Photoaffinity labeling study of the interaction of calmodulin with the plasma membrane Ca2+ pump.

Bovine brain calmodulin was labeled with synthetic peptides corresponding to the calmodulin-binding domain of the erythrocyte plasma membrane Ca(2+)-ATPase. One 20-amino acid peptide and two 28-amino acid peptides were used, carrying L-4'-(1-azi-2,2,2-trifluoroethyl)phenylalanine residues in position 9 (peptides C20W* and C28W*) and position 25 (peptide C28WC*), respectively. The localization of the contact regions between calmodulin and the N- and C-terminal portions of the peptides was the aim of this study. The three peptides were N-terminally blocked with a 3H-labeled acetyl group to facilitate the identification of labeled fragments after isolation and digestion. The binding site for phenylalanine 25 was identified in the N-terminal domain of calmodulin while the phenylalanine derivative in position 9 labeled the C-terminal domain. Fluorescence studies using the dansylated N- and C-terminal halves of calmodulin and peptide C20W corresponding to the first 20 amino acids of the calmodulin-binding domain showed that only the C-terminal lobe of calmodulin had high affinity for the peptide (KD in the nanomolar range).     

The calmodulin binding domain of the plasma membrane Ca2+ pump interacts both with calmodulin and with another part of the pump

Synthetic peptides corresponding to the calmodulin-binding domain of the human erythrocyte Ca2+ pump were prepared representing residues 2-29 (C28W), 2-21 (C20W), 2-16 (C15W), and 16-29 (C14) of the sequence (James, P., Maeda, M., Fisher, R., Verma, A. K., Krebs, J., Penniston, J. T., and Carafoli, E. (1988) J. Biol. Chem. 263, 2905-2910). Peptides C28W, C20W, and C15W bound to calmodulin with an apparent 1:1 stoichiometry in the presence of Ca2+ and inhibited the activation of the Ca2+ pump by calmodulin, while C14 was ineffective. Substituting tyrosine (C28Y) or alanine (C28A) for the tryptophan residue lowered the affinity for calmodulin. The estimated Kd values for the calmodulin-peptide complexes were 0.1 nM for C28W, 5-15 nM for C20W, C28Y, and C28A, and 700-1700 nM for C15W. The Ca2+ pump in inside-out erythrocyte membrane vesicles was activated by proteolytic removal of the endogenous calmodulin-binding domain. Addition of C20W or C28W then inhibited calmodulin-independent Ca2+ transport, while a calmodulin-binding peptide from another enzyme had no effect. The inhibition of the pump by C20W was purely competitive with Ca2+, while C28W decreased the Vmax and increased the K1/2 for Ca2+, restoring the pump activity nearly to its low basal level. The results suggest that a calmodulin-binding peptide from any enzyme has two kinds of specificity: it shares with peptides from other enzymes the ability to bind to calmodulin, but only it has the specificity to interact with its own (proteolytically activated) enzyme.     

A synthetic 33-residue analogue of bovine brain calmodulin calcium binding site III: synthesis, purification, and calcium binding

The sequential solid-phase synthesis of a peptide analogue of bovine brain calmodulin calcium binding site III covering residues 81-113 of the natural sequence is described. Methionine-109 is replaced by a leucine residue to avoid complications in the synthesis and purification. In an attempt to relate the structure of the calcium binding sites in the naturally occurring calcium binding protein to the calcium affinity of these sites, the synthetic analogue is examined for calcium binding by circular dichroism spectroscopy. The calcium binding characteristics are compared to those of a synthetic analogue of the homologous calcium binding site III in rabbit skeletal troponin C. The Kd of the calmodulin site III fragment for Ca2+ is determined as 878 microM whereas the Kd of the troponin C fragment is 30 times smaller at 28 microM. Structural changes induced in the peptides by Ca2+ and trifluoroethanol are similar. This study supports our contention that the single synthetic calcium binding site is a reasonable model for the study of the structure-activity relationships of the calcium binding sites in calcium-regulated proteins such as calmodulin and troponin C.     

Calcium binding to tryptic fragments of calmodulin

Fragments of scallop testis calmodulin were prepared by tryptic digestion. One peptide consisted of 75 amino acid residues from N-acetylalanine to lysine at position 75 (F12) and the other of 71 residues from aspartic acid at position 78 to C-terminal lysine (F34). Flow dialysis and equilibrium dialysis experiments revealed the existence of two Ca2+ binding sites in each fragment. Half-saturating concentrations of the Ca2+ titration curves were 11 microM for F12 and 3.2 microM for F34, and Hill coefficients were obtained as 1.14 and 1.84, respectively. The results indicate that the high-affinity sites for Ca2+ are located on the C-terminal region of the calmodulin. The sum of the two Ca2+ titration curves of F12 and F34 fits well to the curves of Ca2+ binding to intact calmodulin. This shows that the characteristic of Ca2+ bindings in intact calmodulin did not change after separation of the whole molecule into two domains, F12 and F34. The domains corresponding to F12 and F34 may exist independently from each other in the intact calmodulin molecule.     

Small-angle X-ray scattering study of calmodulin bound to two peptides corresponding to parts of the calmodulin-binding domain of the plasma membrane Ca2+ pump.

The interaction between calmodulin (CaM) and two synthetic peptides, C20W and C24W, corresponding to parts of the calmodulin-binding domain of the Ca2+ pump of human erythrocytes, has been studied by using small-angle X-ray scattering (SAXS). The total length of the CaM-binding domain of the enzyme is estimated to be 28 amino acids. C20W contains the 20 N-terminal amino acids of this domain, C24W the 24 C-terminal amino acids. The experiments have shown that the binding of either peptide results in a complex with a radius of gyration (Rg) smaller than that of CaM. The complex between CaM and C20W revealed an interatomic length distribution function, P(r), similar to that of calmodulin alone, indicating that the complex retains an extended, dumbbell-shaped structure. By contrast, the binding of C24W resulted in the formation of a globular structure similar to those observed with many other CaM-binding peptides.     

Xeroderma pigmentosum group C protein possesses a high affinity binding site to human centrin 2 and calmodulin

Human centrin 2 (HsCen2), a member of the EF-hand superfamily of Ca2+-binding proteins, is commonly associated with centrosome-related structures. The protein is organized in two domains, each containing two EF-hand motifs, but only the C-terminal half exhibits Ca2+ sensor properties. A significant fraction of HsCen2 is localized in the nucleus, where it was recently found associated with the xeroderma pigmentosum group C protein (XPC), a component of the nuclear excision repair pathway. Analysis of the XPC sequence (940 residues), using a calmodulin target recognition software, enabled us to predict two putative binding sites. The binding properties of the two corresponding peptides were investigated by isothermal titration calorimetry. Only one of the peptides (P1-XPC) interacts strongly (Ka = 2.2 x 10(8) m-1, stoichiometry 1:1) with HsCen2 in a Ca2+-dependent manner. This peptide also binds, with a similar affinity (Ka = 1.1 x 10(8) m-1) to a C-terminal construct of HsCen2, indicating that the interaction with the integral protein is mainly the result of the contribution of the C-terminal half. The second peptide (P2-XPC) failed to show any detectable binding either to HsCen2 or to its C-terminal lobe. The two peptides interact with different affinities and mechanisms with calmodulin. Circular dichroism and nuclear magnetic resonance were used to structurally characterize the complex formed by the C-terminal domain of HsCen2 with P1-XPC.     

Regulation of the calcium ion pump of sarcoplasmic reticulum: reversible inhibition by phospholamban and by the calmodulin binding domain of the plasma membrane calcium ion pump.

A 45 amino acid peptide (A45) corresponding to the phospholamban (PLN) binding domain of the sarcoplasmic reticulum (SR) ATPase was synthesized. Circular dichroism experiments have shown that the peptide had a predominantly random-coil conformation but adopted a higher proportion of secondary structure in the presence of a synthetic 32 amino acid peptide corresponding to the hydrophilic portion of PLN. A similar conformational change was induced by the synthetic calmodulin binding domain of the plasma membrane Ca2+ pump (peptide C28W), which acts as an endogenous inhibitor of the pump and is homologous to PLN. Cross-linking experiments have shown that peptide C28W interacted with peptide A45. The Ca(2+)-pumping activity of cardiac SR, which contains endogenous PLN, was stimulated about 30% by peptide A45. The stimulation was maximal at submicromolar Ca2+ levels and tended to disappear at higher Ca2+ concentrations. By contrast, the Ca(2+)-pumping activity of skeletal muscle SR, which lacks endogenous PLN, was unaffected. Peptide C28W strongly inhibited the pumping activity of skeletal muscle SR, and peptide A45 reversed the inhibition. The results suggest that peptide A45 competed with the ATPase for phospholamban or for peptide C28W, removing the inhibition of the pump. Thus, the exogenous inhibitor of the SR Ca(2+)-ATPase, PLN, and the internal inhibitor of the plasma membrane Ca(2+)-ATPase, peptide C28W, are functionally analogous.     

Kinetics of calcium dissociation from calmodulin and its tryptic fragments. A stopped-flow fluorescence study using Quin 2 reveals a two-domain structure

The kinetics of calcium dissociation from bovine testis calmodulin and its tryptic fragments have been studied by fluorescence stopped-flow methods, using the calcium indicator Quin 2. Two distinct rate processes, each corresponding to the release of two calcium ions are resolved for calmodulin at both low and high ionic strength. The effect of 0.1 M KCl is to accelerate the slow process from 9.1 +/- 1.5 s-1 to 24 +/- 6.0 s-1 and to reduce the rate of the fast process from 650 s-1 to 240 +/- 50 s-1 at 25 degrees C. In the presence of 0.1 M KCl it was possible to determine activation parameters for the fast process: delta H# = 41 +/- 5 kJ mol-1 and delta S# = -63 +/- 17 J K-1 mol-1. These values are in good agreement with those obtained by 43Ca NMR. Studies of the tryptic fragments TR1C and TR2C, comprising the N-terminal or C-terminal half of calmodulin, clearly identified Ca2+-binding sites I and II as the low-affinity (rapidly dissociating) sites and sites III and IV as the high-affinity (slowly dissociating) sites. The kinetic properties of the two proteolytic fragments are closely similar to the fast and slowly dissociating sites of native calmodulin, supporting the idea that calmodulin is constructed from two largely independent domains. The presence of the calmodulin antagonist trifluoperazine markedly decreased the Ca2+ dissociation rates from calmodulin. One of the two high-affinity trifluoperazine-binding sites was found to be located on the N-terminal half and the other on the C-terminal half of calmodulin. The affinity of the C-terminal site is at least one order of magnitude greater.     

Calcium binding to calmodulin and its globular domains

The macroscopic Ca(2+)-binding constants of bovine calmodulin have been determined from titrations with Ca2+ in the presence of the chromophoric chelator 5,5'-Br2BAPTA in 0, 10, 25, 50, 100, and 150 mM KCl. Identical experiments have also been performed for tryptic fragments comprising the N-terminal and C-terminal domains of calmodulin. These measurements indicate that the separated globular domains retain the Ca2+ binding properties that they have in the intact molecule. The Ca2+ affinity is 6-fold higher for the C-terminal domain than for the N-terminal domain. The salt effect on the free energy of binding two Ca2+ ions is 20 and 21 kJ. mol-1 for the N- and C-terminal domain, respectively, comparing 0 and 150 mM KCl. Positive cooperativity of Ca2+ binding is observed within each globular domain at all ionic strengths. No interaction is observed between the globular domains. In the N-terminal domain, the cooperativity amounts to 3 kJ.mol-1 at low ionic strength and greater than or equal to 10 kJ.mol-1 at 0.15 M KCl. For the C-terminal domain, the corresponding figures are 9 +/- 2 kJ.mol-1 and greater than or equal to 10 kJ.mol-1. Two-dimensional 1H NMR studies of the fragments show that potassium binding does not alter the protein conformation.     

Calcium-dependent and -independent binding of soybean calmodulin isoforms to the calmodulin binding domain of tobacco MAPK phosphatase-1

The recent finding of an interaction between calmodulin (CaM) and the tobacco mitogen-activated protein kinase phosphatase-1 (NtMKP1) establishes an important connection between Ca(2+) signaling and the MAPK cascade, two of the most important signaling pathways in plant cells. Here we have used different biophysical techniques, including fluorescence and NMR spectroscopy as well as microcalorimetry, to characterize the binding of soybean CaM isoforms, SCaM-1 and -4, to synthetic peptides derived from the CaM binding domain of NtMKP1. We find that the actual CaM binding region is shorter than what had previously been suggested. Moreover, the peptide binds to the SCaM C-terminal domain even in the absence of free Ca(2+) with the single Trp residue of the NtMKP1 peptides buried in a solvent-inaccessible hydrophobic region. In the presence of Ca(2+), the peptides bind first to the C-terminal lobe of the SCaMs with a nanomolar affinity, and at higher peptide concentrations, a second peptide binds to the N-terminal domain with lower affinity. Thermodynamic analysis demonstrates that the formation of the peptide-bound complex with the Ca(2+)-loaded SCaMs is driven by favorable binding enthalpy due to a combination of hydrophobic and electrostatic interactions. Experiments with CaM proteolytic fragments showed that the two domains bind the peptide in an independent manner. To our knowledge, this is the first report providing direct evidence for sequential binding of two identical peptides of a target protein to CaM. Discussion of the potential biological role of this interaction motif is also provided.     

Mechanism of calmodulin recognition of the binding domain of isoform 1b of the plasma membrane Ca(2+)-ATPase: kinetic pathway and effects of methionine oxidation

Calmodulin (CaM) binds to a domain near the C-terminus of the plasma membrane Ca2+-ATPase (PMCA), causing the release of this domain and relief of its autoinhibitory function. We investigated the kinetics of dissociation and binding of Ca2+-CaM with a 28-residue peptide [C28W(1b)] corresponding to the CaM-binding domain of isoform 1b of PMCA. CaM was labeled with a fluorescent probe on either the N-terminal domain at residue 34 or the C-terminal domain at residue 110. Formation of complexes of CaM with C28W(1b) results in a decrease in the fluorescence yield of the fluorophore, allowing the kinetics of dissociation or binding to be detected. Using a maximum entropy method, we determined the minimum number and magnitudes of rate constants required to fit the data. Comparison of the fluorescence changes for CaM labeled on the C-terminal or N-terminal domain suggests sequential and ordered binding of the C-terminal and N-terminal domains of CaM with C28W(1b). For dissociation of C28W(1b) from CaM labeled on the N-terminal domain, we observed three time constants, indicating the presence of two intermediate states in the dissociation pathway. However, for CaM labeled on the C-terminal domain, we observed only two time constants, suggesting that the fluorescence label on the C-terminal domain was not sensitive to one of the kinetic steps. The results were modeled by a kinetic mechanism in which an initial complex forms upon binding of the C-terminal domain of CaM to C28W(1b), followed by binding of the N-terminal domain, and then formation of a tight binding complex. Oxidation of methionine residues in CaM resulted in significant perturbations to the binding kinetics. The rate of formation of a tight binding complex was reduced, consistent with the poorer effectiveness of oxidized CaM in activating the Ca2+ pump.     

Structure of calmodulin bound to the hydrophobic IQ domain of the cardiac Ca(v)1.2 calcium channel

Ca2+-dependent inactivation (CDI) and facilitation (CDF) of the Ca(v)1.2 Ca2+ channel require calmodulin binding to a putative IQ motif in the carboxy-terminal tail of the pore-forming subunit. We present the 1.45 A crystal structure of Ca2+-calmodulin bound to a 21 residue peptide corresponding to the IQ domain of Ca(v)1.2. This structure shows that parallel binding of calmodulin to the IQ domain is governed by hydrophobic interactions. Mutations of residues I1672 and Q1673 in the peptide to alanines, which abolish CDI but not CDF in the channel, do not greatly alter the structure. Both lobes of Ca2+-saturated CaM bind to the IQ peptide but isoleucine 1672, thought to form an intramolecular interaction that drives CDI, is buried. These findings suggest that this structure could represent the conformation that calmodulin assumes in CDF.     

Sites on calmodulin that interact with the C-terminal tail of Cav1.2 channel

Two fragments of the C-terminal tail of the alpha(1) subunit (CT1, amino acids 1538-1692 and CT2, amino acids 1596-1692) of human cardiac L-type calcium channel (Ca(V)1.2) have been expressed, refolded, and purified. A single Ca(2+)-calmodulin binds to each fragment, and this interaction with Ca(2+)-calmodulin is required for proper folding of the fragment. Ca(2+)-calmodulin, bound to these fragments, is in a more extended conformation than calmodulin bound to a synthetic peptide representing the IQ motif, suggesting that either the conformation of the IQ sequence is different in the context of the longer fragment, or other sequences within CT2 contribute to the binding of calmodulin. NMR amide chemical shift perturbation mapping shows the backbone conformation of calmodulin is nearly identical when bound to CT1 and CT2, suggesting that amino acids 1538-1595 do not contribute to or alter calmodulin binding to amino acids 1596-1692 of Ca(V)1.2. The interaction with CT2 produces the greatest changes in the backbone amides of hydrophobic residues in the N-lobe and hydrophilic residues in the C-lobe of calmodulin and has a greater effect on residues located in Ca(2+) binding loops I and II in the N-lobe relative to loops III and IV in the C-lobe. In conclusion, Ca(2+)-calmodulin assumes a novel conformation when part of a complex with the C-terminal tail of the Ca(V)1.2 alpha(1) subunit that is not duplicated by synthetic peptides corresponding to the putative binding motifs.     

Study of calmodulin binding to the alternatively spliced C-terminal domain of the plasma membrane Ca2+ pump.

The C-terminal regions of the four human plasma membrane Ca2+ pump isoforms 1a-d generated from alternatively spliced RNA have been expressed in Escherichia coli, and the recombinant proteins have been purified to a very high degree. The C-termini of isoforms 1a, 1c, and 1d contain an insert encoded by an alternatively spliced exon which is homologous to the calmodulin binding domain of isoform 1b. In isoforms 1c and 1d (29 and 38 amino acid insertions, respectively), subdomain A of the original calmodulin binding site of isoform 1b is followed by the spliced-in domain, which is then followed by subdomain B of the original calmodulin binding site. The positive charges of histidine residues at positions 27, 28, and 38 of the alternatively spliced sequence are likely to be responsible for the observed pH-dependent calmodulin binding to the novel "duplicated" binding site. The affinity of calmodulin for the C-terminal domains of isoforms 1a, 1c, and 1d, which contain the histidine-rich inserts, is much higher at pH 5.9 than at pH 7.2. A synthetic peptide (I31) containing 31 amino acids of the alternatively spliced sequence (from residue 9 to 40) also binds calmodulin with strong pH dependency. Alternative splicing in the C-terminal domain is proposed to confer pH dependence to the regulation of the activity of Ca2+ pump isoforms.     

Structural features underlying the unusual mode of calmodulin phosphorylation by protein kinase CK2: A study with synthetic calmodulin fragments

To shed light on the paradoxical behaviour of calmodulin, whose phosphorylation is inhibited by the regulatory beta-subunit of protein kinase CK2, a series of peptides encompassing the phosphoacceptor sites of calmodulin have been synthesized and assayed as substrates of CK2 alpha-subunit either alone or combined with the beta-subunit. The shortest peptide whose phosphorylation is reduced instead of being enhanced by the beta-subunit encompasses the sequence 68-106, including the central helix and the Ca2+-binding loop-III. In contrast, the phosphorylation of a peptide encompassing loop II and the central helix (54-92) is stimulated, like that of several shorter peptides, by the beta-subunit. Our data localize to the C-terminal domain of calmodulin the structural elements that are responsible for inverted susceptibility to beta-subunit regulation.     

The gamma-subunit of skeletal muscle phosphorylase kinase contains two noncontiguous domains that act in concert to bind calmodulin

Phosphorylase kinase is a Ca2+-regulated, multisubunit enzyme that contains calmodulin as an integral subunit (termed the delta-subunit). Ca2+-dependent activity of the enzyme is thought to be regulated by direct interaction of the delta-subunit with the catalytic subunit (the gamma-subunit) in the holoenzyme complex. In order to systematically search for putative calmodulin (delta-subunit)-binding domain(s) in the gamma-subunit of phosphorylase kinase, a series of 18 overlapping peptides corresponding to the C terminus of the gamma-subunit was chemically synthesized using a tea bag method. The calmodulin-binding activity of each peptide was tested for its ability to inhibit Ca2+/calmodulin-dependent activation of myosin light chain kinase. Data were obtained indicating that two distinct regions in the gamma-subunit, one spanning residues 287-331 (termed domain-N) and the other residues 332-371 (domain-C), are capable of binding calmodulin with nanomolar affinity. Peptides from both of these two domains also inhibited calmodulin-dependent reactivation of denatured gamma-subunit. The interactions of peptides from both domain-N and domain-C with calmodulin were found to be Ca2+-dependent. Dixon plots obtained using mixtures of peptides from domain-N and domain-C indicate that these two domains can bind simultaneously to a single molecule of calmodulin. Multiple contacts between the gamma-subunit and calmodulin (delta-subunit), as indicated by our data, may help to explain why strongly denaturing conditions are required to dissociate these two subunits, whereas complexes of calmodulin with most other target enzymes can be readily dissociated by merely lowering Ca2+ to submicromolar concentrations. Comparison of the sequences of the two calmodulin-binding domains in the gamma-subunit of phosphorylase kinase with corresponding regions in troponin I indicates similarities that may have functional and evolutionary significance.     

Peptides related to the calcium binding domains II and III of calmodulin. Synthesis and calmodulin-like features

Three hexadecapeptides which correspond to the putative Ca2+ binding domains II and III of calmodulin were synthesized employing solid phase methodology. One of the peptides contained an internal cystine bridge which was formed while the corresponding linear peptide was still attached to the polymeric carrier. The interaction of the synthetic peptides with calcium ions was investigated using Tb3+-mediated fluorescence. Binding was of the order Ca12 greater than Ca13 greater than Ca13C (Fig. 1) with binding constants KTb3+ = 0.68 X 10(-5), 0.54 X 10(-5), and 0.21 X 10(-5) M-1 respectively. Biological activity of the compounds was assessed by measuring their stimulatory effect on erythrocyte membrane (Ca2+ + Mg2+)-ATPase activity. For 50% activity as compared with CaM, the concentration of peptides required was for Ca12, Ca13 and Ca13C, 50, 100 and 167 times higher than CaM, respectively. The results suggest that the three synthetic peptides possess certain calmodulin-like features.     

Calcium-dependent binding of calmodulin to neuronal gap junction proteins

We examined the interactions of calmodulin with neuronal gap junction proteins connexin35 (Cx35) from perch, its mouse homologue Cx36, and the related perch Cx34.7 using surface plasmon resonance. Calmodulin bound to the C-terminal domains of all three connexins with rapid kinetics in a concentration- and Ca2+-dependent manner. Dissociation was also very rapid. K(d)'s for calmodulin binding at a high-affinity site ranged from 11 to 72 nM, and K(1/2)'s for Ca2+ were between 3 and 5 microM. No binding to the intracellular loops was observed. Binding competition experiments with synthetic peptides mapped the calmodulin binding site to a 10-30 amino acid segment at the beginning of the C-terminal domain of Cx36. The micromolar K(1/2)'s and rapid on and off rates suggest that this interaction may change dynamically in neurons, and may occur transiently when Ca2+ is elevated to a level that would occur in the near vicinity of an activated synapse.     

NMR solution structure of a complex of calmodulin with a binding peptide of the Ca2+ pump.

The three-dimensional structure of the complex between calmodulin (CaM) and a peptide corresponding to the N-terminal portion of the CaM-binding domain of the plasma membrane calcium pump, the peptide C20W, has been solved by heteronuclear three-dimensional nuclear magnetic resonance (NMR) spectroscopy. The structure calculation is based on a total of 1808 intramolecular NOEs and 49 intermolecular NOEs between the peptide C20W and calmodulin from heteronuclear-filtered NOESY spectra and a half-filtered experiment, respectively. Chemical shift differences between free Ca(2+)-saturated CaM and its complex with C20W as well as the structure calculation reveal that C20W binds solely to the C-terminal half of CaM. In addition, comparison of the methyl resonances of the nine assigned methionine residues of free Ca(2+)-saturated CaM with those of the CaM/C20W complex revealed a significant difference between the N-terminal and the C-terminal domain; i.e., resonances in the N-terminal domain of the complex were much more similar to those reported for free CaM in contrast to those in the C-terminal half which were significantly different not only from the resonances of free CaM but also from those reported for the CaM/M13 complex. As a consequence, the global structure of the CaM/C20W complex is unusual, i.e., different from other peptide calmodulin complexes, since we find no indication for a collapsed structure. The fine modulation in the peptide protein interface shows a number of differences to the CaM/M13 complex studied by Ikura et al. [Ikura, M., Clore, G. M., Gronenborn, A. M., Zhu, G., Klee, C. B., and Bax, A. (1992) Science 256, 632-638]. The unusual binding mode to only the C-terminal half of CaM is in agreement with the biochemical observation that the calcium pump can be activated by the C-terminal half of CaM alone [Guerini, D., Krebs, J., and Carafoli, E. (1984) J. Biol. Chem. 259, 15172-15177].     

Agonist and antagonist properties of calmodulin fragments

Limited proteolysis of calmodulin with trypsin in the presence of ethylene glycol bis(beta-aminoethyl ether)-N, N,N',N'-tetracetic acid (EGTA) or Ca2+ was performed according to a modification of the method of Drabikowski et al. (Drabikowski, W., Kuznicki, J., and Grabarek, Z. (1977) Biochim. Biophys. Acta 485, 124-133). The resulting peptides were purified by reverse-phase high performance liquid chromatography. Tryptic digests in EGTA yielded peptides 1-106, 1-90, and 107-148 with yields of 9, 47, and 61%, respectively. The digests performed with Ca2+ yielded peptides 1-77 and 78-148 in 35 and 45% yield. Analysis by high performance liquid chromatography indicated that the purified fragments contained less than 0.1% contamination by calmodulin, thus allowing a definitive study of the ability of these fragments to activate, or interact with, calmodulin-regulated enzymes and anti-calmodulin drugs. Each of the fragments, except 107-148, bound to a phenothiazine affinity column in a Ca2+-dependent manner. Thus, calmodulin contains two interaction sites for phenothiazines: one on the NH2-terminal half (fragment 1-77) and one on the COOH-terminal half (fragment 78-148). None of the fragments activates the protein phosphatase, calcineurin, or prevents its stimulation by calmodulin, nor does any of the fragments stimulate Ca2+-dependent cAMP phosphodiesterase. A single cleavage in the middle of the calmodulin molecule results in the rapid dissociation of the two resultant fragments and a loss of ability to activate cAMP phosphodiesterase. One fragment, 78-148, interacts with phosphodiesterase and prevents its activation by calmodulin (Ki: 1.5 +/- 0.4 X 10(-6) M). The same fragment, 78-148, can fully activate phosphorylase kinase but with a lower affinity than calmodulin (Kuznicki, J., Grabarek, Z., Brzeska, H., Drabikowski, W., and Cohen, P. (1981) FEBS Lett. 130, 141-145). Thus, peptide 78-148 behaves as a calmodulin agonist or antagonist or as neither, depending on the enzyme under study.