Wild-type Calmodulin Research Articles

Neurite outgrowth of neuroblastoma cells overexpressing α and β isoforms of Ca 2+/calmodulin-dependent protein kinase II—effects of protein kinase inhibitors

Ca 2+/calmodulin-dependent protein kinase II (CaM kinase II) is one of the most abundant protein kinases in the brain and has a broad substrate specificity [M.K. Bennett, N.E. Erondu, M.B. Kennedy, Purification and characterization of a calmodulin-dependent protein kinase that is highly concentrated in brain, J. Biol. Chem. 258 (1983) 12735–12744 [1]; J.R. Goldenring, B. Gonzalez, J.S. McGuire, Jr., R.J. DeLorenzo, Purification and characterization of a calmodulin-dependent kinase from rat brain cytosol able to phosphorylate tubulin and microtubule-associated proteins, J. Biol.Chem. 258 (1983) 12632–12640 [4]; M.B. Kennedy, P. Greengard, Two calcium/calmodulin-dependent protein kinases, which are highly concentrated in brain, phosphorylate protein I at distinct sites, Proc. Natl. Acad. Sci. U.S.A. 78 (1981) 1293–1297 [10]; T. Yamauchi, H. Fujisawa, Evidence for three distinct forms of calmodulin-dependent protein kinases from rat brain, FEBS Lett. 116 (1980) 141–144 [20]; T. Yamauchi, H. Fujisawa, Purification and characterization of the brain calmodulin-dependent protein kinase (kinase II), which is involved in the activation of tryptophan 5-monooxygenase, Eur. J. Biochem. 132 (1983) 15–21 [21]]. The α and β isoforms of CaM kinase II are known to be expressed almost exclusively in the brain [P.I. Hanson, H. Schulman, Ca 2+/calmodulin-dependent protein kinases, Annu. Rev. Biochem. 61 (1992) 559–601 [7]]. To elucidate the cellular function of CaM kinase II, we introduced cDNA of wild-type CaM kinase II α- or β-isoform, and of mutant α-isoform (Ala-286 kinase) into two different types of neuroblastoma, Neuro2a (Nb2a) and NG108-15, thus generating cell lines stably producing elevated levels of these kinases. The mutant α-isoform is markedly suppressed in its autophosphorylation by replacement of Thr-286 with Ala [Y.-L. Fong, W.L. Taylor, A.R. Means, T.R. Soderling, Studies of the regulatory mechanism of Ca 2+/calmodulin-dependent protein kinase II. Mutation of threonine 286 to alanine and aspartate, J. Biol. Chem. 264 (1989) 16759–16763 [3]; P.I. Hanson, M.S. Kapiloff, L.L. Lou, M.G. Rosenfeld, H. Schulman, Expression of a multifunctional Ca 2+/calmodulin-dependent protein kinase and mutational analysis of its autoregulation, Neuron 3 (1989) 59–70 [6]; S. Ohsako, H. Nakazawa, S. Sekihara, A. Ikai, T. Yamauchi, Role of Threonine-286 as autophosphorylation site for appearance of Ca 2+-independent activity of calmodulin-dependent protein kinase II α subunit, J. Biochem. 109 (1991) 137–143 [15]]. We provided evidence that CaM kinase II played a role in regulating neurite outgrowth and growth cone motility in these cells, and that the autophosphorylation is essential for the kinase to sufficiently exert its cellular function in vivo [Y. Goshima, S. Ohsako, T. Yamauchi, Overexpression of Ca 2+/calmodulin-dependent protein kinase II in Neuro2a and NG108-15 neuroblastoma cell lines promotes neurite outgrowth and growth cone motility, J. Neurosci. 13 (1993) 559–567 [5]]. Neurite outgrowth was further stimulated by treatment with 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) or chelerythrine, inhibitors of protein kinase C [T. Nomura, K. Kumatoriya, Y. Yoshimura, T. Yamauchi, Overexpression of α and β isoforms of Ca 2+/calmodulin-dependent protein kinase II in neuroblastoma cells—H-7 promotes neurite outgrowth, Brain Res. 766 (1997) 129–141 [14]]. The morphological change stimulated with protein kinase inhibitors was rapid and was greater in the β than α cells. Some substrates of CaM kinase II related to neurite outgrowth were detected in cells overexpressing the kinase stimulated with H-7. These results suggest that CaM kinase II and protein kinase C play an important role in the control of cell change. Themes: Development and regeneration Topics: Process outgrowth, growth cones, and sprouting

Calcium binding sites of calmodulin and electron transfer by neuronal nitric oxide synthase.

Although binding of calmodulin (CaM) to neuronal nitric oxide synthase (nNOS) has been demonstrated to act as the trigger necessary for electron transfer and catalytic activity, the exact manner in which this is achieved is unclear. By using a series of single point mutants of Drosophila melanogaster CaM, the role that each Ca2+ binding site plays in the transfer of electrons within nNOS has been examined. In these mutants, the bidentate glutamic acid (E) residue which coordinates Ca2+ at the -Z position in each site has been mutated to a glutamine (Q), preventing Ca2+ binding at that site. The results demonstrate that Ca2+ binding at site I of CaM is critical for all electron transfer reactions. All nNOS activities measured (citrulline formation, NADPH oxidation, and cytochrome c reduction) in the presence of the site I CaM mutant (denoted B1Q) were only 2% of the nNOS activity with wild-type CaM. The B2Q and B4Q mutants activated nNOS to similar levels. These two mutants, however, affected nNOS heme-dependent activities to a greater extent than they affected activities independent of the nNOS heme. The site III CaM mutant (B3Q) activated nNOS to levels similar to activities measured with wild-type CaM. Rates of formation of the ferrous-CO complex were also obtained with each of the mutant CaMs. The relative binding affinities of these mutants do not correlate with the observed differences in electron transfer rates. These results demonstrate that, although binding of CaM to nNOS is necessary for catalysis, specific interactions between the two proteins exist which are required for efficient electron transfer.

The role of beta-sheet interactions in domain stability, folding, and target recognition reactions of calmodulin.

Single-residue mutations have been made of the hydrophobic Ile or Val residue in position 8 of each of the four calcium-binding loop sequences (sites I-IV) of Drosophila calmodulin. These highly conserved residues are part of the hydrophobic core of either calmodulin domain and are involved in the structural link of two calcium-binding sites via a short antiparallel beta-sheet. In the apo-form, the replacement of Ile (or Val) by Gly causes a significant destabilization, shown by the unfolding of the secondary structure of the domain carrying the mutation. In the presence of calcium, the deficiency in alpha-helical structure at 20 degrees C is restored for the mutants at site I, II, or III but not at site IV, which requires the further binding of a high-affinity target peptide to re-establish the native conformation. The extent of the destabilization is seen in the depression of the melting temperature of individual domains, which can be as large as 80 degrees C in the case of Ca4-CaM(V136G). However, because of low values of the unfolding enthalpy for calmodulin domains, only relatively low values of <2 kcal/mol are implied for DeltaDeltaG, the free energy of destabilization due to mutation. Consistent with this, the secondary structure of any unfolded mutant domain is highly sensitive to solvent composition and is largely refolded in the presence of 12.5% (v/v) aqueous trifluoroethanol. Compared to wild-type calmodulin, the affinities of the mutants for calcium and target peptides from sk-MLCK at 20 degrees C are significantly reduced but the effects are relatively small. These results indicate that the conformation of calmodulin can be dramatically altered by mutation of a single highly conserved residue but that changes in solvent or the binding of a target sequence can readily compensate for this, restoring the wild-type properties. The results also suggest that the integrity of both the apo- and holo-forms of calmodulin is important for the maintenance of its biological function and confirm the importance of conserving the structural function of the residues involved in the beta-sheet interactions.

Phosphorylation of Calmodulin by Myosin Light Chain Kinase Is Altered by Exchange or Duplication of EF-Hand Pairs

We have previously demonstrated that under certain conditions, myosin light chain kinase can phosphorylate its activator, calmodulin. In this study we show that myosin light chain kinase from chicken gizzard can phosphorylate recombinant calmodulins in which the EF-hand pairs (Ca2+-binding domains) are duplicated or exchanged. Three mutants were used CaMNN (the amino-terminal EF-hand pair is duplicated), CaMCC (the carboxy-terminal EF-hand pair is duplicated) and CaMCN (the carboxy- and amino-terminal EF-hand pairs are switched). Myosin light chain kinase phosphorylated CaMNN and CaMCN to a greater extent than wild-type CaM but did not phosphorylate CaMCC. While CaMCC is a competitive inhibitor of myosin light chain kinase-catalyzed phosphorylation of myosin light chains, it did not prevent the phosphorylation of native calmodulin under the conditions employed in these studies. These data suggest that, although the carboxy- and amino-terminal EF-hand pairs are similar, their orientation can be distinguished by chicken gizzard myosin light chain kinase.

NMR studies of the E140Q mutant of the carboxy-terminal domain of calmodulin reveal global conformational exchange in the Ca2+-saturated state.

In the present investigation, the Ca2+ activation of the C-terminal domain of bovine calmodulin and the effects of replacing the bidentate Ca2+-coordinating glutamic acid residue in the 12th and last position of loop IV with a glutamine are studied by NMR spectroscopy. The mutation E140Q results in sequential Ca2+ binding in this domain and has far-reaching effects on the structure of (Ca2+)2 TR2C, thereby providing further evidence for the critical role of this glutamic acid residue for the Ca2+-induced conformational change of regulatory EF-hand proteins. Analyses of the NOESY spectra of the mutant under Ca2+-saturated conditions, such that 97% of the protein is in the (Ca2+)2 form, revealed two sets of mutually exclusive NOEs. One set of NOEs is found to be consistent with the closed structure observed in the apo state of the C-terminal domain of the wild-type protein, while the other set supports the open structure observed in the Ca2+-saturated state. In addition, several residues in the hydrophobic core exhibit broadened resonances. We conclude that the (Ca2+)2 form of the mutant experiences a global conformational exchange between states similar to the closed and open conformations of the C-terminal domain of wild-type calmodulin. A population of 65 +/- 15% of the open conformation and an exchange rate of (1-7) x 10(4) s(-1) were estimated from the NMR data and the chemical shifts of the wild-type protein. From a Ca2+ titration of the 15N-labeled mutant, the macroscopic binding constants [log(K1) = 4.9 +/- 0.3 and log(K2) = 3.15 +/- 0.10] and the inherent chemical shifts of the intermediate (Ca2+)1 form of the mutant were determined using NMR. Valuable information was also provided on the mechanism of the Ca2+ activation and the roles of the structural elements in the two Ca2+-binding events. Comparison with the wild-type protein indicates that the (Ca2+)1 conformation of the mutant is essentially closed but that some rearrangement of the empty loop IV toward the Ca2+-bound form has occurred.

Regulation of type I adenylyl cyclase by calmodulin kinase IV in vivo.

Type I adenylyl cyclase is a neurospecific enzyme that is stimulated by Ca2+ and calmodulin (CaM). This enzyme couples the Ca2+ and cyclic AMP (cAMP) regulatory systems in neurons, and it may play an important role for some forms of synaptic plasticity. Mutant mice lacking type I adenylyl cyclase show deficiencies in spatial memory and altered long-term potentiation (Z. Wu, S. A. Thomas, Z. Xia, E. C. Villacres, R. D. Palmiter, and D. R. Storm, Proc. Natl. Acad. Sci. USA 92:220-224, 1995). Although type I adenylyl cyclase is synergistically stimulated by Ca2+ and G-protein-coupled receptors in vivo, very little is known about mechanisms for inhibition of the enzyme. Here, we report that type I adenylyl cyclase is inhibited by CaM kinase IV in vivo. Expression of constitutively active or wild-type CaM kinase IV inhibited Ca2+ stimulation of adenylyl cyclase activity without affecting basal or forskolin-stimulated activity. Type I adenylyl cyclase has two CaM kinase IV consensus phosphorylation sequences near its CaM binding domain at Ser-545 and Ser-552. Conversion of either serine to alanine by mutagenesis abolished CaM kinase IV inhibition of adenylyl cyclase. This suggests that the activity of this enzyme may be directly inhibited by CaM kinase IV phosphorylation. Type VIII adenylyl cyclase, another enzyme stimulated by CaM, was not inhibited by CaM kinase II or IV. We propose that CaM kinase IV may function as a negative feedback regulator of type I adenylyl cyclase and that CaM kinases may regulate cAMP levels in some cells.

The linker of calmodulin lacking Glu84 is elongated in solution, although it is bent in the crystal.

The solution structure of a mutant calmodulin (des84) lacking Glu84 in the central helix linking the two calmodulin lobes is substantially different from its crystal structure. As determined by small-angle X-ray scattering, the radius of gyration and the maximum linear dimension of des84 in the presence of 0.1 mM calcium are 20.8 A and 62.5 A, respectively. These respective dimensions are larger than those expected from the crystal structure of des84, 18.5 A and 55.0 A, and smaller than those expected from the crystal structure of wild type, 22.8 A and 67.5 A. The distance distribution function of des84 indicates that it assumes an elongated, dumbbell shape in solution. The solution scattering profile of des84 is indistinguishable from that of wild-type calmodulin. The calcium-dependent binding of melittin to des84 causes a change in its shape from elongated to spherical, as seen with other calmodulins. In comparison with calcium-saturated des84, calcium-free des84 is slightly elongated; a slight compaction is observed with native calmodulin. The observed differences between the averaged solution structure and the crystal structure of des84 suggests that an ensemble of structures is available to calmodulin in solution and that its target need not induce a change in its conformation. These results support the theory that the linker region of the central helix of calmodulin functions as a flexible tether.

The Role of Phe-92 in the Ca2+-induced Conformational Transition in the C-terminal Domain of Calmodulin

Recent studies have shown that substitution of Ala for one or more Phe residues in calmodulin (CaM) imparts a temperature-sensitive phenotype to yeast (Ohya, Y., and Botstein, D. (1994) Science 263, 963-966). The Phe residue immediately preceding the first Ca(2+) ligand in site III of CaM (Phe-92) was found to be of particular importance because the mutation at this position alone was sufficient to induce this phenotype. In the present work we have studied the functional and structural consequences of the Phe-92 --> Ala mutation in human liver calmodulin. We found that the mutant (CaMF92A) is incapable of activating phosphodiesterase, and the maximal activation of calcineurin is reduced by 40% as compared with the wild type CaM. Impaired regulatory properties of CaMF92A are accompanied by an increase in affinity for Ca(2+) at the C-terminal domain. To investigate the structural consequences of the F92A mutation, we constructed four recombinant C-terminal domain fragments (C-CaM) of calmodulin (residues 78-148): 1) wild type (C-CaMW); 2) Ala substituted for Phe-92 (C-CaMF92A); 3) cysteine residues introduced at position 85 and 112 to lock the domain with a disulfide bond in the Ca(2+)-free (closed) conformation (C-CaM85/112); and 4) mutations 2 and 3 combined (C-CaM85/112F92A). The Cys-containing mutants readily form intramolecular disulfide bonds regardless whether Phe or Ala is present at position 92. The F92A mutation causes a decrease in stability of the domain in the absence of Ca(2+) as indicated by an 11.8 degree C shift in the far UV circular dichroism thermal unfolding curve. This effect is reversed by the disulfide bond in the C-CaM85/112F92A mutant. The C-CaMW peptide shows a characteristic Ca(2+)-dependent increase in solvent-exposed hydrophobic surface which was monitored by an increase in the fluorescence of the hydrophobic probe 1,1'-bis(4-anilino)-naphthalene-5,5'-disulfonic acid. The fluorescence increase induced by C-CaMF92A is approximately 45% lower than that induced by C-CaMW suggesting that the F92A mutation causes a decrease in the accessibility of several hydrophobic side chains in the C-terminal domain of CaM in the presence of Ca(2+). The Cys-85-Cys-112 disulfide bond causes a 10- or 5.9-fold decrease in Ca(2+) affinity depending on whether Phe or Ala is present at position 92, respectively, suggesting that coupling between Ca(2+) binding and the conformational transition is weaker in the absence of the phenyl ring at position 92. Our results indicate that Phe-92 makes an important contribution to the Ca(2+)-induced transition in the C-terminal domain of CaM. This is most likely the reason for the severely impaired regulatory properties of the CaM mutants having Ala substituted for Phe-92.

Differential Stimulation of NAD Kinase and Binding of Peptide Substrates by Wild-Type and Mutant Plant Calmodulin Isoforms

Calmodulin fromArabidopsis thalianaconsists of at least four isoforms, which differ in their deduced amino acid sequences by as many as six conservative substitutions. To determine whether these differences are biochemically significant, cDNAs encoding three of the four isoforms were engineered to produce recombinant proteins inEscherischia coli,purified to apparent homogeneity, and assayed for their abilities to activate pea leaf NAD kinasein vitro.The CaM-2 isoform was a significantly more efficient activator of NAD kinase compared with the CaM-4 and -6 isoforms based on both the apparentVmaxit elicited and theK0.5activation. These results are consistent with the hypothesis that theArabidopsisCaM isoforms have evolved to optimize the protein's interaction with different Ca2+/CaM-regulated target enzymes. The ability to activate NAD kinase was also investigated for a carboxy-terminal nonsense mutant of CaM-6 (CaM-6M), which substituted 14 hydrophilic amino acids for a region of seven amino acids that normally form an exposed hydrophobic surface whenwtCaM-6 binds Ca2+. CaM-6M-activated NAD kinase displayed an apparentVmaxthat was reduced 40% and aK0.5that was an order of magnitude greater than the CaM-6-activated enzyme. The Ca2+-dependence of CaM-6M to activate NAD kinase was identical to that of CaM-6, but CaM-6M bound synthetic peptide substrates with lower apparent affinity than did CaM-6. Thus, the carboxy-terminal hydrophobic domain of CaM appears to be critical for its interaction with NAD kinase. In contrast, amino-terminal fusions of a hydrophilic, α-helical 12-residuec-mycepitope tag to CaM-2 and -4 yielded proteins that activated NAD kinase to apparentVmaxvalues within 10% of those obtained with the wild-type CaM proteins.

Calcium-induced interactions of calmodulin domains revealed by quantitative thrombin footprinting of Arg37 and Arg106.

Calcium-dependent conformational states of calmodulin (CaM) were probed by thrombin to determine quantitative differences in the susceptibility of two bonds: Arg37-Ser38 (R37-S38, near site I in the N-terminal domain) and Arg106-His107 (R106-H107, near site III in the C-terminal domain). Quantitative thrombin footprinting of a discontinuous equilibrium calcium titration of wild-type calmodulin showed that the R37-S38 bond of the apoprotein was cleaved at a barely detectable level while the R106-H107 bond was maximally susceptible. Calcium binding to sites III and IV monotonically protected R106-H107 from proteolysis; concomitantly, the susceptibility of R37-S38 increased. However, calcium binding to sites I and II protected R37-S38 from cleavage, yielding a peaked biphasic profile composed of equal and opposite transitions. Both bonds were fully protected when calmodulin was saturated with calcium. Susceptibility profiles resolved from the fractional abundance of primary cleavage products (peptides 1-37, 38-148, 1-106, 107-148) were interpreted as directly reflecting calcium-induced conformational changes in whole calmodulin; free energies of calcium binding and cooperativity were estimated. Secondary cleavage was never observed; both R37 and R106 were sites of thrombinolysis in whole calmodulin only. In studies of E140Q-CaM (having a mutation in site IV), the susceptibility of R37-S38 decreased monotonically. Thus, the biphasic character of cleavage of R37 in helix B was not intrinsic to that domain but depended on propagation of effects of calcium-induced changes in the C-terminal domain. The observed patterns of susceptibility indicated that partially saturated wild-type calmodulin adopts at least one intermediate conformation whose structure is determined by calcium-mediated interactions between the domains.

Ca2+ Binding to Calmodulin and Its Role in Schizosaccharomyces pombe as Revealed by Mutagenesis and NMR Spectroscopy

As a first step toward identifying the important structural elements of calmodulin from Schizosaccharomyces pombe, we examined the ability of heterologous calmodulins and Ca(2+)-binding site mutant S. pombe calmodulins to replace the essential cam1+ gene. A cDNA encoding vertebrate calmodulin allows growth of S. pombe. However, calmodulin from Saccharomyces cerevisiae does not support growth even though the protein is produced at high levels. With one exception, all mutant S. pombe calmodulins with one or more intact Ca(2+)-binding sites allow growth at 21 degrees C. A mutant containing only an intact Ca(2+)-binding site 3 fails to support growth, as does S. pombe calmodulin with all four Ca(2+)-binding sites mutated. Several of the mutant proteins confer a temperature-sensitive phenotype. Analysis of the degree of temperature sensitivity allows the Ca(2+)-binding sites to be ranked by their ability to support fission yeast proliferation. Site 2 is more important than site 1, which is more important than site 4, which is more important than site 3. A visual colony color screen based on the fission yeast ade1+ gene was developed to perform these genetic analyses. To compare the Ca(2+)-binding properties of individual sites to their functional importance for viability, Ca2+ binding to calmodulin from S. pombe was studied by 1H NMR spectroscopy. NMR analysis indicates a Ca(2+)-binding profile that differs from those previously determined for vertebrate and S. cerevisiae calmodulins. Ca(2+)-binding site 3 has the highest relative affinity for Ca2+, while the affinities of sites 1, 2, and 4 are indistinguishable. A combination of an in vivo functional assay and an in vitro physical assay reveals that the relative affinity of a site for Ca2+ does not predict its functional importance.

Paramecium Na+ channels activated by Ca2+-calmodulin: Calmodulin is the Ca2+ sensor in the channel gating mechanism

Paramecium Na+ channels, which were Ca(2+)-calmodulin activated, were studied in the inside-out mode of patch clamp. After excision of the membrane patch, they were active in the presence of 10(-5) to 10(-3) M Ca+ in the bath. They became much less active in the presence of 10(-6) M Ca2+, and their activity subsided completely at 10(-8) M Ca2+. A Hill plot showed a dissociation constant of 6 microM for Ca2+ binding. This dissociation constant shifted to a submicromolar range in the presence of 1 mM Mg2+. The channels also exhibited a mild voltage dependence. When exposed to 10(-8) M Ca2+ for an extended period of 2-4 min, channels were further inactivated even after bath Ca2+ was restored to 10(-4) M. Whereas neither high voltage (+100 mV) nor high Ca2+ (10(-3) M) was effective in reactivation of the inactive channels, addition of Paramecium wild-type calmodulin together with high Ca2+ to the bath restored channel activity without a requirement of additional Mg2+ and metabolites such as ATP. The channels reactivated by calmodulin had the same ion conductance, ion selectivity and Ca2+ sensitivity as those prior to inactivation. These inactivation and reactivation of the channels could be repeated, indicating that the direct calmodulin effect on the Na+ channel was reversible. Thus, calmodulin is a physiological factor critically required for Na+ channel activation, and is the Ca2+ sensor of the Na(+)-channel gating machinery.

Recovery of native structure by calcium binding site mutants of calmodulin upon binding of sk-MLCK target peptides.

The calcium-dependent binding of two synthetic 18-residue peptides derived from the calmodulin binding region of skeletal myosin light chain kinase to wild-type Drosophila melanogaster calmodulin and four calcium binding site calmodulin mutants has been investigated using optical spectroscopy. The WFF peptide (with W4 and F17) and the FFW peptide (with F4 and W17) both bind to wild-type calmodulin with 1:1 stoichiometry and Kd values of < or = 0.2 and 1.6 nM, respectively. Near-UV CD spectra of the protein-peptide complexes suggest that both peptides bind in the same orientation, with the side chain of residue 4 interacting with the C-domain of calmodulin and that of residue 17 with the N-domain [as in the structure of the calmodulin-M13 peptide complex determined by Ikura et al. [Ikura, M., Clore, G. M., Gronenborn, A. M., Zhu, G., Klee, C. B., & Bax, A. (1992) Science 256, 632-638]]. Both peptides have lower affinities for all the mutant calmodulins than for the wild-type protein. Fluorescence measurements suggest that mutation of calcium binding site 2 in the N-domain does not affect the interaction of the W4 side chain of the WFF peptide with the C-domain of calmodulin. However, the E67Q (B2Q) but not the E67K (B2K) mutation (site 2, N-domain) alters the interaction of W17 of the FFW peptide with the protein. In contrast, the E140K (B4K) mutation has a much greater effect than the E140Q (B4Q) mutation (site 4, C-domain) on the interaction of calmodulin with both peptides.(ABSTRACT TRUNCATED AT 250 WORDS)

A Protein/Peptide Assay Using Peptide-Resin Adduct: Application to the Calmodulin/RS20 Complex

To obtain equilibrium and kinetic constants of a protein/peptide complex, we have developed a rapid procedure which uses peptides specifically linked to a resin. With this peptide-resin adduct, bound and free 125I-labeled protein could be easily separated by simple centrifugation. The feasibility of the method was demonstrated with the calmodulin/RS20 complex, where RS20 is the putative calmodulin binding peptide of the smooth muscle myosin light chain kinase (smMLCK). In addition to the wild-type calmodulin (SYNCAM) expressed in Escherichia coli, we also examined calmodulin mutants with charge reversals called SYNCAM12A (DEE 118-120 → KKK) and SYNCAM18A (EEE 82-84 → KKK and DEE 118-120 → KKK). The kinetic analysis of the interaction between SYNCAM and RS20 associated with titration experiments allowed us to measure dissociation constants (KD) in the range of 10−9 M, in good agreement with previously published data. Moreover, the binding assays showed that SYNCAM18A did not interact with RS20, whereas SYNCAM12A did with a KD around 10−8 M. The lack of binding of SYNCAM18A to RS20 provides an explanation for the lack of smMLCK activation by SYNCAM18A. Altogether, these results demonstrate that peptide-resin can be used as a tool for separating bound from free protein, thus enabling a rapid and reliable quantification of the protein/peptide interaction.

The effect of Met–>Leu mutations on calmodulin's ability to activate cyclic nucleotide phosphodiesterase.

Calmodulin (CaM) has two hydrophobic surface patches that are particularly rich in Met residues, and these are the major contact areas where CaM interacts with its target enzymes. The amino acid Leu has been introduced by site-directed mutagenesis to replace all the Met residues in CaM. All nine individual Met-->Leu mutants of CaM as well as some double and quadruple mutants were expressed in Escherichia coli. All mutants could be purified by calcium-dependent hydrophobic affinity chromatography, indicating that they still expose their hydrophobic surfaces upon binding calcium. Each single Met-->Leu mutation in the C-terminal domain of the protein had little effect on its ability to activate phosphodiesterase (PDE), while a quadruple mutant with four C-terminal Leu residues instead of Met has a significantly lower affinity for PDE. The M36L mutant is a poor activator compared with the other three N-terminal single Met-->Leu mutants, which have a slightly lower affinity for PDE than wild-type CaM. The introduction of a positively charged Arg for Met-145 resulted in an almost complete loss of CaM's ability to activate PDE. Nuclear magnetic resonance spectroscopy was used to show that most CaM mutants retain their overall three-dimensional structure. Thus, the altered activation properties appear to arise from differences in the flexibility and polarizability of the Met and Leu sidechains, rather than from structural perturbations.

Two-dimensional NMR Studies of Selenomethionyl Calmodulin

Calmodulin (CaM) is a ubiquitous calcium regulatory protein that can interact with almost 30 different target proteins. The majority of the CaM-binding domains of the target proteins are believed to interact with two hydrophobic surfaces on Ca2+-CaM; these two regions are very rich in Met residues. To obtain more information about the role of these residues, we have biosynthetically incorporated selenomethionine (SeMet) in place of the nine Met residues of CaM. Amino acid analysis shows that the SeMet-CaM contains 15% Met and 85% SeMet. SeMet-CaM retains many of the properties of the wild-type protein; it activates the enzyme cyclic nucleotide phosphodiesterase, it binds to phenyl-Sepharose and myosin light chain kinase (MLCK) in a calcium-dependent manner, and it experience a calcium-dependent band shift during SDS-gel electrophoresis. Moreover, by comparing the natural abundance (1H, 13C)-heteronuclear multiple quantum coherence (HMQC) spectra of the calcium, apo and target peptide-bound forms of wild-type CaM and SeMet-CaM, we have found that the two proteins have very similar, if not identical, structures. Thus, the substitution of SeMet for Met does not cause a change in the conformation and function of CaM, in agreement with the results obtained for other proteins. The apo, calcium and target peptide-bound forms of SeMet-CaM were subsequently studied by natural abundance (1H, 77 Se)-heteronuclear multiple bond correlation (HMBC) and (1H, 13C)-HMQC NMR. Nine well-resolved 77Se resonances could be observed. Substitution of SeMet for Met gave rise to the same 1H and 13C chemical shift changes for each individual Met residue, this facilitated making the assignments from known 1 H, 13C assignments of the Met residues. Some of these assignments were confirmed by studying Met→Leu mutants of CaM. With the exception of Met76, which always remains solvent exposed, all resonances experienced large 77Se chemical shift changes upon the addition of Ca2+ and the MLCK peptide. The large shift changes indicate that the electron distribution in the SeMet side-chain can be adjusted for the different states of CaM, suggesting that the polarizability of sulfur or selenium may be important for the proper functioning of CaM. This study also shows that the natural abundance (1H, 77Se)-HMBC experiment provides a sensitive approach for the study of SeMet protein

A calmodulin-target peptide hybrid molecule with unique calcium-binding properties.

This paper describes the production and properties of a hybrid protein comprising the full length of the Xenopus laevis calmodulin (CaM) sequence, followed, through a glycylglycine linker, by the 26-residue CaM-binding region of myosin light-chain kinase (M13). This hybrid molecule appears to have high thermal stability (Tm > 75 degrees C in the presence of Ca2+) as well as unusual Ca(2+)-binding properties: (i) a wide-range biphasic Ca(2+)-binding response (extending over pCa 4.8-7.4) and (ii) a high apparent binding constant (pCa50% = 6.3, a 10-fold increase from that of wild-type CaM). NMR and CD data indicate that the CaM-M13 hybrid molecule exists in equilibrium in an approximate 1:1 ratio between two major conformations, one of which is similar to the compact globular structure of the CaM-M13 complex [M.Ikura, G.M. Clore, A.M. Gronenborn, G. Zhu, C.B. Klee and A. Bax (1992) Science, 256, 632-638] and the other to the dumb-bell-like structure of the wild type CaM [Y.S. Babu, C.E. Bugg and W.J. Cook (1988) J. Mol. Biol., 204, 191-204]. The biphasic Ca(2+)-binding curve can be interpreted using a linear combination of two Hill binding curves with significantly different dissociation constants (2 x 10(-9) M and 8 x 10(-8) M), which can be attributed to the two conformations in equilibrium. The present study has opened an avenue to engineer proteins with higher Ca(2+)-binding affinities using the known CaM structures as a template.

The essential mitotic target of calmodulin is the 110-kilodalton component of the spindle pole body in Saccharomyces cerevisiae.

Two independent methods identified the spindle pole body component Nuf1p/Spc110p as the essential mitotic target of calmodulin. Extragenic suppressors of cmd1-1 were isolated and found to define three loci, XCM1, XCM2, and XCM3 (extragenic suppressor of cmd1-1). The gene encoding a dominant suppressor allele of XCM1 was cloned. On the basis of DNA sequence analysis, genetic cosegregation, and mutational analysis, XCM1 was identified as NUF1/SPC110. Independently, a C-terminal portion of Nuf1p/Spc110p, amino acid residues 828 to 944, was isolated as a calmodulin-binding protein by the two-hybrid system. As assayed by the two-hybrid system, Nuf1p/Spc110p interacts with wild-type calmodulin and triple-mutant calmodulins defective in binding Ca2+ but not with two mutant calmodulins that confer a temperature-sensitive phenotype. Deletion analysis by the two-hybrid system mapped the calmodulin-binding site of Nuf1p/Spc110p to amino acid residues 900 to 927. Direct binding between calmodulin and Nuf1p/Spc110p was demonstrated by a modified gel overlay assay. Furthermore, indirect immunofluorescence with fixation procedures known to aid visualization of spindle pole body components localized calmodulin to the spindle pole body. Sequence analysis of five suppressor alleles of NUF1/SPC110 indicated that suppression of cmd1-1 occurs by C-terminal truncation of Nuf1p/Spc110p at amino acid residues 856, 863, or 881, thereby removing the calmodulin-binding site.

The essential mitotic target of calmodulin is the 110-kilodalton component of the spindle pole body in Saccharomyces cerevisiae.

Two independent methods identified the spindle pole body component Nuf1p/Spc110p as the essential mitotic target of calmodulin. Extragenic suppressors of cmd1-1 were isolated and found to define three loci, XCM1, XCM2, and XCM3 (extragenic suppressor of cmd1-1). The gene encoding a dominant suppressor allele of XCM1 was cloned. On the basis of DNA sequence analysis, genetic cosegregation, and mutational analysis, XCM1 was identified as NUF1/SPC110. Independently, a C-terminal portion of Nuf1p/Spc110p, amino acid residues 828 to 944, was isolated as a calmodulin-binding protein by the two-hybrid system. As assayed by the two-hybrid system, Nuf1p/Spc110p interacts with wild-type calmodulin and triple-mutant calmodulins defective in binding Ca2+ but not with two mutant calmodulins that confer a temperature-sensitive phenotype. Deletion analysis by the two-hybrid system mapped the calmodulin-binding site of Nuf1p/Spc110p to amino acid residues 900 to 927. Direct binding between calmodulin and Nuf1p/Spc110p was demonstrated by a modified gel overlay assay. Furthermore, indirect immunofluorescence with fixation procedures known to aid visualization of spindle pole body components localized calmodulin to the spindle pole body. Sequence analysis of five suppressor alleles of NUF1/SPC110 indicated that suppression of cmd1-1 occurs by C-terminal truncation of Nuf1p/Spc110p at amino acid residues 856, 863, or 881, thereby removing the calmodulin-binding site.

Calmodulin is essential for assembling links necessary for exocytotic membrane fusion in Paramecium.

Calmodulin has long been suspected to be involved in calcium-regulated exocytosis but its precise site(s) of action has not yet been identified. In Paramecium, a genetic approach to the problem is possible as in vivo-selected mutations in the calmodulin gene that prevent the activation of some channels have been characterized. Three of these calmodulin mutants were examined for exocytotic capacity and the mutant cam1 was found to be defective for exocytosis at 35 degrees C. The loss of exocytotic capacity in cam1 cells can be restored by transformation with the wild-type calmodulin gene, demonstrating that its exocytotic lesion is indeed due to the mutation in the calmodulin gene. The cam1 mutant displays abnormal exocytotic sites at the non-permissive temperature: it lacks the links ('rosettes' of intramembranous particles in the plasma membrane and the fibrous 'connecting material') which normally connect plasma and trichocyst membranes. Upon shift of cam1 cells from the permissive to a non-permissive temperature, performed sites remain functional. These results demonstrate that calmodulin is necessary for the assembly of these links at the exocytotic site. These results do not, however, exclude the possibility of calmodulin also being involved in Ca(2+)-dependent steps of the stimulus-exocytosis coupling.