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PubMed1996

Characterization of the calmodulin gene family in wheat: structure, chromosomal location, and evolutionary aspects.

Calmodulin is a ubiquitous transducer of calcium signals in eukaryotes. In diploid plant species, several isoforms of calmodulin have been described. Here, we report on the isolation and characterization of calmodulin cDNAs corresponding to 10 genes from hexaploid (bread) wheat (Triticum aestivum). These genes encode three distinct calmodulin isoforms; one isoform is novel in that it lacks a conserved calcium binding site. Based on their nucleotide sequences, the 10 cDNAs were classified into four subfamilies. Using subfamily-specific DNA probes, calmodulin genes were identified and the chromosomal location of each subfamily was determined by Southern analysis of selected aneuploid lines. The data suggest that hexaploid wheat possesses at least 13 calmodulin-related genes. Subfamilies 1 and 2 were both localized to the short arms of homoeologous-group 3 chromosomes; subfamily 2 is located on all three homoeologous short arms (3AS, 3BS and 3DS), whereas subfamily 1 is located only on 3AS and 3BS but not on 3DS. Further analysis revealed that Aegilops tauschii, the presumed diploid donor of the D-genome of hexaploid wheat, lacks a subfamily-1 calmodulin gene homologue, whereas diploid species related to the progenitors of the A and B genomes do contain such genes. Subfamily 3 was localized to the short arm of homoeologous chromosomes 2A, 2B and 2D, and subfamily 4 was mapped to the proximal regions of 4AS, 4BL and 4DL. These findings suggest that the calmodulin genes within each subfamily in hexaploid wheat represent homoeoallelic loci. Furthermore, they also suggest that calmodulin genes diversified into subfamilies before speciation of Triticum and Aegilops diploid species.

Amino Acid Sequence↗
PubMed1988

1,25 Dihydroxyvitamin D3 affects calmodulin distribution among subcellular fractions of skeletal muscle.

1,25 Dihydroxyvitamin D3 has been shown to stimulate calcium fluxes across skeletal muscle membranes. The involvement of calmodulin in the effects of the metabolite was investigated. Primary cultures of chick embryo skeletal muscle myoblasts and soleus muscles from vitamin D-deficient or 1,25 (OH)2D3-treated chicks were used. Culture of myoblasts and vitamin D-deficient soleus with 1,25 (OH)2D3 (0.05 ng/ml) for 24 and 1 hour, respectively, significantly increased 45Ca uptake by the preparations. In the presence of the calmodulin antagonists flufenazine or compound 48/80 in the uptake medium, no differences between control and treated cultures were observed. The calmodulin content of myoblasts and soleus homogenates and subcellular fractions derived therefrom was estimated by measuring their capacity to stimulate calmodulin-depleted cAMP phosphodiesterase. No changes in total calmodulin cellular content could be detected in response to 1,25(OH)2D3. However, the sterol produced an increase in calmodulin levels of microsomes, mitochondria, and crude myofibrillar fraction and a proportional decrease in cytosolic calmodulin concentration. The 1,25(OH)2D3-dependent changes in calmodulin distribution among subcellular fractions of soleus muscle were observed either in vivo or in vitro. The effects in vitro were already detectable after 5 minutes of treatment with the sterol and parallel 1,25(OH)2D3-dependent changes in tissue Ca uptake. The results suggest that changes in calmodulin intracellular distribution may underly part of the mechanism by which 1,25(OH)2D3 affects muscle calcium transport.

Animals↗
PubMed1987

Mechanism of calmodulin inhibition of cAMP-dependent protein kinase activation of phosphorylation kinase.

The activation of phosphorylase kinase (EC 2.7.1.38; ATP:phosphorylase b phosphotransferase) by the catalytic subunit of cAMP-dependent protein kinase (EC 2.7.1.37; ATP:protein phosphotransferase) is inhibited by calmodulin. The mechanism of that inhibition has been studied by kinetic measurements of the interactions of the three proteins. The binding constant for calmodulin with phosphorylase kinase was found to be 90 nM when measured by fluorescence polarization spectroscopy. Glycerol gradient centrifugation studies indicated that 1 mol of calmodulin was bound to each phosphorylase kinase. Phosphorylation of the phosphorylase kinase did not reduce the amount of calmodulin bound. Kinetic studies of the activity of the catalytic subunit of cAMP-dependent protein kinase on phosphorylase kinase as a function of phosphorylase kinase and calmodulin concentrations were performed. The results of those studies were compared with mathematical models of four different modes of inhibition: competitive, noncompetitive, substrate depletion, and inhibition by a complex between phosphorylase kinase and calmodulin. The data conform best to the model in which the inhibitory species is a complex of phosphorylase kinase and calmodulin. The complex apparently competes with the substrate, phosphorylase kinase, which does not have exogenous calmodulin bound to it. In contrast, the phosphorylation of the synthetic phosphate acceptor peptide, Kemptide, is not inhibited by calmodulin.

Adenosine Triphosphate↗
PubMed1982

Association of calmodulin inhibition, erythrocyte membrane stabilization and pharmacological effects of drugs.

The present study was designed to determine whether there is an association of drug-induced inhibition of calmodulin functions, drug-induced membrane stabilization (protection against osmotic lysis), and pharmacological effects of drugs. First, data on drugs which have been studied for both calmodulin inhibition and membrane antihemolysis were collected from the literature and an association of the two properties was established. Second, ten additional drugs were selected for study of all three properties. Four drugs, with known antihemolytic effects, were studied for calmodulin inhibition. One drug, which was a known calmodulin inhibitor, was studied for antihemolysis. Our results show that membrane-stabilizing drugs are usually calmodulin inhibitors, and vice versa; that drugs in certain therapeutic classes inhibit calmodulin-activated functions and protect against osmotic lysis; and finally, that there is a significant correlation (P less than 0.01) in terms of potency between these two actions of drugs. Data from the literature which bear on these mechanisms of drug actions suggest that the interactions between drugs and calmodulin, and drugs and the membrane, appear to be hydrophobic in nature. At this point, we do not know whether there is some causal relationship between calmodulin inhibition and the antihemolytic effect of drugs, or whether the two are simply a result of hydrophobic properties of drugs. Similarly, the roles of calmodulin inhibition and/or membrane antihemolysis in producing therapeutic efficacy are unknown.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1994

Regulation of calmodulin binding to the ATP extractable 110 kDa protein (myosin I) from chicken duodenal brush border by 1,25-(OH)2D3.

In earlier studies we observed that the active vitamin D metabolite 1,25-(OH)2D3 increased the calmodulin content of purified duodenal brush-border membrane vesicles where it bound principally to the 110 kDa protein myosin I. In this study we further evaluated the regulation of calmodulin binding to ATP releasable myosin I. Whole brush borders (BB) or purified brush-border membrane vesicles (BBMV) were prepared from duodena of vitamin D-deficient rachitic chicks treated 12-18 h before killing with either 625 pmol 1,25-(OH)2D3 or vehicle. The ATP extractable myosin I from BB resulted in an 1.6-fold increase of calmodulin binding to the 110 kDa band after treatment with 1,25-(OH)2D3. In contrast to BB, ATP extraction of myosin I from purified BBMV required alamethicin for ATP entry. As for BB extracts, calmodulin binding to the 110 kDa band in BBMV extracts was also increased about 2.4-fold by 1,25-(OH)2D3. It was concluded that both intact BB and purified BBMV showed the same type of increase in calmodulin binding to ATP releasable myosin I by 1,25-(OH)2D3. To see whether 1,25-(OH)2D3 increased the intrinsic affinity of calmodulin binding to myosin I, the ATP extractable myosin I from BB was purified from rachitic chicks treated with 1,25-(OH)2D3 or vehicle. In contrast to ATP extracts of BB or BBMV, calmodulin binding to the purified myosin I was not different between preparations from 1,25-(OH)2D3- or vehicle-treated chicks. We conclude that 1,25-(OH)2D3 does not change the affinity of calmodulin binding to myosin I but increases the amount of myosin I in the membrane or alters its ATP releasability. It was further investigated whether phosphorylation is involved in these 1,25-(OH)2D3 dependent posttranslational changes of myosin I. Phosphorylation of brush-border membrane proteins in vivo was performed by incubation of [32P]P(i) in the lumen of a ligated duodenal loop in situ for 15 min. Brush-border membrane proteins were phosphorylated in vitro by incubating BB or BBMV with [gamma-32P]ATP for 1 min. Incubation experiments in vivo and in vitro in fact resulted in phosphorylation of several proteins including 110 kDa proteins. However, there was no specific effect of 1,25-(OH)2D3 on phosphorylation of 110 kDa proteins. We conclude that the effects of 1,25-(OH)2D3 on protein phosphorylation are minimal and not likely to explain 1,25-(OH)2D3 stimulated calmodulin binding to ATP extractable brush-border membrane myosin I and 1,25-(OH)2D3 stimulated changes of calcium uptake across the brush-border membrane.

Adenosine Triphosphate↗
PubMed1979

Calmodulin resolution of multiple peaks of activity by preparative electrofocusing.

When the supernatant fractions from rat brain homogenates were subjected to preparative electrofocusing in a bed of Sephadex G75, several peaks of calmodulin were resolved. A minor peak representing free calmodulin migrated with a pI of 3.8 --4.4. Other peaks of calmodulin activity were observed with isoelectric points at pH 4.8, 5.2, 6.0 and 6.8. The peak of calmodulin activity at 5.2 co-migrated with phosphodiesterase activity which was stimulated 1.8-fold by calcium. A second peak of phosphodiesterase activity detected at pH 8.0 was stimulated 1.2-fold by calcium and occurred in an area where no calmodulin activity could be detected. If isoelectric focusing was done in the presence of 8 M urea only one peak of calmodulin activity was observed with a pI of 4.0--4.4. It is suggested that the multiple peaks of calmodulin resolved by electrofocusing represent calmodulin associated with various proteins which are subject to modulation by calmodulin and calcium.

Animals↗
PubMed1983

Inhibition of calmodulin activity by insect venom peptides.

Several peptides found in insect venom, including melittin, apamin and mastoparan, inhibited the activity of calmodulin-stimulated phosphodiesterase at concentrations that had no appreciable effect on basal phosphodiesterase activity; the Ki value of melittin for inhibiting calmodulin activity was 30 nM. Acetylation of the peptides reduced their inhibitory effect on calmodulin, suggesting that a net positive charge was an important determinant of anti-calmodulin activity. An examination of other structural features of these peptides suggested that the most potent inhibitors of calmodulin had an alpha-helical conformation. Equilibrium dialysis experiments showed that melittin inhibited the calcium-dependent binding of 3H-chlorpromazine to calmodulin (IC50 0.9 microM); kinetic analyses of these data indicated that this inhibition was non-competitive, suggesting that melittin and chlorpromazine act at different sites on calmodulin. Since calmodulin regulates a number of processes that these peptides inhibit, our results raise the possibility that the inhibition of calmodulin activity by these insect venom peptides may explain some of their biochemical or toxicological effects.

Animals↗
PubMed1988

Verapamil, diltiazem and nifedipine interactions with calmodulin stimulated (Ca2+ + Mg2+)-ATPase.

The functional interactions of the three prototype Ca2+ antagonists, verapamil, diltiazem and nifedipine, were examined in relation to the calmodulin regulated plasma membrane Ca2+ pump ATPase. For this we used low ionic strength derived, calmodulin depleted, human red cell ghost membranes. Exogenously added calmodulin activated basal (Ca2+ + Mg2+)-ATPase in a concentration-dependent manner. Half-maximal activation by 6 nM calmodulin was antagonized by 10(-3) M verapamil and 10(-3) M diltiazem 25.1 and 12.1% respectively. The inhibition appeared to be specific for calmodulin activation since basal activity was not affected by these agents. Nifedipine had no effects on basal or calmodulin stimulated (Ca2+ + Mg2+)-ATPase activity. Unlike dihydropyridine modulation of verapamil and diltiazem binding at high affinity channel sites, nifedipine in this system did not alter the inhibitory responses of verapamil and diltiazem. The calmodulin directed antagonism of the two drugs was shown to be strictly additive over a full range of calmodulin concentrations and appeared to change predominantly the Vmax and, to a lesser degree, the affinity of calmodulin for the (Ca2+ + Mg2+)-ATPase. It is concluded that this model system provides evidence for additional functional discrepancies among the various classes of Ca2+ antagonists.

Ca(2+) Mg(2+)-ATPase↗
PubMed1990

Plant and fungus calmodulins are polyubiquitinated at a single site in a Ca2(+)-dependent manner.

In plants Ca2+ plays a crucial role as second messenger. Thus calmodulin is one of the most important signal transducing molecules for metabolic regulation in plants. Previously we showed that bovine testis calmodulin can be covalently coupled at one site to ubiquitin in a Ca2(+)-dependent manner in the presence of ATP/Mg2+ by ubiquityl-calmodulin synthetase. Since calmodulin from spinach has 13 amino acid sequence differences to bovine calmodulin - two of them in Ca2(+)-binding loops - it was unclear, whether a conjugation of ubiquitin to this molecule would be possible. In this paper it is shown that calmodulin from spinach and a similar calmodulin from the mold Neurospora crassa can be covalently conjugated to ubiquitin in a Ca2(+)-dependent manner. It is shown that higher molecular mass conjugates containing up to three ubiquitin molecules per calmodulin are obtained. Experiments with methylated ubiquitin demonstrate that, as with vertebrate calmodulins, only one lysine residue is linked to ubiquitin and that the incorporation of additional ubiquitin molecules leads to a polyubiquitin chain.

Animals↗
PubMed1984

Interaction of alpha adrenergic antagonists with calmodulin.

Several alpha-adrenergic antagonists inhibited the activation of calmodulin-stimulated phosphodiesterase at concentrations that had little or no effect on basal phosphodiesterase activity. The most potent of these compounds were phenoxybenzamine and dibenamine (IC50 values of about 1 microM); the amino acid ergot alkaloids ergocryptine, ergocristine, ergotamine and their dihydrogenated derivatives were less potent calmodulin-inhibitors (IC50 values of 35-80 microM). The amino ergot alkaloids ergonovine and methysergide were essentially devoid of inhibitory activity. A variety of other alpha 1-antagonists (phentolamine, tolazoline and prazosin), an alpha 2-antagonist (yohimbine), alpha-agonists (norepinephrine, phenylephrine and clonidine), beta-adrenergic antagonists (propranolol and practolol) and the beta-adrenergic agonist methoxyphenamine displayed little or no anti-calmodulin activity (IC50 values greater than 300 microM). Similarly, the alkylating agents chlorambucil and mechlorethamine also failed to inhibit calmodulin activity. Phenoxybenzamine and dibenamine inhibited calmodulin activity irreversibly, whereas the inhibition caused by other alpha adrenergic blocking agents was reversible. Phenoxybenzamine inhibited calmodulin activity by binding directly to it. This binding was calcium-dependent and irreversible. The irreversible binding and inhibition of calmodulin activity by phenoxybenzamine (or dibenamine) may serve as a useful tool for studying the sites at which drugs bind to calmodulin and may also be useful for studying the distribution and turnover of calmodulin.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1981

Plant and fungal calmodulin: Ca2+-dependent regulation of plant NAD kinase.

Although little is known about the role(s) of second messengers, including free Ca2+, in plant cells there has been increasing evidence for a role for Ca2+ in metabolic regulation in plants. The recent demonstration that the Ca2+-binding protein, calmodulin exists in extracts of higher plants and basidiomycete fungi provides a basis for understanding Ca2+-dependent metabolic regulation in plant cells. In this review we summarize the similarities and differences of plant, fungal and mammalian calmodulin. We also discuss the known in vitro functions of calmodulin in higher plants. A Ca2+-calmodulin-dependent NAD kinase has been purified to homogeneity from extracts of pea seedlings and shown to be absolutely dependent upon calmodulin and microM levels of free Ca2+ for activity. The available evidence suggest that this Ca2+-calmodulin-dependent NAD kinase is the major form of plant NAD kinase and that this regulatory enzyme is localized in the chloroplast. A model is presented which predicts that the rate of photosynthesis is regulated by a receptor-mediated change in the level of chloroplastic free Ca2+ upon illumination. Free Ca2+, acting as a second messenger, forms a Ca2+-calmodulin complex thus converting calmodulin to its active conformation. This Ca2+-calmodulin complex then activates chloroplastic NAD kinase resulting in an increased NADP/NAD ratio.

Adenylyl Cyclases↗
PubMed1988

Interactions of spin-labeled calmodulin with trifluoperazine and phosphodiesterase in the presence of Ca(II), Cd(II), La(III), Tb(III), and Lu(III).

Bovine calmodulin analogues, spin-labeled at methionine and tyrosine residues, have been utilized in electron paramagnetic resonance (EPR) studies designed to investigate calmodulin interactions with the antipsychotic drug trifluoperazine and the calmodulin-binding protein 3',5'-cyclic nucleotide phosphodiesterase. Trifluoperazine titrations of spin-labeled calmodulin analogues were carried out in the presence of Ca(II), Cd(II), and Tb(III). Similar experiments were performed with the phosphodiesterase in the presence of Ca(II), Cd(II), La(III), Tb(III), and Lu(III). EPR signals from the methionine-directed probe proved to be more sensitive to the binding of target molecules than signals from the tyrosine-directed probe, perhaps indicating that the spin-labeled methionine is at a site close to the target molecule binding site. While the binding of TFP, as monitored by EPR spectral changes in the methionine spin-labeled calmodulin, was in evidence with Ca(II), Cd(II), and all the lanthanides examined, no binding of phosphodiesterase to calmodulin could be detected in the presence of the lanthanide ions, perhaps due to inactivation of the phosphodiesterase by lanthanide ion binding. The abilities of the spin-labeled calmodulins to activate phosphodiesterase were also investigated. The spin-labeled tyrosine calmodulin was able to activate phosphodiesterase as well as native calmodulin, while a lower degree of activation was found when the spin-labeled methionine analogue was used.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1983

Involvement of calmodulin in the regulation of adenylate cyclase activity in guinea-pig enterocytes.

The involvement of calmodulin as an activator of adenylate cyclase activity was examined in isolated guinea-pig enterocytes and in a membrane preparation. In enterocytes, which responded to prostaglandin E1, vasoactive intestinal peptide and cholera toxin with a significant increase in the rate of cAMP formation trifluoperazine, a calmodulin antagonist, completely inhibited cAMP formation. In a membrane preparation adenylate cyclase activity was stimulated 10-20-fold by the GTP analog, guanosine 5'-[beta-imido]5'-triphosphate (Gpp[NH]p). Prostaglandin E1 and vasoactive intestinal peptide enhanced cAMP formation in this system by 2-3- and 1.2-1.6-fold. respectively. Addition of 200 nM calmodulin to membranes, in which endogenous calmodulin was decreased from 1.4 microgram/mg protein to 0.5 microgram/mg protein by washing with buffer containing EGTA and EDTA, resulted in a 3-4-fold increase of adenylate cyclase activity. The absolute increment in adenylate cyclase activity caused by calmodulin (10-15 pmol cAMP/min per mg protein) was approximately the same in the absence or presence of Gpp[NH]p. The apparent Ka for Gpp[NH]p (6 . 10-7 M) was not significantly changed by the addition of calmodulin. Although endogenous calcium (approx. 10 microM) in the enzyme assay was adequate to affect stimulation by calmodulin, a maximal effect was observed at a calcium concentration of 100 microM. These findings indicate that a calmodulin-sensitive form of adenylate cyclase is present in guinea-pig enterocytes, and that stimulation of cAMP formation in the intestinal mucosa may involve a calmodulin-mediated mechanism.

1-Methyl-3-isobutylxanthine↗
PubMed1995

Immunocytochemical localization of calmodulin in PC12 cells and its possible interaction with histones.

The subcellular localization of calmodulin, a multi-functional calcium-binding regulatory protein, was examined immunocytochemically in undifferentiated PC12 rat pheochromocytoma cells and cells differentiated with nerve growth factor (NGF) and dibutyryl cyclic AMP. In undifferentiated PC12 cells, diffuse immunostaining for calmodulin was observed in the cytoplasm, and weak, patch-like staining was found in the nucleus. In differentiated cells, intense immunostaining for calmodulin was observed in the cytoplasm, while nuclear immunostaining was still evident. Immunoreactivity for calmodulin was also observed along newly-formed neuritic processes, with strong staining in varicosity-like structures and growth cones. Using double-label immunochemistry, the relative intensity of immunostaining for calmodulin among the nuclei was found to correlate with the relative intensity of immunostaining for histones in the same nuclei. A comparison of a profile of 125I-calmodulin binding in the nuclear fraction from PC12 cells to that of immunoblotting for histones in the same fraction indicated that some of the histones are calmodulin-binding proteins in PC12 cells. These results show that the level and subcellular distribution of calmodulin are altered during the course of nerve cell differentiation and suggest the possibility that histones may function as major nuclear binding proteins for calmodulin.

Animals↗
PubMed1986

Contamination of commercial preparations of calmodulin by phospholipase A2.

In the course of studies of the possible regulation of cellular phospholipase A2 activities by calcium and calmodulin, it was observed that some of the commercial preparations of calmodulin contained significant phospholipase A2 activity. Six commercially available calmodulin sources were compared for the presence of contaminating phospholipase A2 activity, relative purity by SDS-gel electrophoresis, and relative biological activity in stimulating calmodulin-deficient phosphodiesterase. One of the commercial calmodulin sources contained a relatively high specific phospholipase A2 activity (1.30 +/- 0.11 nmol [1-14C]arachidonic acid released/mg protein per h) and yielded two major bands in SDS-gel electrophoresis. Two of the calmodulin sources tested were relatively free of phospholipase A2 activity, were quite pure (one band on SDS-gel) and had high biological activity in stimulating calmodulin-deficient phosphodiesterase. Thus, investigators using commercially available preparations of calmodulin should be aware of the contamination of some of these sources by phospholipase A2 activity. These findings may be of importance to investigators considering the role of calmodulin in activating a variety of calcium-dependent enzymes, including phospholipase A2.

Animals↗
PubMed1988

Alterations in calmodulin content in fractions of rat hippocampal slices during tetanic- and calcium-induced long-term potentiation.

The content of cytosolic and membrane-bound calmodulin was radioimmunologically determined in fractions of rat hippocampal slices 5 min to 7 hours after long-term potentiation (LTP) had been induced by tetanization or exposure of slices to 4 mM Ca++. In light of concepts presuming multistage dynamics in LTP development as reflecting different cellular mechanisms, similar patterns of calmodulin alterations were observed with both models: The alterations in calmodulin content occurred during the early phase(s) of LTP development and continued for two and one hours during tetanic- and calcium-induced LTP, respectively. Thus, 5-30 min after LTP elicitation, membrane-bound calmodulin increased while cytosolic calmodulin diminished and, inversely, 30 min later an increase in cytosolic and decrease in membrane-bound calmodulin were observed. Consequently, the present results indicate that calmodulin was involved in the early phases(s) of LTP development in terms of a two-step translocation sequence. Hence, calmodulin translocation within both intracellular compartments may reflect the involvement of Ca++-calmodulin-dependent intraneuronal metabolic processes which might induce and/or temporarily maintain neuronal functional changes occurring immediately after repeated or intense stimulation of synaptic functions.

Action Potentials↗
PubMed2004

Structure, dynamics and interaction with kinase targets: computer simulations of calmodulin.

Calmodulin (CaM) is a small protein involved in calcium signaling; among the targets of CaM are a number of kinases, including myosin light chain kinases (MLCK), various CaM-dependent kinases and phosphorylase kinase. We present results of molecular dynamics (MD) simulations of 4-ns length for calmodulin in its three functional forms: calcium-free, calcium-loaded, and in complex with both calcium and a target peptide, a fragment of the smooth muscle MLCK. The simulations included explicit water under realistic conditions of constant temperature and pressure, the presence of counterions and Ewald summation of electrostatic forces. Our simulation results present a more complete description of calmodulin structure, dynamics and interactions in solution than previously available. The results agree with a wide range of experimental data, including X-ray, nuclear magnetic resonance (NMR), fluorescence, cross-linking, mutagenesis and thermodynamics. Additionally, we are able to draw interesting conclusions about microscopic properties related to the protein's biological activity. First, in accord with fluorescence data, we find that calcium-free and calcium-loaded calmodulin exhibit significant structural flexibility. Our simulations indicate that these motions may be described as rigid-body translations and rotations of the N- and C-terminal domains occurring on a nanosecond time scale. Our second conclusion deals with the standard model of calmodulin action, which is that calcium binding leads to solvent exposure of hydrophobic patches in the two globular domains, which thus become ready to interact with the target. Surprisingly, the simulation results are inconsistent with the activation model when the standard definitions of the hydrophobic patches are used, based on hydrophobic clefts found in the X-ray structure of calcium-loaded calmodulin. We find that both experimental and simulation results are consistent with the activation model after a redefinition of the hydrophobic patches as those residues which are actually involved in peptide binding in the experimental structure of the calmodulin-peptide complex. The third conclusion is that the calmodulin-peptide interactions in the complex are very strong and are dominated by hydrophobic effects. Using quasi-harmonic entropy calculations, we find that these strong interactions induce a significant conformational strain in the protein and peptide. This destabilizing entropic contribution leads to a moderate overall binding free energy in the complex. Our results provide interesting insights into calmodulin binding to its kinase targets. The flexibility of the protein may explain the fact that CaM is able to bind many different targets. The large loss of conformational entropy upon CaM:peptide binding cancels the entropy gain due to hydrophobic interactions. This explains why the observed entropic contribution to the binding free energy is small and positive, and not large and negative as expected for a complex with such extensive hydrophobic contacts.

Animals↗
PubMed2003

Indolicidin, a 13-residue basic antimicrobial peptide rich in tryptophan and proline, interacts with Ca(2+)-calmodulin.

Indolicidin, ILPWKWPWWPWRR-NH(2), a short 13-residue antimicrobial and cytolytic peptide characterized from bovine neutrophils, has the calmodulin-recognition 1-5-10 hydrophobic pattern (indicated by amino acids in bold), is cationic, and thereby fulfills the requirements to interact with calmodulin. Hence, we have investigated the calmodulin-binding properties of indolicidin. Indolicidin interacted with calmodulin with fairly high affinity in a Ca(2+)-dependent manner. However, when bound, the peptide did not adopt helical conformation. Indolicidin also inhibited calmodulin-stimulated phosphodiesterase activity with IC(50) values in the nanomolar range. Replacement of either the proline residues of indolicidin with alanines or tryptophan residues with phenylalanines did not affect binding to calmodulin. However, these replacements had distinctive effects on the conformations of the bound peptides. While the alanine analog of indolicidin adopted predominantly alpha-helical conformation, the phenylalanine analog remained largely unordered. Differences in the ability of these analogs to inhibit the calmodulin-stimulated phosphodiesterase activity were observed. While the alanine analog was capable of inhibiting the activity with IC(50) values comparable to that of indolicidin, the phenylalanine analog did not inhibit the activity. Our results indicate that ability to adopt amphiphilic alpha-helical structure is not a prerequisite for binding to calmodulin and also binding does not necessarily result in inhibition of calmodulin-stimulated enzyme activities.

Amino Acid Sequence↗