the secondary structure in its surrounding and also to hinder the physiological bending of the central helix of CaM through an alteration of the hydrogen bond network established by the side chain of residue 81. Using quantum mechanical methods to predict the CD spectra for the frames obtained during the MD simulations, we are able to reproduce the relative experimental intensities in the far-UV CD spectra for our peptides. Similar conformational changes that take place in CaM [54-106] upon Ca(2+) binding and phosphorylation may occur in the full-length CaM.     

Calcium binding to complexes of calmodulin and calmodulin binding proteins

The free energy of coupling for binding of Ca2+ and the calmodulin-sensitive phosphodiesterase to calmodulin was determined and compared to coupling energies for two other calmodulin binding proteins, troponin I and myosin light chain kinase. Free energies of coupling were determined by quantitating binding of Ca2+ to calmodulin complexed to calmodulin binding proteins with Quin 2 to monitor free Ca2+ concentrations. The geometric means of the dissociation constants (-Kd) for Ca2+ binding to calmodulin in the presence of equimolar rabbit skeletal muscle troponin I, rabbit skeletal muscle myosin light chain kinase, and bovine heart calmodulin sensitive phosphodiesterase were 2.1, 1.1, and 0.55 microM. The free-energy couplings for the binding of four Ca2+ and these proteins to calmodulin were -4.48, -6.00, and -7.64 kcal, respectively. The Ca2+-independent Kd for binding of the phosphodiesterase to calmodulin was estimated at 80 mM, indicating that complexes between calmodulin and this enzyme would not exist within the cell under low Ca2+ conditions. The large free-energy coupling values reflect the increase in Ca2+ affinity of calmodulin when it is complexed to calmodulin binding proteins and define the apparent positive cooperativity for Ca2+ binding expected for each system. These data suggest that in vitro differences in free-energy coupling for various calmodulin-regulated enzymes may lead to differing Ca2+ sensitivities of the enzymes.     

Ion channel regulation by calmodulin binding

While many ion channels are modulated by phosphorylation, there is growing evidence that they can also be regulated by Ca²⁺-calmodulin, apparently through direct binding. In some cases, this binding activates channels; in others, it modulates channel activities. These phenomena have been documented in Paramecium, in Drosophila, in vertebrate photoreceptors and olfactory receptors, as well as in ryanodine receptor Ca²⁺-release channels. Furthermore, studies on calmodulin mutants in Paramecium have shown a clear bipartite distribution of two groups of mutations in the calmodulin gene that lead to opposite behavioral and electrophysiological phenotypes. These results indicate that the N-lobe of calmodulin specifically interacts with one class of ion-channel proteins and the C-lobe with another.     

Phosphofructokinase is a calmodulin binding protein

A trial to purify myosin light chain kinase from crude myosin led to the isolation of a Mr 85 000 calmodulin binding protein different from this enzyme. Because it showed inherent phosphofructokinase activity we investigated its relation to this enzyme. We demonstrated identity to phosphofructokinase by a close to identical amino acid composition, by antigenic identity and a set of completely identical peptide maps. The calmodulin binding property was also shown for a fraction of the enzyme prepared by standard methods. First experiments show that Ca2+--calmodulin is a potent regulator of phosphofructokinase polymerization.     

Calcium release from intact calmodulin and calmodulin fragment 78-148 measured by stopped-flow fluorescence with 2-p-toluidinylnaphthalene sulfonate. Effect of calmodulin fragments on cardiac sarcoplasmic reticulum

Calcium release from high and low-affinity calcium-binding sites of intact bovine brain calmodulin (CaM) and from the tryptic fragment 78-148, purified by high-pressure liquid chromatography, containing only the high-affinity calcium-binding sites, was determined by fluorescence stopped-flow with 2-p-toluidinylnaphthalene sulfonate (TNS). The tryptic fragments 1-77 and 78-148 each contain a calcium-dependent TNS-binding site, as shown by the calcium-dependent increase in TNS fluorescence. The rate of the monophasic fluorescence decrease in endogenous tyrosine on calcium dissociation from intact calcium-saturated calmodulin (kobs 10.8 s-1 and 3.2 s-1 at 25 degrees C and 10 degrees C respectively) as well as the rate of equivalent slow phase of the biphasic decrease in TNS fluorescence (kobsslow 10.6 s-1 and 3.0 s-1 at 25 degrees C and 10 degrees C respectively) and the rate of the solely monophasic decrease in TNS fluorescence, obtained with fragment 78-148 (kobs 10.7 s-1 and 3.5 s-1 at 25 degrees C and 10 degrees C respectively), were identical, indicating that the rate of the conformational change associated with calcium release from the high-affinity calcium-binding sites on the C-terminal half of calmodulin is not influenced by the N-terminal half of the molecule. The fast phase of the biphasic decrease of TNS fluorescence, observed by the N-terminal half of the molecule. The fast phase of the biphasic decrease of TNS fluorescence, observed with intact calmodulin only (kobsfast 280 s-1 at 10 degrees C) but not with fragment 78-148, is most probably due to the conformational change associated with calcium release from low-affinity sites on the N-terminal half. The calmodulin fragments 1-77 and 78-148 neither activated calcium/calmodulin-dependent protein kinase of cardiac sarcoplasmic reticulum nor inhibited calmodulin-dependent activation at a concentration approximately 1000-fold greater (5 microM) than that of the calmodulin required for half-maximum activation (5.9 nM at 0.8 mM Ca2+ and 5 mM Mg2+) of calmodulin-dependent phosphoester formation.     

Al(3+) interaction sites of calmodulin and the Al(3+) effect on target binding of calmodulin

The interaction between calmodulin (CaM) and Al(3+) was studied by spectroscopic methods. Heteronuclear two-dimensional NMR data indicated that peaks related to the both lobes and middle of the central helix of CaM are largely affected by Al(3+). But chemical shift perturbation suggested that overall conformation of Ca(2+)-loaded CaM is not changed by Al(3+) binding. It is thought that Al(3+) interaction to the middle of the central helix is a key for the property of CaM's target recognition. If the structure and/or flexibility of the central helix are/is changed by Al(3+), target affinity to CaM must be influenced by Al(3+). Thus, we performed surface plasmon resonance experiments to observe the effect of Al(3+) on the target recognition by CaM. The data clearly indicated that target affinity to CaM is reduced by addition of Al(3+). All the results presented here support a hypothesis that Al(3+) may affect on the Ca(2+) signaling pathway in cells.     

Quantitative aspects of the development of a hydrophobic binding site on calmodulin by calcium binding

The interactive binding by calmodulin of Ca²⁺ and 1-anilinonaphthalene-8-sulfonate (1,8-ANS) has been examined. In the presence of saturating levels of Ca²⁺, calmodulin develops one moderately strong binding site for 1,8-ANS, plus one or more weaker sites. The binding of 1,8-ANS by unliganded, or singly liganded, calmodulin is slight; the development of a strong binding site, as well as the characteristic fluorescence enhancement and CD spectrum, requires the binding of two Ca²⁺ ions. Little further change occurs on binding additional Ca²⁺ ions.     

High affinity binding of the mastoparans by calmodulin

Calmodulin exhibits high affinity, calcium-dependent binding of the mastoparans — a group of cytoactive tetradecapeptides. The dissociation constants for the peptide-calmodulin complexes determined in 0.20 N KCl, 1.0 mM CaCl₂, pH 7.3 are ～0.3 nM for mastoparan, ～0.9 nM for mastoparan X, and ～3.5 nM for $\text{Polistes}$ mastoparan. The dissociation constant for the mastoparan-calmodulin complex is the smallest known for any calmodulin binding protein or peptide, suggesting that some type of peptide-calmodulin interaction could be physiologically significant.     

Binding of amphiphilic peptides to a carboxy-terminal tryptic fragment of calmodulin

Calmodulin (CaM) fragments 1-77 (CaM 1-77) and 78-148 (CaM 78-148) were prepared by tryptic cleavage of CaM. CaM 78-148 exhibited Ca2+-dependent binding to mastoparan X, Polistes mastoparan, and melittin with apparent dissociation constants less than 0.2 microM as judged from changes in the fluorescence spectrum and anisotropy of the single tryptophan residue of each of these cationic, amphiphilic peptides. This interaction was accompanied by a large spectral blue shift of the peptide fluorescence spectrum. These findings are consistent with earlier results [Malencik, D.A., & Anderson, S.R. (1984) Biochemistry 23, 2420-2428] on the binding of mastoparan X to CaM fragment 72-148. The binding of the peptide to CaM 78-148 also caused a significant loss of the accessibility of the peptide tryptophan to the fluorescence quencher acrylamide. The CaM 78-148 induced effects on the fluorescence spectra and tryptophan accessibility of the peptides were most pronounced for mastoparan X, a peptide with tryptophan on the apolar face of the putative amphiphilic helix. The data were comparable with results from parallel experiments on the Ca2+-dependent interaction of these peptides with intact CaM. Difference circular dichroic spectra suggested that binding to CaM 78-148 was associated with the induction of considerable degrees of helicity in the amphiphilic peptides, which by themselves have predominantly random coil structures in aqueous solution. This finding is also reminiscent of the interaction of these peptides with intact CaM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding of La3+ to calmodulin and its effects on the interaction between calmodulin and calmodulin binding peptide, polistes mastoparan

Binding of La(3+) to calmodulin (CaM) and its effects on the complexes of CaM and CaM-binding peptide, polistes mastoparan (Mas), were investigated by nuclear magnetic resonance (NMR) spectroscopy, fluorescence and circular dichroism spectroscopy, and by the fluorescence stopped-flow method. The four binding sites of La(3+) on CaM were identified as the same as the binding sites of Ca(2+) on CaM through NMR titration of La(3+) to uniformly (15)N-labeled CaM. La(3+) showed a slightly higher affinity to the binding sites on the N-terminal domain of CaM than that to the C-terminal. Large differences between the (1)H-(15)N heteronuclear single quantum coherence (HSQC) spectra of Ca(4)CaM and La(4)CaM suggest conformational differences between the two complexes. Fluorescence and CD spectra also exhibited structural differences. In the presence of Ca(2+) and La(3+), a hybrid complex, Ca(2)La(2)CaM, was formed, and the binding of La(3+) to the N-terminal domain of CaM seemed preferable over binding to the C-terminal domain. Through fluorescence titration, it was shown that La(4)CaM and Ca(2)La(2)CaM had similar affinities to Mas as Ca(4)CaM. Fluorescence stopped-flow experiments showed that the dissociation rate of La(3+) from the C-terminal domain of CaM was higher than that from the N-terminal. However, in the presence of Mas, the dissociation rate of La(3+) decreased and the dissociation processes from both global domains were indistinguishable. In addition, compared with the case of Ca(4)CaM-Mas, the slower dissociations of Mas from La(4)CaM-Mas and Ca(2)La(2)CaM-Mas complexes indicate that in the presence of La(3+), the CaM-Mas complex became kinetically inert. A possible role of La(3+) in the Ca(2+)-CaM-dependent pathway is discussed.     

Regulation of calmodulin binding to P-57. A neurospecific calmodulin binding protein

P-57 is a neural-specific calmodulin binding protein with novel calmodulin binding properties. P-57 exhibits higher affinity for calmodulin-Sepharose in the absence of free Ca2+ than in the presence of Ca2+ (Andreasen, T.J., Luetje, C.W., Heideman, W. & Storm, D.R. (1983) Biochemistry 22, 4615-4618; Cimler, B. M., Andreasen, T.J., Andreasen, K.I. & Storm, D.R. (1985) J. Biol. Chem. 260, 10784-10788). In this study, the dissociation constants for P-57 and immunopurified 5-[[(iodoacetylamino)ethyl]-amino]-1-naphthalenesulfonic acid-labeled calmodulin (AEDANS-CaM) were determined under low and high ionic strength conditions. In the absence of added KCl, the dissociation constants for the P-57 X AEDANS-CaM complex were 2.3 X 10(-7) +/- 6 X 10(-8) M and 1.0 X 10(-6) +/- 3 X 10(-7) M in the presence and absence of excess Ca2+ chelator. The addition of KCl to 150 mM increased the Ca2+-independent and -dependent dissociation constants to 3.4 X 10(-6) +/- 9 X 10(-7) M and 3.0 X 10(-6) +/- 9 X 10(-7) M, respectively. The association of P-57 with AEDANS-CaM under low Ca2+ conditions was determined as a function of KCl concentrations. By taking into account the amount of P-57 found in brain and its affinity for calmodulin, it is concluded that most or all of the CaM would be complexed to P-57 in unstimulated cells. P-57 was phosphorylated by the Ca2+-phospholipid-dependent protein kinase (protein kinase C) with a phosphate:protein molar ratio of 1.3. Phosphoamino acid analysis demonstrated phosphorylation at a serine residue. CaM decreased the rate of phosphorylation of P-57 by protein kinase C, and phosphorylation prevented P-57 binding to calmodulin-Sepharose. P-57 was not phosphorylated by the catalytic subunit of the cAMP-dependent protein kinase. It is proposed that P-57 binds and localizes calmodulin at specific sites within the cell and that free calmodulin is released locally in response to phosphorylation of P-57 by protein kinase C and/or to increases in intracellular free Ca2+. This regulatory mechanism, which appears to be specific to brain, would serve to decrease the response time for Ca2+-calmodulin-regulated processes.     

Energetics of target peptide binding by calmodulin reveals different modes of binding

Thermodynamic parameters of interactions of calcium-saturated calmodulin (Ca(2+)-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, DeltaC(p). The calmodulin-dependent protein kinase I, constitutive (cerebellar) nitric-oxide synthase, and melittin peptides have DeltaC(p) 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 DeltaC(p) 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 Ca(2+)-CaM can be either enthalpically or entropically driven. The difference in the energetics of peptide/Ca(2+)-CaM complex formation appears to be due to the coupling of peptide/Ca(2+)-CaM complex formation to the coil-helix transition of the peptide. The binding of a helical peptide to Ca(2+)-CaM is dominated by favorable entropic effects, which are probably mostly due to hydrophobic interactions between nonpolar groups of the peptide and Ca(2+)-CaM. Applications of these findings to the design of potential CaM inhibitors are discussed.     

1H NMR studies of mastoparan binding by calmodulin

Association with the cytoactive tetradecapeptide mastoparan perturbs the downfield 1H NMR spectrum of the calmodulin-Ca42+ complex. Changes occur in the resonances assigned to His-107 and Tyr-138. Composite peaks assigned to Phe-16 and Phe-89 and to Phe-68 and Tyr-99 are also affected. Both the upfield and downfield 1H NMR spectra contain evidence for spectroscopically distinct intermediates in Ca2+ binding by the calmodulin-mastoparan complex.     

IP3 receptors and their regulation by calmodulin and cytosolic Ca2+

Inositol 1,4,5-trisphosphate (IP(3)) receptors are tetrameric intracellular Ca(2+) channels, the opening of which is regulated by both IP(3) and Ca(2+). We suggest that all IP(3) receptors are biphasically regulated by cytosolic Ca(2+), which binds to two distinct sites. IP(3) promotes channel opening by controlling whether Ca(2+) binds to the stimulatory or inhibitory sites. The stimulatory site is probably an integral part of the receptor lying just upstream of the pore region. Inhibition of IP(3) receptors by Ca(2+) probably requires an accessory protein, which has not yet been unequivocally identified, but calmodulin is a prime candidate. We speculate that one lobe of calmodulin tethers it to the IP(3) receptor, while the other lobe can bind Ca(2+) and then interact with a second site on the receptor to cause inhibition.     

Co-operative ligand binding in a protein composed of subunits

The mechanism of the co-operative ligand binding in a protein composed of subunits is studied on a realistic assumption that the strain energy due to the ligand binding is stored in weak contacts between the subunits. In a pair of subunits, which is the minimum co-operative unit, two competitive mechanisms operate; the expansion or contraction of the weak contacts results in negative co-operativity, and positive co-operativity can be produced by the alternation of the contacts.Emphasizing the above role of the weak contacts, and taking into account the widespread interaction which correlates the alternation of the contacts in one interface of subunits with that in another interface, we construct a general partition function to describe the co-operative effects in proteins composed of more than two subunits. The partition function thus obtained includes those of the two allosteric models (Monod, Wyman & Changeux, 1965; Koshland, Nemethy & Filmer, 1966) in the two extreme cases of the strength of the widespread interaction.The partition function is applied to the analysis of the molecular mechanism of heme-heme interaction by the curve fitting to the experimental data for hemoglobin solution under various conditions. The comparison of the parameter values thus determined with the molecular structure leads us to one interpretation that the widespread interaction originates from contact connecting three subunits. This interpretation suggests that the strength of the widespread interaction can be changed by modification of an amino acid residue in the intermediate subunit, and that this interaction plays an essential role in the manifestation of the co-operativity. The reduced co-operativity in mutant hemoglobins can also be explained along these lines.     

Modifying Mg2+ binding and exchange with the N-terminal of calmodulin

To follow Mg2+ binding to the N-terminal of calmodulin (CaM), we substituted Phe in position 19, which immediately precedes the first Ca2+/Mg2+ binding loop, with Trp, thus making F19WCaM (W-Z). W-Z has four acidic residues in chelating positions, two of which form a native Z-acid pair. We then generated seven additional N-terminal CaM mutants to examine the role of chelating acidic residues in Mg2+ binding and exchange with the first EF-hand of CaM. A CaM mutant with acidic residues in all of the chelating positions exhibited Mg2+ affinity similar to that of W-Z. Only CaM mutants that had a Z-acid pair were able to bind Mg2+ with physiologically relevant affinities. Removal of the Z-acid pair from the first EF-hand produced a dramatic 58-fold decrease in its Mg2+ affinity. Additionally, removal of the Z-acid pair led to a 1.8-fold increase in the rate of Mg2+ dissociation. Addition of an X- or Y-acid pair could not restore the high Mg2+ binding lost with removal of the Z-acid pair. Therefore, the Z-acid pair in the first EF-hand of CaM supports high Mg2+ binding primarily by increasing the rate of Mg2+ association.