Search

Calmodulin (CaM) is the universal calcium sensor in eukaryotes, regulating the function of numerous proteins. Crystallography and NMR show that free CaM-4Ca(2+) exists in an extended conformation with significant interdomain separation, but clamps down upon target peptides to form a highly compact structure. NMR has revealed substantial interdomain motions in CaM-4Ca(2+), enabled by a flexible linker. In one instance, CaM-4Ca(2+) has been crystallized in a compact configuration; however, no direct evidence for transient interdomain contacts has been observed in solution, and little is known about how large-scale interdomain motions contribute to biological function. Here, we use paramagnetic relaxation enhancement (PRE) to characterize transient compact states of free CaM that are too sparsely populated to observe by traditional NMR methods. We show that unbound CaM samples a range of compact structures, populated at 5-10%, and that Ca(2+) dramatically alters the distribution of these configurations in favor of states resembling the peptide-bound structure. In the absence of Ca(2+), the target peptide binds only to the C-terminal domain, and the distribution of compact states is similar with and without peptide. These data suggest an alternative pathway of CaM action in which CaM remains associated with its kinase targets even in the resting state. Only CaM-4Ca(2+), however, shows an innate propensity to form the physiologically active compact structures, suggesting that Ca(2+) activates CaM not only through local structural changes within each domain but also through more global remodeling of interdomain interactions. Thus, these findings illustrate the subtle interplay between conformational selection and induced fit.

Caldesmon is an actin- and myosin-binding protein found in smooth muscle that inhibits actin activation of myosin ATPase activity. The activity of caldesmon is controlled by phosphorylation and by binding to Ca(2+)-calmodulin. We investigated the effects of phosphorylation by p(21)-activated kinase 3 (PAK) and calmodulin on the 22 kDa C-terminal fragment of caldesmon (CaD22). We substituted the major PAK sites, Ser-672 and Ser-702, with either alanine or aspartic acid to mimic nonphosphorylated and constitutively phosphorylated states of caldesmon, respectively. The aspartic acid mutation of CaD22 weakened Ca(2+)-calmodulin binding but had no effect on inhibition of ATPase activity. Phosphorylation of the aspartic acid mutant with PAK resulted in the slow phosphorylation of Thr-627, Ser-631, Ser-635, and Ser-642. Phosphorylation at these sites weakened Ca(2+)-calmodulin binding further and reduced the inhibitory activity of CaD22 in the absence of Ca(2+)-calmodulin. Phosphorylation of these sites of the alanine mutant of CaD22 had no effect on Ca(2+)-calmodulin binding but did reduce inhibition of ATPase activity. Thus, the region between residues 627 and 642 may contribute to the overall regulation of caldesmon's activity., (Copyright © 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Calcium signaling in neurons as in other cell types mediates changes in gene expression, cell growth, development, survival, and cell death. However, neuronal Ca(2+) signaling processes have become adapted to modulate the function of other important pathways including axon outgrowth and changes in synaptic strength. Ca(2+) plays a key role as the trigger for fast neurotransmitter release. The ubiquitous Ca(2+) sensor calmodulin is involved in various aspects of neuronal regulation. The mechanisms by which changes in intracellular Ca(2+) concentration in neurons can bring about such diverse responses has, however, become a topic of widespread interest that has recently focused on the roles of specialized neuronal Ca(2+) sensors. In this article, we summarize synaptotagmins in neurotransmitter release, the neuronal roles of calmodulin, and the functional significance of the NCS and the CaBP/calneuron protein families of neuronal Ca(2+) sensors.

Cardiac myocyte intracellular calcium varies beat-to-beat and calmodulin (CaM) transduces Ca2+ signals to regulate many cellular processes (e.g. via CaM targets such as CaM-dependent kinase and calcineurin). However, little is known about the dynamics of how CaM targets process the Ca2+ signals to generate appropriate biological responses in the heart. We hypothesized that the different affinities of CaM targets for the Ca2+-bound CaM (Ca2+-CaM) shape their actions through dynamic and tonic interactions in response to the repetitive Ca2+ signals in myocytes. To test our hypothesis, we used two fluorescence resonance energy transfer-based biosensors, BsCaM-45 (Kd = approximately 45 nm) and BsCaM-2 (Kd = approximately 2 nm), to monitor the real time Ca2+-CaM dynamics at low and high affinity CaM targets in paced adult ventricular myocytes. Compared with BsCaM-2, BsCaM-45 tracks the beat-to-beat Ca2+-CaM alterations more closely following the Ca2+ oscillations at each myocyte contraction. When pacing frequency is raised from 0.1 to 1.0 Hz, the higher affinity BsCaM-2 demonstrates significant elevation of diastolic Ca2+-CaM binding compared with the lower affinity BsCaM-45. Biochemically detailed computational models of Ca2+-CaM biosensors in beating cardiac myocytes revealed that the different Ca2+-CaM binding affinities of BsCaM-2 and BsCaM-45 are sufficient to predict their differing kinetics and diastolic integration. Thus, data from both experiments and computational modeling suggest that CaM targets with low versus high Ca2+-CaM affinities (like CaM-dependent kinase versus calcineurin) respond differentially to the same Ca2+ signal (phasic versus integrating), presumably tuned appropriately for their respective and distinct Ca2+ signaling pathways.

Ca(2+) binds to calmodulin (CaM) and triggers the interaction of CaM with its target proteins; CaM binding proteins (CaMBPs) can also regulate the metal binding to CaM. In the present paper, La(3+) binding to CaM was studied in the presence of the CaM binding peptides, Mastoparan (Mas) and Mas X, using ultrafiltration and titration of fluorescence. Ca(2+) binding was used as an analog to understand La(3+) binding in intact CaM and isolated N/C-terminal CaM domain of metal-CaM binary system and metal-CaM-CaMBPs ternary system. Mas/Mas X increased binding affinity of La(3+) to CaM by 0.5 approximately 3 orders magnitude. The metal ions binding affinity to the C-terminal or the N-terminal CaM domain suggested that in the first phase of binding process both Ca(2+) and La(3+) bind to C-terminal of CaM in the presence of Mas/Mas X. In the presence of CaM binding peptides, La(3+) binding preference was substantially altered from the metal-CaM binary system where La(3+) slightly preferred binding to the N-terminal sites of CaM. Our results will be helpful in understanding La(3+) interactions with CaM in the biological systems.

We have performed a kinetic analysis of Ca2+-dependent switching in the complex between calmodulin (CaM) and the IQ domain from neuromodulin, and have developed detailed kinetic models for this process. Our results indicate that the affinity of the C-ter Ca2+-binding sites in bound CaM is reduced due to a approximately 10-fold decrease in the Ca2+ association rate, while the affinity of the N-ter Ca2+-binding sites is increased due to a approximately 3-fold decrease in the Ca2+ dissociation rate. Although the Ca2+-free and Ca2+-saturated forms of the CaM-IQ domain complex have identical affinities, CaM dissociates approximately 100 times faster in the presence of Ca2+. Furthermore, under these conditions CaM can be transferred to the CaM-binding domain from CaM kinase II via a ternary complex. These properties are consistent with the hypothesis that CaM bound to neuromodulin comprises a localized store that can be efficiently delivered to neuronal proteins in its Ca2+-bound form in response to a Ca2+ signal.

The relationship between the free Ca2+ concentration and the apparent dissociation constant for the complex between calmodulin (CaM) and the neuromodulin IQ domain consists of two phases. In the first phase, Ca2+ bound to the C-ter EF hand pair in CaM increases the Kd for the complex from the Ca2+-free value of 2.3 +/- 0.1 microM to a value of 14.4 +/- 1.3 microM. In the second phase, Ca2+ bound to the N-ter EF hand pair reduces the Kd for the complex to a value of 2.5 +/- 0.1 microM, reversing the effect of the first phase. Due to energy coupling effects associated with these phases, the mean dissociation constant for binding of Ca2+ to the C-ter EF hand pair is increased approximately 3-fold, from 1.8 +/- 0.1 to 5.1 +/- 0.7 microM, and the mean dissociation constant for binding of Ca2+ to the N-ter EF hand pair is decreased by the same factor, from 11.2 +/- 1.0 to 3.5 +/- 0.6 microM. These characteristics produce a bell-shaped relationship between the apparent dissociation constant for the complex and the free Ca2+ concentration, with a distance of 5-6 microM between the midpoints of the rising and falling phases. Release of CaM from the neuromodulin IQ domain therefore appears to be promoted over a relatively narrow range of free Ca2+ concentrations. Our results demonstrate that CaM-IQ domain complexes can function as biphasic Ca2+ switches through opposing effects of Ca2+ bound sequentially to the two EF hand pairs in CaM.

Fesselin is a proline-rich actin-binding protein that was isolated from avian smooth muscle. Fesselin bundles actin and accelerates actin polymerization by facilitating nucleation. We now show that this polymerization of actin can be regulated by Ca(2+)-calmodulin. Fesselin was shown to bind to immobilized calmodulin in the presence of Ca(2+). The fesselin-calmodulin interaction was confirmed by a Ca(2+)-dependent increase in 2-(4-maleimidoanilino)naphthalene-6-sulfonic acid (MIANS) fluorescence upon addition of fesselin to MIANS-labeled wheat germ calmodulin. The affinity was estimated to be approximately 10(9) M(-1). The affinity of Ca(2+)-calmodulin to the fesselin F-actin complex was approximately 10(8) M(-1). Calmodulin binding to fesselin appeared to be functionally significant. In the presence of fesselin and calmodulin, the polymerization of actin was Ca(2+)-dependent. Ca(2+)-free calmodulin either had no effect or enhanced the ability of fesselin to accelerate actin polymerization. Ca(2+)-calmodulin not only reversed the stimulatory effect of fesselin but reduced the rate of polymerization below that observed in the absence of fesselin. While Ca(2+)-calmodulin had a large effect on the interaction of fesselin with G-actin, the effect on F-actin was small. Neither the binding of fesselin to F-actin nor the subsequent bundling of F-actin was greatly affected by Ca(2+)-calmodulin. Fesselin may function as an actin-polymerizing factor that is regulated by Ca(2+) levels.

We have used a highly environment-sensitive fluorescent probe 6-bromoacetyl-2-dimethylaminonaphthalene (badan) to study the interaction between calmodulin (CaM) and a CaM-binding peptide of the ryanodine receptor (CaMBP) and its sub-fragments F1 and F4. Badan was attached to the Thr34Cys mutant of CaM (CaM-badan). Ca(2+) increase in a physiological range of Ca(2+) (0.1-2 microM) produced about 40 times increase in the badan fluorescence. Upon binding to CaMBP, the badan fluorescence of apo-CaM showed a small increase at a slow rate; whereas that of Ca-CaM showed a large decrease at a very fast rate. Upon binding of CaM to the badan-labeled CaMBP, the badan fluorescence showed a small and slow increase at low Ca(2+), and a large and fast increase at high Ca(2+). Thus, the badan probe attached to CaM Cys(34) can be used to monitor conformational changes occurring not only in CaM, but also those in the CaM-CaMBP interface. Based on our results we propose that both the interaction interface and the global conformation of the CaM-CaMBP complex are altered by calcium.

The calcium hypothesis of Alzheimer's disease (AD) invokes the disruption of calcium signaling as the underlying cause of neuronal dysfunction and ultimately apoptosis. As a primary calcium signal transducer, calmodulin (CaM) responds to cytosolic calcium fluxes by binding to and regulating the activity of target CaM-binding proteins (CaMBPs). Ca(2+)-dependent CaMBPs primarily contain domains (CaMBDs) that can be classified into motifs based upon variations on the basic amphiphilic alpha-helix domain involving conserved hydrophobic residues at positions 1-10, 1-14 or 1-16. In contrast, an IQ or IQ-like domain often mediates Ca(2+)-independent CaM-binding. Based on these attributes, a search for CaMBDs reveals that many of the proteins intimately linked to AD may be calmodulin-binding proteins, opening new avenues for research on this devastating disease.

A variety of viral and signal transduction proteins are known to be myristoylated. Although the role of myristoylation in protein-lipid interaction is well established, the involvement of myristoylation in protein-protein interactions is less well understood. CAP-23/NAP-22 is a brain-specific protein kinase C substrate protein that is involved in axon regeneration. Although the protein lacks any canonical calmodulin (CaM)-binding domain, it binds CaM with high affinity. The binding of CAP-23/NAP-22 to CaM is myristoylation dependent and the N-terminal myristoyl group is directly involved in the protein-protein interaction. Here we show the crystal structure of Ca2+-CaM bound to a myristoylated peptide corresponding to the N-terminal domain of CAP-23/NAP-22. The myristoyl moiety of the peptide goes through a hydrophobic tunnel created by the hydrophobic pockets in the N- and C-terminal domains of CaM. In addition to the myristoyl group, several amino-acid residues in the peptide are important for CaM binding. This is a novel mode of binding and is very different from the mechanism of binding in other CaM-target complexes.

Synthetic RS20 peptide and a set of its point-mutated peptide analogs have been used to analyze the interactions between calmodulin (CaM) and the CaM-binding sequence of smooth-muscle myosin light chain kinase both in the presence and the absence of Ca(2+). Particular peptides, which were expected to have different binding strengths, were chosen to address the effects of electrostatic and bulky mutations on the binding affinity of the RS20 sequence. Relative affinity constants for protein/ligand interactions have been determined using electrospray ionization and Fourier transform ion cyclotron resonance mass spectrometry. The results evidence the importance of electrostatic forces in interactions between CaM and targets, particularly in the presence of Ca(2+), and the role of hydrophobic forces in contributing additional stability to the complexes both in the presence and the absence of Ca(2+).

Receptor-mediated activation of leukocytes by inflammatory stimuli elicits Ca2+ ion influx as a common and important activation mechanism that has been well established in the literature for over a decade. Inhibiting such receptor-operated Ca2+ influx channels is a potentially attractive strategy for developing anti-inflammatory drugs to attenuate leukocyte activation. Until very recently, the molecular identity of these channels has been unknown, which has hampered drug development in this area. However, the recent explosion of molecular information about one particular family of non-voltage-activated Ca2+ channels, the transient receptor potential (TRP) channels, together with emerging knowledge of their distribution, function and regulation, suggests that they represent a key subgroup of these channels and are therefore potentially attractive drug targets.

The mammalian gene products, transient receptor potential (trp)1 to trp7, are related to the Drosophila TRP and TRP-like ion channels, and are candidate proteins underlying agonist-activated Ca(2+)-permeable ion channels. Recently, the TRP4 protein has been shown to be part of native store-operated Ca(2+)-permeable channels. These channels, most likely, are composed of other proteins in addition to TRP4. In the present paper we report the direct interaction of TRP4 and calmodulin (CaM) by: (1) retention of in vitro translated TRP4 and of TRP4 protein solubilized from bovine adrenal cortex by CaM-Sepharose in the presence of Ca(2+), and (2) TRP4-glutathione S-transferase pull-down experiments. Two domains of TRP4, amino acid residues 688-759 and 786-848, were identified as being able to interact with CaM. The binding of CaM to both domains occurred only in the presence of Ca(2+) concentrations above 10 microM, with half maximal binding occurring at 16.6 microM (domain 1) and 27.9 microM Ca(2+) (domain 2). Synthetic peptides, encompassing the two putative CaM binding sites within these domains and covering amino acid residues 694-728 and 829-853, interacted directly with dansyl-CaM with apparent K(d) values of 94-189 nM. These results indicate that TRP4/Ca(2+)-CaM are parts of a signalling complex involved in agonist-induced Ca(2+) entry.

Calcium is essential for pollen germination and pollen tube growth. A large body of information has established a link between elevation of cytosolic Ca(2+) at the pollen tube tip and its growth. Since the action of Ca(2+) is primarily mediated by Ca(2+)-binding proteins such as calmodulin (CaM), identification of CaM-binding proteins in pollen should provide insights into the mechanisms by which Ca(2+) regulates pollen germination and tube growth. In this study, a CaM-binding protein from maize pollen (maize pollen calmodulin-binding protein, MPCBP) was isolated in a protein-protein interaction-based screening using (35)S-labeled CaM as a probe. MPCBP has a molecular mass of about 72 kDa and contains three tetratricopeptide repeats (TPR) suggesting that it is a member of the TPR family of proteins. MPCBP protein shares a high sequence identity with two hypothetical TPR-containing proteins from Arabidopsis. Using gel overlay assays and CaM-Sepharose binding, we show that the bacterially expressed MPCBP binds to bovine CaM and three CaM isoforms from Arabidopsis in a Ca(2+)-dependent manner. To map the CaM-binding domain several truncated versions of the MPCBP were expressed in bacteria and tested for their ability to bind CaM. Based on these studies, the CaM-binding domain was mapped to an 18-amino acid stretch between the first and second TPR regions. Gel and fluorescence shift assays performed with CaM and a CaM-binding synthetic peptide further confirmed MPCBP binding to CaM. Western, Northern, and reverse transcriptase-polymerase chain reaction analysis have shown that MPCBP expression is specific to pollen. MPCBP was detected in both soluble and microsomal proteins. Immunoblots showed the presence of MPCBP in mature and germinating pollen. Pollen-specific expression of MPCBP, its CaM-binding properties, and the presence of TPR motifs suggest a role for this protein in Ca(2+)-regulated events during pollen germination and growth.

Neurogranin (NG) binding of calmodulin (CaM) at its IQ domain is sensitive to Ca(2+) concentration and to modifications by protein kinase C (PKC) and oxidants. The PKC phosphorylation site of NG is within the IQ domain whereas the four oxidant-sensitive Cys residues are outside this region. These Cys residues were oxidized forming two pairs of intramolecular disulfides, and could also be glutathiolated by S-nitrosoglutathione resulting in the incorporation of four glutathiones per NG. Circular dichroism (CD) showed that modification of NG by phosphorylation, oxidation forming intramolecular disulfides, or glutathiolation did not affect the alpha-helical content of this protein. Mutation of the four Cys residues [Cys(-)-NG] to Gly and Ser did not affect the alpha-helical content either. Interaction of CaM with the reduced (red)-, glutathiolated (GS)-, or Cys(-)-NG in the Ca(2+)-free solution resulted in an increase in the alpha-helicity determined by their CD spectra, but relatively little change was seen with the oxidized NG (ox-NG) or phosphorylated NG (PO(4)-NG). The binding affinities between the various modified forms of NG and CaM were determined by CD spectrometry and sedimentation equilibrium: their affinities were Cys(-)-NG > red-NG, GS-NG > ox-NG > PO(4)-NG. Unlike Cys(-)-, red-, and GS-NG, neither ox- nor PO(4)-NG bound to a CaM-affinity column. Thus, both oxidation of NG to form intramolecular disulfides and phosphorylation of NG by PKC are effective in modulating the intracellular level of CaM. These results indicate that modification of NG to form intramolecular disulfides outside the IQ domain provides an alternative mechanism for regulation of its binding affinity to CaM.

Kinesin-like calmodulin-binding protein (KCBP), a novel kinesin-like protein from plants, is unique among kinesins and kinesin-like proteins in having a calmodulin-binding domain adjacent to its motor domain. KCBP localizes to mitotic microtubule (MT) arrays including the preprophase band, the spindle apparatus, and the phragmoplast, suggesting a role for KCBP in establishing these MT arrays by bundling MTs. To determine if KCBP bundles MTs, we expressed C-terminal motor and N-terminal tail domains of KCBP, and used the purified proteins in MT bundling assays. The 1.5 C protein with the motor and calmodulin-binding domains induced MT bundling. The 1.5 C-induced bundles were dissociated in the presence of Ca(2+)/calmodulin. Similar results were obtained with a 1.4 C protein, which lacks much of the coiled-coil region present in 1.5 C protein and does not form dimers. The N-terminal tail of KCBP, which contains an ATP-independent MT binding site, is also capable of bundling MTs. These results, together with the KCBP localization data, suggest the involvement of KCBP in establishing mitotic MT arrays during different stages of cell division and that Ca(2+)/calmodulin regulates the formation of these MT arrays., (Copyright 2000 Academic Press.)

Recently we discovered a tobacco protein (designated NtCBP4) that modulates heavy-metal tolerance in transgenic plants. Structurally, NtCBP4 is similar to mammalian cyclic-nucleotide-gated non-selective cation channels containing six putative transmembrane domains, a predicted pore region, a conserved cyclic-nucleotide-binding domain, and a high-affinity calmodulin-binding site that coincides with its cyclic-nucleotide-binding domain. Transgenic tobacco expressing the plasma-membrane-localized NtCBP4 exhibit improved tolerance to Ni(2+) and hypersensitivity to Pb(2+), which are associated with a decreased accumulation of Ni(2+) and an enhanced accumulation of Pb(2+) respectively. Transgenic plants expressing a truncated version of NtCBP4, from which regulatory domains had been removed, have a different phenotype. Here we describe our approach to studying the involvement of NtCBP4 in heavy-metal tolerance and to elucidate its physiological role.

The ubiquitous Ca2+-binding regulatory protein calmodulin (CaM) binds and activates a wide range of regulatory enzymes. The binding is usually dependent on the binding of Ca2+ to CaM; however, some target proteins interact with CaM in a calcium-independent manner. In this work, we have studied the interactions between CaM and a 20-residue synthetic peptide encompassing the major calmodulin-binding domain of cyclic nucleotide phosphodiesterase (PDE1A2). The binding was studied in the absence and presence of Ca2+ by far-UV and near-UV circular dichroism, fluorescence, and infrared spectroscopy. In addition, two-dimensional heteronuclear NMR studies with 13C-methyl-Met-CaM and uniformly 15N-labeled CaM were performed. Competition assays with smooth muscle myosin light chain kinase revealed a Kd of 224 nM for peptide binding to Ca2+-CaM, while binding of the peptide to apo-CaM is weaker. The peptide binds with an alpha-helical structure to both lobes of Ca2+-saturated CaM, and the single Trp residue is firmly anchored into the C-terminal lobe of CaM. In contrast, the Trp residue plays a minor role in the binding to the apo-protein. Moreover, when bound to apo-CaM, the PDE peptide is only partially helical, and it interacts solely with the C-terminal lobe of CaM. These results show that the Ca2+-induced activation of PDE involves a significant change in the structure and positioning of the CaM-bound PDE peptide domain.

Kinesin-like calmodulin-binding protein (KCBP) is a recently identified novel kinesin-like protein that appears to be unique to and ubiquitous in plants. KCBP is distinct from all other known KLPs in having a calmodulin-binding domain adjacent to its motor domain. We have used different regions of KCBP to study its interaction with tubulin subunits and the regulation of this interaction by Ca(2+)-calmodulin. The results show that the carboxy-terminal part of the KCBP, with or without calmodulin-binding domain, binds to tubulin subunits and this binding is sensitive to nucleotides. In the presence of Ca(2+)-calmodulin the motor with calmodulin-binding domain does not bind to tubulin. This Ca(2+)-calmodulin modulation is abolished in the presence of antibodies specific to the calmodulin-binding domain of KCBP. Similar binding studies with the carboxy-terminal part of KCBP lacking the calmodulin-binding domain show no effect of Ca(2+)-calmodulin. These results indicate that Ca(2+)-calmodulin modulates the interaction of KCBP with tubulin subunits and this modulation is due to the calmodulin-binding domain in the KCBP. Calcium-dependent calmodulin modulation of KCBP interaction with tubulin suggests regulation of KCBP function by calcium, the first such regulation of a kinesin heavy chain among all the known kinesin-like proteins.

Brush border myosin I (BBMI) is a single-headed molecular motor. Its catalytic domain exhibits extensive sequence homology to the catalytic domain of myosin II, while its tail lacks the coiled-coil nature of myosin II. The BBMI tail domain contains at least three IQ motifs and binds calmodulin. Addition of calcium removes one of these calmodulin light chains, with effects on ATPase activity and motility in in vitro assays. Using the techniques of cryoelectron microscopy and helical image analysis we have calculated three-dimensional (3D) maps of BBMI-decorated actin filaments prepared in the presence and absence of calcium. The 3D maps describe a BBMI catalytic domain that is strikingly similar to the catalytic domain of myosin II subfragment 1 (S1), with the exception of a short amino-terminal region of the heavy chain, which is absent from BBMI. The tail domains of BBMI and S1 are highly divergent in structure, continuing on from their respective motor domains with very different geometries. Addition of calcium to BBMI, and the concomitant loss of a calmodulin light chain, results in an extensive reorganization of mass in the tail domain.

The mechanism of dissociation reactions induced by calcium chelators has been studied for complexes of Drosophila calmodulin with target peptides, including four derived from the skeletal muscle myosin light chain kinase target sequence. Reactions were monitored by fluorescence stopped-flow techniques using a variety of intrinsic probes and the indicator Quin2. For most of the complexes, apparently biphasic kinetics were observed in several fluorescence parameters. The absence of any obvious relationship between dissociation rates and peptide affinities implies kinetic control of the dissociation pathway. A general mechanism for calcium and peptide dissociation was formulated and used in numerical simulation of the experimental data. Unexpectedly, the rate of the slowest step decreases with increasing [peptide]/[calmodulin] ratio. Numerical simulation shows this step could contain a substantial contribution from a reversible relaxation process (involving the species Ca2-calmodulin-peptide), convolved with the following step (loss of C-terminal calcium ions). The results indicate the potentially key kinetic role of the partially calcium-saturated intermediate species. They show that subtle changes in the peptide sequence can have significant effects on both the dissociation rates and also the dissociation pathway. Both effects could contribute to the variety of regulatory behavior shown by calmodulin with different target enzymes.

Protein phosphorylation by protein kinases plays a central regulatory role in cellular processes and these kinases are themselves tightly regulated. One common mechanism of regulation involves Ca2+-binding proteins (CaBP) such as calmodulin (CaM). Here we report a Ca2+-effector mechanism for protein kinase activation by demonstrating the specific and >1,000-fold activation of the myosin-associated giant protein kinase twitchin by Ca2+/S100A1(2). S100A1(2) is a member of a large CaBP family that is implicated in various cellular processes, including cell growth, differentiation and motility, but whose molecular actions are largely unknown. The S100A1(2)-binding site is a part of the autoregulatory sequence positioned in the active site that is responsible for intrasteric autoinhibition of twitchin kinase; the mechanism of autoinhibition based on the crystal structures of two twitchin kinase fragments is described elsewhere. Ca2+/S100 represents a likely physiological activator for the entire family of giant protein kinases involved in muscle contractions and cytoskeletal structure.

We describe the properties of a hybrid protein comprising the full length of the Xenopus laevis calmodulin sequence, followed by a pentapeptide linker (GGGGS), and residues 3-26 of M13, the calmodulin binding region of skeletal muscle myosin light chain kinase. The properties of the hybrid protein are compared with those of the complex formed between Drosophila calmodulin and a peptide corresponding to residues 1-18 of the M13 sequence. The addition of calcium to the hybrid protein produces pronounced changes in the near- and far-UV CD spectra, in the fluorescence emission spectrum of the single tryptophan residue at position 4 in the M13 sequence, and in the accessibility of this tryptophan residue to acrylamide quenching. These changes are consistent with the tryptophan residue being immobilized in a hydrophobic environment and with the hybrid protein adopting a more alpha-helical structure when calcium is bound. The increased alpha-helicity derives from changes in both the calmodulin and peptide regions of the hybrid protein. Changes in the circular dichroism and fluorescence properties of the hybrid protein as a function of the calcium to hybrid protein ratio are consistent with the fact that these changes parallel the cooperative binding of all four calcium ions. The hybrid protein shows greatly increased affinity (>250-fold) for calcium compared with calmodulin itself. Macroscopic calcium binding constants (K(1)-K(4)) were determined from calcium titrations performed in the presence of the calcium chelator Quin 2. Values for log(K(1)K(2)) and log(K(3)K(4)) were determined to be 15.4 +/- 0.2 and 15.59 +/- 0.22 (20 degrees C). The corresponding values for Drosophila calmodulin alone are 11.65 +/- 0.15 and 9.66 +/- 0.25. Consistent with this increased affinity for calcium stopped-flow kinetic studies suggest that the dissociation rate for the N-terminal calcium ions is reduced to at least 0.77 s(-1), compared with approximately 700 s(-1) for Drosophila calmodulin in the absence of peptide. This hybrid protein illustrates the principle whereby the binding of a peptide sequence covalently attached to calmodulin can enhance the average calcium affinity by more than 2 orders of magnitude. Conversely, the target sequence in the hybrid protein undergoes a calcium-induced conformational change to bind to the calmodulin in a conformation very similar to that of the corresponding dissociable target sequence binding to calmodulin, but with a greatly enhanced affinity due to its physical proximity to the binding site. This avoidance of the energetic penalty of dissociation may be a key contributory factor in determining the high affinity and specificity of the complex multiple interactions involved in recognition of biological targets by calmodulin.

The protein phosphatase activity of calcineurin (CaN) is activated through calcium binding to both calmodulin and the B subunit of CaN. The purpose of this study was to determine which domain(s) in the CaN B subunit is required for either binding to the CaN A subunit or for transducing the effects of B subunit Ca2+ binding to the stimulation of the CaN A subunit phosphatase activity. We have previously demonstrated that interaction of CaN B regulatory subunit with the CaN A catalytic subunit requires hydrophobic residues within the CaN A sequence 328-390 [Watanabe Y., Perrino, B.A., Chang, B.H., & Soderling, T.R. (1995) J. Biol. Chem. 270, 456-460]. In the present study, selected hydrophobic residues within the B subunit were mutated to Glu to Gln. CaN B subunit mutants BE-1 (Val115/Leu116 to Glu), BE-2 (Val156/157/168/169 to Glu), and BQ-2 (Val156/157/168/169 to Gln) were expressed and purified. The three mutant B subunits bound 45Ca2+ normally. Mutants BE-2 and BQ-2 interacted with a GST fusion protein containing the B subunit binding domain of the CaN A subunit (residues 328-390), and they stimulated the phosphatase activity of the CaN A subunit in an in vitro reconstitution assay. Mutant BE-1 had a 3-fold reduced affinity for binding CaN A, and this mutant, even at saturating concentrations, gave very little stimulation of CaN A phosphatase activity. We conclude that residues Val115/Leu116 in the B subunit participate in high-affinity binding to the A subunit and are required for transducing the effects [i.e., decrease Km and increase Vmax; Perrino, B.A., Ng, L.Y., & Soderling, T.R. (1995) J. Biol. Chem. 270, 340-346] of B subunit Ca2+ binding to stimulation of CaN A phosphatase activity.

Perchlorate is one of the most potent activators of skeletal muscle excitation-contraction (E-C) coupling reported in the literature, but the detailed mechanism of its action remains to be elucidated. In an attempt to further resolve the mode of perchlorate action, the effects of increasing concentrations of perchlorate on the voltage-dependent (T-tubule-mediated) and voltage-independent portions of Ca2+ release were investigated using the isolated triad model. Low concentrations of perchlorate (< or = 10 mM) activated SR Ca2+ release only when the T-tubule moiety was chemically depolarized. Higher concentrations of perchlorate (30-100 mM), on the other hand, produced significant activation of SR Ca2+ release, regardless of whether or not the T-tubule was depolarized. In order to gain further insights, we monitored the conformational change in the junctional foot protein (JFP), which presumably is an important intermediate step in E-C coupling [Yano, M., El-Hayek, R., & Ikemoto, N. (1995) J. Biol. Chem. 270, 3017-3021], using the fluorescently labeled triad preparation. Again, low concentrations of perchlorate (< or = 10 mM) produced a preferential activation of voltage-dependent protein conformational change, while higher concentrations of perchlorate produced significant activation of voltage-independent protein conformational change. An increase in the ryanodine binding by perchlorate occurred only in the higher concentration range where the voltage-independent protein conformational change was activated. These results suggest that perchlorate activates E-C coupling by acting on at least two different steps: at lower concentrations, on the T-tubule-to-JFP signal transmission step; at higher concentrations, on the JFP directly.

Two neuronal protein kinase C substrates, RC3/neurogranin and GAP-43/neuromodulin, preferentially bind to calmodulin (CaM) when Ca2+ is absent. We examine RC3.CaM and GAP-43.CaM interactions by circular dichroism spectroscopy using purified, recombinant RC3 and GAP-43, sequence variants of RC3 displaying qualitative and quantitative differences in CaM binding affinities, and overlapping peptides that cumulatively span the entire amino acid sequence of RC3. We conclude that CaM stabilizes a basic, amphiphilic alpha-helix within RC3 and GAP-43 under physiological salt concentrations only when Ca2+ is absent. This provides structural confirmation for two binding modes and suggests that CaM regulates the biological activities of RC3 and GAP-43 through an allosteric, Ca(2+)-sensitive mechanism that can be uncoupled by protein kinase C-mediated phosphorylation. More generally, our observations imply an alternative allosteric regulatory role for the Ca(2+)-free form of CaM.

Myosin is an ATPase enzyme with the unique property that the hydrolysis and release of Pi and ADP is coupled to movement via a cyclic interaction between myosin and actin filaments. Recent evidence indicates that for all myosin and myosin-like molecules, from slime mould and spinach vacuole to man, the mechanism of the molecular motor is essentially the same. It is now appropriate to ask general questions about how these motors are regulated by Ca2+. Is regulation the same throughout nature or are there different proteins in different phyla independently evolved? It is possible to define two basic mechanisms. Myosin may be regulated by EF hand Ca2+ binding proteins interacting with the regulatory domain or the thin filament activity may be regulated by accessory proteins. In this review I have analysed examples of myosin and actin-linked regulatory systems in order to determine the basic principles of the mechanism of these protein switches. I propose three principles common to all myosin-linked regulatory systems: (1) the regulatory proteins inhibit the cycling of a constitutively active myosin motor domain; (2) a regulatory domain in the myosin molecule has several special motifs ("IQ motif") which form binding sites for regulatory proteins; and (3) the regulatory proteins bound to the heavy chain are "EF hand" proteins related to calmodulin. I also propose a common set of principles for actin-linked regulatory systems: (1) the actin filament is normally capable of interacting with myosin to produce movement and the regulatory proteins inhibit the interaction; (2) inhibitory proteins are controlled by interaction with Ca(2+)-binding "EF hand" proteins; and (3) regulation is cooperative; the inhibitory proteins act as allosteric effectors of actin-tropomyosin state. The elongated tropomyosin propagates signals over many actins. It seems likely that myosin-linked regulation is of ancient origin. The origin of thin filament regulation is not clear. Such regulation has only been detected in animals but tropomyosin, which is a prerequisite for thin filament based regulation, is also found in protozoa and fungi, perhaps even in plants.

Calcineurin (CaN) contains an autoinhibitory element (residues 457-482) 43 residues COOH-terminal of the calmodulin-binding domain (Hashimoto, Y., Perrino, B. A., and Soderling, T. R. (1990) J. Biol. Chem. 265, 1924-1927) that regulates the Ca(2+)-dependent activation of its phosphatase activity. Substitution of Arg476 and Arg477 or Asp467 to Ala in the autoinhibitory peptide 457-482 significantly decreased its inhibitory potency. CaN A subunits with these residues mutated to Ala were coexpressed with the Ca(2+)-binding B subunit using the baculovirus/Sf9 cell system. Kinetic analysis showed that although the purified mutants had no activity in the absence of calcium, they were less dependent than the wild-type enzyme on calcium and calmodulin for activity. To determine if additional autoinhibitory motifs were present in the COOH terminus of calcineurin, the A subunit was truncated at residues 457 or 420 and co-expressed with B subunit. The Vmax values of both truncation mutants with or without Ca2+ were increased relative to wild-type calcineurin. The increased Ca(2+)-independent activity of CaN420 relative to CaN457 indicates the presence of additional autoinhibitory element(s) within residues 420-457. CaN420 had similar high Vmax values with or without Ca2+, but the Km value for peptide substrate was increased 5-fold to 125 microM in the absence of Ca2+. The Km values of all the expressed calcineurin species were increased in the absence of Ca2+. The CaN A or CaN A420 subunits alone have low Vmax and high Km (115 microM) values even in the presence of Ca2+. These results indicate that 1) there are several autoinhibitory motifs between the CaM-binding domain and the COOH terminus that are relieved by Ca2+ binding to CaM and the B subunit, 2) Ca2+ binding to the B subunit also regulates enzyme activity by lowering the Km of the catalytic subunit for substrate, 3) binding of the B subunit is required for high Vmax values even after removal of the autoinhibitory domain. These results are consistent with synergistic activation of calcineurin by Ca2+ acting through both CaM and the B subunit.

We measured Ca(2+)-calmodulin binding to expressed human caldesmon fragments by three techniques: tryptophan fluorescence enhancement, change in fluorescence of TA-calmodulin, and cosedimentation with calmodulin-Sepharose. Ca(2+)-calmodulin bound with similar affinity to peptide M73 (C714SMWEKGNVFSSPGF727, N terminus of domain 4b), to all the fragments of caldesmon containing this peptide, and also to H9 (Thr726-Val793), which did not contain this peptide (Kd = 0.2-0.8 microM). We conclude that Ca(2+)-calmodulin binds at two sites on caldesmon; site A is the sequence 715MWEKGNVFS723 previously identified by Zhan et al. (Zhan, Q., Wong, S. S., and Wang, C.-L.A. (1991) J. Biol. Chem. 266, 21810-21814), and site B is located nearer the C terminus of caldesmon. Ca(2+)-calmodulin binding at site B is coupled to reversal of caldesmon inhibition of actin-tropomyosin activated myosin MgATPase, while calmodulin binding at site A has no detectable function. H9 did not displace M73 from Ca(2+)-calmodulin, while the other fragments did. High concentrations of M73 (> 1000 x Kd) could not displace H9 bound to Ca(2+)-calmodulin-Sepharose. Thus sites A and B in calmodulin are functionally separate. Analysis of overlapping expressed fragments indicates that site B is located in the sequence Thr726-Leu767, which includes Trp749. The minimal Ca(2+)-calmodulin binding sequence could be 744SRINEWLTK752.

Caldesmon from chicken gizzard muscle has been examined for its ability to interact with caltropin using affinity chromatography and the fluorescent probe acrylodan. The action of caltropin on the inhibitory effect of caldesmon on actomyosin ATPase was also studied. Like calmodulin, caltropin could release the inhibitory effect of caldesmon in the presence of Ca2+. Complete reversal was obtained when 1 mol of caltropin was added per mol of caldesmon. When caldesmon was applied to caltropin-Sepharose in the presence of Ca2+, most of the caldesmon was bound to the column and could be eluted with EGTA, indicating that there is a direct interaction between caldesmon and caltropin. Acrylodan-labeled caldesmon, when excited at 375 nm, had an emission maximum at 504 nm. Addition of caltropin in the presence of Ca2+ resulted in a nearly 50% increase in fluorescence intensity, and this was accompanied by a blue shift in the emission maximum (i.e., lambda em,max 492 nm), suggesting that the probe now occupies a more nonpolar environment. Titration of caltropin with labeled caldesmon indicated a strong affinity for this protein (Kd was in the order of 8 x 10(-8)-2 x 10(-7) M). However, when caltropin was added to labeled caldesmon in the presence of EGTA, there was no indication of any interaction. Caltropin was at least as potent as calmodulin, if not better, in reversing the inhibitory effect of caldesmon in the presence of calcium, making it a potential Ca2+ factor in regulating caldesmon in smooth muscle.

The interaction between calmodulin and synthetic peptides corresponding to the calmodulin binding domain of the plasma membrane Ca2+ pump has been studied by measuring Ca2+ binding to calmodulin. The largest peptide (C28W) corresponding to the complete 28 amino acid calmodulin binding domain enhanced the Ca2+ affinity of calmodulin by more than 100 times, implying that the binding of Ca2+ increased the affinity of calmodulin for the peptide by more than 10(8) times. Deletion of the 8 C-terminal residues from peptide C28W did not decrease the affinity of Ca2+ for the high-affinity sites of calmodulin, but it decreased that for the low-affinity sites. A larger deletion (13 residues) decreased the affinity of Ca2+ for the high-affinity sites as well. The data suggest that the middle portion of peptide C28W interacts with the C-terminal half of calmodulin. Addition of the peptides to a mixture of tryptic fragments corresponding to the N- and C-terminal halves of calmodulin produced a biphasic Ca2+ binding curve, and the effect of peptides was different from that on calmodulin. The result shows that one molecule of peptide C28W binds both calmodulin fragments. Interaction of the two domains of calmodulin through the central helix is necessary for the high-affinity binding of four Ca2+ molecules.

Endogenous calmodulin (CaM) in the EGTA-washed cerebral-cortical synaptosomal membrane (SM) preparation was estimated below 3 micrograms/ml protein by the semiquantitative immunoblot analysis (Natsukari, N., Ohta, H. and Fujita, M. (1989) J. Immunol. Methods 125, 159-166). Membrane-bound CaM was immunoelectron-microscopically demonstrated in EGTA-washed, non-treated (control), and Ca(2+)-treated cerebral-cortical synaptosomal membranes (SM) as well as for the SM enriched with added CaM. The density of CaM increased in the above order. CaM-dependent adenylate cyclase and CaM-dependent protein kinase II (CaM-kinase II) activities were restored, whereas the phosphodiesterase (PDE) activity was not affected by exogenous CaM over all the Ca2+ concentrations tested. Adenylate cyclase at pCa 6.2 was synergistically activated either by GTP and CaM or by CaM and beta-adrenergic agonist, (+/-)-isoproterenol, reflecting the intactness of signal transduction pathway in the SM. Also demonstrated were the presence of protein kinase A, CaM-kinase II, and their endogenous substrates in the SM. Based on 32P-autoradiography and 125I-CaM overlay data certain CaM-binding proteins such as CaM-kinase II and synapsin I were identified on SDS-PAGE. Ca(2+)-dependent and -independent CaMBPs were distinguished by 125I-CaM gel overlay with and without Ca2+. The former had bigger molecular size (greater than or equal to 49 kDa) than the latter (less than or equal to 34 kDa). Yield of Ca(2+)-dependent CaMBPs was not affected by Ca2+ concentration during preparation of the SM while that of Ca(2+)-independent CaMBPs was reduced by exposure to 100 microM Ca2+. In contrast with the CaMBPs of brain SM, those of enterocyte and eyrthrocyte plasma membranes especially, microvillous membrane of the enterocyte, showed quite distinct CaMBP profiles. The present findings suggested that the EGTA-washed SM preparation made a useful system for studying the role of CaM in the brain SM.

We have initiated a systematic study of Ca2+/calmodulin-regulated enzymes in the cellular slime mold Dictyostelium discoideum. Using 125I-labelled D. discoideum calmodulin (CaM) as a functional probe, several Ca2+/CaM-binding proteins were detected in crude cell lysates. Proteins with apparent molecular weights of 22 kDa and 78-80 kDa, respectively, were found in the soluble fraction. In addition, membrane-bound high molecular weight CaM-binding proteins were identified. Binding of CaM to all of the proteins required the presence of Ca2+ ions and competed efficiently with nonradioactive CaM from both Dictyostelium and bovine brain. The CaM antagonists melittin, W-7 and R24571 inhibited CaM binding. With a functional cloning approach, we previously obtained cDNA clones by screening a lambda gt11 lysogen expression library; in this paper, we report the analysis of CaM-binding activity by one of the recombinant cDNA clones in Escherichia coli. When rabbit antiserum was raised against it, the antiserum recognized a 78-80-kDa protein in Dictyostelium extracts which comigrated on SDS-polyacrylamide gels with 78-80-kDa CaM-binding activity.

Thiol titrations of bovine brain calcineurin, phosphatase with Ellman's reagent revealed the presence of 5 exposed sulfhydryl groups on the native protein, and 10 sulfhydryl groups on the denatured protein. Attempts were made to identify the location of the free thiols within the catalytic and regulatory domains of the enzyme. Our data indicates that free sulfhydryl groups are absent from the vicinity of the Mg2+ and calmodulin binding sites as well as from the active site of the enzyme. However, the fact that the number of free thiols decreased in the presence of Ca2+ and Mn2+, to 4 and 2 respectively, possibly indicates that either free thiols are at or near these domains or become inaccessible as a consequence of conformational changes induced by the metal ions. The Ca2+ and Ca2+/Mg2+ stimulated activities of calcineurin were monitored during modification with Ellman's reagent, iodoacetate and iodoacetamide. Upon modification of 1-2 of the free thiols the activity of the enzyme increased 1.3 to 10.5-fold depending on the thiol reagent and the stimulatory metal ions employed. Modification of the remainder of the free thiols resulted in a decrease in activity. These results suggest that 1-2 thiols are essential for the full expression of calcineurin activity.