The interaction of calmodulin with melittin

Studies utilizing the interaction of melittin with the 1-106 fragment of calmodulin, the protection of calmodulin from tryptic digestion by melittin, and the interaction of the carbocyanine dye Stains-all with the calmodulin-melittin complex have indicated that complex formation of calmodulin with melittin involves the alpha-helical connecting bridge joining the N- and C-terminal lobes of calmodulin.     

The interaction of calmodulin with amphiphilic peptides

Calmodulin has recently been shown to form exceptionally tight, calcium-dependent complexes with several natural peptides (Kdiss greater than 10(-7) M). These peptides were demonstrated to be capable of forming basic, amphiphilic alpha-helices. To further illustrate the importance of this structural feature for calmodulin binding, several other amphiphilic alpha-helical peptides were tested for their ability to bind calmodulin. To monitor complexes of high affinity (greater than 10(8) M-1), a new competition assay was devised with Sepharose 4B-conjugated melittin. Stoichiometries were assessed by electrophoresis and equilibrium size exclusion chromatography. Three peptides, which were designed to form idealized amphiphilic alpha-helices were tested. The basic peptides, N alpha-9-fluorenylmethoxycarboxyl-(FMOC)-(Leu-Lys-Lys-Leu-Leu-Lys-L eu)1 and FMOC-(Leu-Lys-Lys-Leu-Leu-Lys-Leu)2 bind calmodulin in a 1:1 complex with dissociation constants of 150 and 3 nM, respectively. The acidic peptide, FMOC-(Leu-Glu-Glu-Leu-Leu-Glu-Leu)2 failed to bind calmodulin, even at micromolar concentrations. Complex formation between calmodulin and the 14-residue basic peptide leads to an increase in the helicity of the complex which is attributed to an increase of about 50% in the helicity of the peptide. Calmodulin also interacts with the neutral alpha-helical peptide toxin delta-hemolysin. Concomitant with binding, the fluorescence maximum of the unique Trp residue increases 2-fold and is blue-shifted. A dissociation constant could not be unambiguously estimated though, since delta-hemolysin has a strong tendency to self-aggregate. The above data support our hypothesis that a basic, amphiphilic alpha-helix is a structural feature which underlies the calmodulin-binding properties common to a variety of peptides.     

The interaction of cyclosporin and calmodulin

The interaction of cyclosporin A and dansyl cyclosporin A with bovine and wheat germ calmodulin has been monitored by measurements of induced changes in dansyl and bound toluidinyl naphthalene sulfonate fluorescence. The interaction is Ca2(+)-dependent and 1:1. Measurements of the efficiency of radiationless energy transfer from bound dansyl cyclosporin A to an acceptor group located on Cys-27 of wheat germ calmodulin suggest that the primary binding site is not located on the N-terminal lobe (residues 1-65). However, studies with proteolytic fragments of calmodulin indicate that elements of the N-terminal half-molecule (residues 1-77) may be involved in the stabilization of the binding site. The binding of cyclosporin alters the physical properties of calmodulin and, in particular, reduces the localized rotational mobility of a fluorescent probe.     

Calcium-dependent interaction of transducin with calmodulin Sepharose

Affinity chromatography on calmodulin Sepharose showed that transducin, the G protein of bovine retinal rod outer segments, interacts with the Ca²⁺-calmodulin complex. This may mean that in the dark, rod outer segment calmodulin is largely in the bound state. It was assumed that photoactivation of rods induces a change in the calmodulin concentration in the cytoplasm of rod outer segments and this may be one of the processes leading to light adaptation of the photoreceptor.     

The interaction of calmodulin with the carbocyanine dye (Stains-all)

The dye "Stains-all" combines with calmodulin to yield a series of complex species whose absorption and circular dichroism spectra are sensitive to the presence of Ca2+ or Mg2+. At high dye:calmodulin ratios, the dominant complex formed is characterized by a strong absorption band at 600-650 nm, which is associated with a biphasic circular dichroism band. These spectral features are abolished in the presence of Ca2+.     

Calcium negatively modulates calmodulin interaction with IQGAP1

IQGAP1 regulates cytoskeletal dynamics through interactions with the Rho family GTPases Rac1 and Cdc42, F-actin, and beta-catenin. Calmodulin interaction with IQ motifs of IQGAP1 negatively influences these IQGAP1 interactions. Although, calmodulin interacts with IQGAP1 in the absence of Ca(2+) and was suggested to exhibit reduced binding when Ca(2+) bound, recent reports show substantially greater binding when Ca(2+) is present. Binding evaluations have primarily relied on IQGAP1 interaction with calmodulin conjugated to Sepharose 4B. In this study we evaluated the Ca(2+)-dependence of calmodulin interaction with native IQGAP1 using a series of independent biochemical approaches. We found the apparent binding of calmodulin to IQGAP1 was Ca(2+)-independent, being between 5- and 20-fold greater in the absence than in the presence of Ca(2+). In addition, calmodulin interaction with IQGAP1 was negatively regulated by buffer [Ca(2+)] (IC(50)=3.4x10(-7)M). Regulation was specific to Ca(2+), as Ba(2+) was approximately 400-fold less effective than Ca(2+) at modulating the interaction. Moreover, testing of calmodulin mutants demonstrated that apocalmodulin tightly binds IQGAP1 and that the N- and C-terminal pair of EF hands are important for Ca(2+) sensitivity. These data indicate that calmodulin may disassemble from IQGAP1 to facilitate IQGAP1 interaction with effectors of cytoskeletal reorganization during conditions of cell activation that promote increased cytosolic [Ca(2+)].     

Sites of interaction of calmodulin with trifluoperazine and glucagon

The combination of glucagon with calmodulin alters the microenvironment of Tyr-99, but not Tyr-138, and is blocked by the binding of trifluoperazine by calmodulin. Trp-25 of glucagon is probably involved in the zone of interaction, which may also overlap one or more strong binding sites for trifluoperazine. From energy transfer measurements, one strong binding site for trifluoperazine probably involves the N-terminal region of binding domain III. Energy transfer and other evidence suggest that the zone of contact with glucagon involves the N-terminal region of binding domain III.     

Regulatory interaction of sodium channel IQ-motif with calmodulin C-terminal lobe

An increasing number of ion channels have been found to be regulated by the direct binding of calmodulin (CaM), but its structural features are mostly unknown. Previously, we identified the Ca(2+)-dependent and -independent interactions of CaM to the voltage-gated sodium channel via an IQ-motif sequence. In this study we used the trypsin-digested CaM fragments (TR(1)C and TR(2)C) to analyze the binding of Ca(2+)-CaM or Ca(2+)-free (apo) CaM with a sodium channel-derived IQ-motif peptide (NaIQ). Circular dichroic spectra showed that NaIQ peptide enhanced alpha-helicity of the CaM C-terminal lobe, but not that of the CaM N-terminal lobe in the absence of Ca(2+), whereas NaIQ enhanced the alpha-helicity of both the N- and C-terminal lobes in the presence of Ca(2+). Furthermore, the competitive binding experiment demonstrated that Ca(2+)-dependent CaM binding of target peptides (MLCKp or melittin) with CaM was markedly suppressed by NaIQ. The results suggest that IQ-motif sequences contribute to prevent target proteins from activation at low Ca(2+) concentrations and may explain a regulatory mechanism why highly Ca(2+)-sensitive target proteins are not activated in the cytoplasm.     

The interaction of calmodulin with alternatively spliced isoforms of the type-I inositol trisphosphate receptor

A 592-amino acid segment of the regulatory domain of the neuronal type-I inositol 1,4,5-trisphosphate receptor (IP(3)R) isoform (type-I long, amino acids1314-1905) and the corresponding 552-amino acid alternatively spliced form present in peripheral tissues (type-I short, amino acids 1693-1733 deleted) were expressed as glutathione S-transferase fusion proteins. These domains encompass a putative calmodulin (CaM) binding domain and two protein kinase A phosphorylation sites. Both long and short fusion proteins retained the ability to bind CaM in a Ca(2+)-dependent manner as measured by CaM-Sepharose chromatography or a dansyl-CaM fluorescence assay. Both assays indicated that the short fusion protein bound twice the amount of CaM than the long form at saturating concentrations of CaM. In addition, the binding of the short form to CaM-Sepharose was inhibited by phosphorylation with protein kinase A, whereas the binding of the long form was unaffected. Full-length cDNAs encoding type-I long, type-I short, and type-III IP(3)R isoforms were expressed in COS cells, and the Ca(2+) sensitivity of [(3)H]IP(3) binding to permeabilized cells was measured. The type-I long isoform was more sensitive to Ca(2+) inhibition (IC(50) = 0.55 microM) than the type-I short (IC(50) = 5.7 microM) or the type-III isoform (IC(50) = 3 microM). In agreement with studies on the fusion proteins, the full-length type-I short bound more CaM-Sepharose, and this binding was inhibited to a greater extent by protein kinase A phosphorylation than the type-I long IP(3)R. Although type-III IP(3)Rs did not bind directly to CaM-Sepharose, hetero-oligomers of type-I/III IP(3)Rs retained the ability to interact with CaM. We conclude that the deletion of the SII splice site in the type-I IP(3)R results in the differential regulation of the alternatively spliced isoforms by Ca(2+), CaM, and protein kinase A.     

Systematic delineation of a calmodulin peptide interaction

We present a comprehensive profile of amino acid side-chain constraints in a calmodulin (CaM) peptide complex. These data were obtained from the analysis of calmodulin binding to an array of all single substitution analogues as well as N- and C-terminal truncations of the skMLCK derived M13 peptide ligand. The experimentally derived binding data were evaluated with respect to the known 3D-structure of the CaM/M13 complex. Besides an almost perfect agreement between the measured affinities and the structural data, the unexpected high-affine Asn5Ala variant of the M13(*) peptide described by Montigiani et al. could be verified. In contrast to other reports our data clearly support the postulate of the minor and major hydrophobic anchors of this calcium dependent interaction.     

Effects of interaction with calcineurin on the reactivities of calmodulin lysines

Calmodulin was trace labeled by acetylation with [3H]acetic anhydride in the presence and absence of a 30% molar excess of the phosphatase calcineurin; phenylalanine was included in the reaction mixtures as an internal standard. The level of 3H acetylation of each of the 7 lysines was determined and corrected for differences arising from reaction conditions using the labeling of the internal standard, following procedures that are closely similar to those used in a previous study of the interaction of calmodulin with myosin light chain kinase (Jackson, A. E., Carraway, K. L., III, Puett, D., and Brew, K. (1986) J. Biol. Chem. 261, 12226-12232). The interaction with calcineurin was found to produce a 10-fold reduction in the acetylation of lysine 75, with lesser but significant effects on lysines 21 and 148. A small but reproducible perturbation of lysine 77 was also observed. The results are similar to those that are produced by the interaction with myosin light chain kinase. However, when they are compared with two recent reports between which there are major discrepancies (Manalan, A. S., and Klee, C. B. (1987) Biochemistry 26, 1382-1390; Winkler, M. A., Fried, V. A., Merat, D. L., and Cheung, W. Y. (1987) J. Biol. Chem. 262, 15466-15471), our results are in good agreement with those obtained in the former study. From the location of the perturbed groups in the three-dimensional structure of calmodulin, it appears that the interaction site on calmodulin for calcineurin, as well as for myosin light chain kinase, is very extended and may include hydrophobic pockets at homologous sites near the carboxyl-terminal ends of the two halves of the molecule.     

The interaction of calmodulin with the C-terminal M5 peptide of myosin light chain kinase

The interaction with calmodulin of the 17-residue C-terminal fragment M5 of myosin light chain kinase has been studied by several physical techniques. Circular dichroism measurements suggest that M5 exists within the complex primarily as an alpha-helix. Fluorescence intensity measurements of the single tryptophan of M5 (Trp-4) indicate that it is in a relatively nonpolar environment and is shielded from solvent. Dynamic measurements of fluorescence anisotropy decay indicate that Trp-4 changes from a freely rotating fluorophore to one which is largely immobilized upon complex formation. Static fluorescence measurements show that 2,6-TNS is displaced from its binding site on calmodulin by M5. The binding of M5 also partially inhibits the proteolytic scission by trypsin of the bond between residues 77 and 78.     

Models of delta-hemolysin membrane channels and crystal structures

Molecular modeling and energy calculations have been used to study how delta-hemolysin and melittin helices may aggregate on membrane surfaces and insert through membranes to form channels. In these models adjacent antiparallel amphipathic helices form planar "raft" structures, in which one surface is hydrophobic and the other hydrophilic. Models of delta-hemolysin crystal structure were developed using these "rafts." These models are based on the unit cell constants and the crystal symmetry obtained from the preliminary crystal data. Energy calculations favor channel models of delta-hemolysin with six or eight monomers per channel.     

Affinity purification of seminalplasmin and characterization of its interaction with calmodulin.

Bull seminalplasmin antagonizes with high potency and selectivity the activating effect of calmodulin on target enzymes [Gietzen & Galla (1985) Biochem. J. 230, 277-280]. In the present paper we establish that seminalplasmin forms a 1:1, Ca2+-dependent and urea-resistant complex with calmodulin. The dissociation constant equals 1.6 nM. In the absence of Ca2+ a low-affinity complex is formed that is disrupted by 4 M-urea. On the basis of these properties, a fast affinity purification of seminalplasmin was developed. The high specificity of seminalplasmin as a calmodulin antagonist was demonstrated for the multipathway-regulated adenylate cyclase of bovine cerebellum. Far-u.v. c.d. properties are consistent with a random form of seminalplasmin in aqueous solution; 23% alpha-helix is induced on interaction with calmodulin. The fluorescence properties of the single tryptophan residue of seminalplasmin are markedly changed on formation of the complex. These studies allowed us to locate tentatively the peptide segment that interacts with calmodulin, and to ascertain the structural homology between seminalplasmin and other calmodulin-binding peptides. Additional material, showing the inhibition of calmodulin-mediated activation of bovine brain phosphodiesterase by melittin and seminalplasmin and also the near-u.v. spectrum of affinity-purified seminalplasmin, has been deposited as supplement SUP 50135 (4 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies may be obtained on the terms indicated in Biochem. J. (1986) 233, 5.     

The effect of media pH and autocatalytic phosphorylation on the interaction of kinase phosphorylase with calmodulin

Using calmodulin covalently labeled with dansyl, the Ca2(+)-dependent interaction of phosphorylase kinase with calmodulin has been studied. It has been shown that at pH 6.8 the (alpha beta gamma delta) protomer of the enzyme binds 2.1 +/- 0.8 mol of calmodulin with Kd = (6.67 +/- 1.77).10(-8) M. The enzyme activation induced by the pH increase up to 8.2 does not affect the enzyme interaction with calmodulin [2.14 +/- 0.58 mol calmodulin per mol of (alpha beta gamma delta)]; Kd = (4.14 +/- 1.22).10(-8) M. However, the enzyme activation during its autocatalytic phosphorylation eliminates this effect practically completely.     

Phenothiazine antagonism of calmodulin: a structurally-nonspecific interaction

Phenothiazine antagonism of calmodulin-stimulated (Mg²⁺ + Ca²⁺)-ATPase activity in erythrocyte ghosts was examined to determine the structural specificity of the interaction. Four analogs of chlorpromazine, differing in the position of the chlorine substitution of the aromatic ring, were about equally potent in antagonizing calmodulin activation, while only the 2-chloro analog (chlorpromazine) has tranquilizer activity and antagonizes dopamine-sensitive adenylate cyclase. As all four analogs have similar hydrophobicity and surface activity, the results indicate that the antagonism of calmodulin by phenothiazines is unrelated to their pharmacological specificity and occurs at a structurally-nonspecific hydrophobic site on the protein.     

Model for the interaction of amphiphilic helices with troponin C and calmodulin

Crystals of troponin C are stabilized by an intermolecular interaction that involves the packing of helix A from the N-terminal domain of one molecule onto the exposed hydrophobic cleft of the C-terminal domain of a symmetry related molecule. Analysis of this molecular recognition interaction in troponin C suggests a possible mode for the binding of amphiphilic helical molecules to troponin C and to calmodulin. From the template provided by this troponin C packing, it has been possible to build a model of the contact region of mastoporan as it might be bound to the two Ca2+ binding proteins. A possible binding mode of melittin to calmodulin is also proposed. Although some of the characteristics of binding are similar for the two amphiphilic peptides, the increased length of melittin requires a significant bend in the calmodulin central helix similar to that suggested recently for the myosin light chain kinase calmodulin binding peptide (Persechini and Kretsinger: Journal of Cardiovascular Pharmacology 12:501-512, 1988). Not only are the hydrophobic interactions important in this model, but there are several favorable electrostatic interactions that are predicted as a result of the molecular modeling. The regions of troponin-C and calmodulin to which amphiphilic helices bind are similar to the regions to which the neuroleptic drugs such as trifluoperazine have been predicted to bind (Strynadka and James: Proteins 3:1-17, 1988).     

Sequential events in calmodulin on binding with calcium and interaction with target enzymes

khe conformational and functional events in calmodulin (CaM) are disproportionate to the mean saturation by Ca2+. The enhancement of intrinsic tyrosine fluorescence closely follows the appearance of species CaM X Can greater than or equal to 1; the exposure of the hydrophobic patch at the surface of CaM coincides with the appearance of CaM X Can greater than or equal to 2. For the activation of four different target enzymes, i.e., brain phosphodiesterase and adenylate cyclase, red blood cell Ca,Mg-ATPase, and skeletal muscle phosphorylase b kinase, CaM X Can greater than or equal to 3 is required. The different enzymes have the same affinity for the active species. The direct interaction of CaM with Ca2+ and phosphorylase b kinase has been analyzed according to the theory of energy coupling: whereas the first two stoichiometric calcium-binding constants in the complex are not significantly different from those of free CaM, the third Ca2+ binds with an affinity at least 10(6)-fold higher to enzyme-bound CaM than to free CaM, which corresponds to a free energy coupling of -7 kcal/mol CaM. The similarities in the activation mechanism of different enzymes suggest the existence of one unique CaM-binding domain. The characteristics of the interaction between CaM and melittin, a small amphiphatic cytotoxin, led us to propose melittin as a model for such a CaM-binding domain.     

Elucidation of the interaction of calmodulin with the IQ motifs of IQGAP1

Calmodulin regulates the function of numerous proteins by binding to short regions on the target molecule. IQ motifs, which are found in over 100 human proteins, appear in tandem repeats and bind calmodulin in the absence of Ca(2+). One of these IQ-containing proteins, IQGAP1, interacts with several targets, including Cdc42, beta-catenin, E-cadherin, and actin, in a calmodulin-regulated manner. To elucidate the molecular mechanism by which apocalmodulin and Ca(2+)/calmodulin differentially regulate IQGAP1, a series of constructs of IQGAP1 with selected point mutations of the four tandem IQ motifs were generated. Mutating the basic charged arginine residues in all four IQ motifs abrogated binding of IQGAP1 to apocalmodulin, but had no effect on its interaction with Ca(2+)/calmodulin. Analysis of IQGAP1 constructs with point mutations in single, double, or triple IQ motifs revealed that apocalmodulin bound only to IQ3 and IQ4. By contrast to the arginine mutant constructs, mutation of selected hydrophobic residues in the IQ motifs produced an IQGAP1 protein incapable of binding either apocalmodulin or Ca(2+)/calmodulin. These results, which differ from the conventional model of Ca(2+)-independent binding of calmodulin to IQ motifs, provide insight into the complexity of the molecular interactions between calmodulin and IQ motifs.