Regulation of RYR1 activity by Ca(2+) and calmodulin

The skeletal muscle calcium release channel (RYR1) is a Ca(2+)-binding protein that is regulated by another Ca(2+)-binding protein, calmodulin. The functional consequences of calmodulin's interaction with RYR1 are dependent on Ca(2+) concentration. At nanomolar Ca(2+) concentrations, calmodulin is an activator, but at micromolar Ca(2+) concentrations, calmodulin is an inhibitor of RYR1. This raises the question of whether the Ca(2+)-dependent effects of calmodulin on RYR1 function are due to Ca(2+) binding to calmodulin, RYR1, or both. To distinguish the effects of Ca(2+) binding to calmodulin from those of Ca(2+) binding to RYR1, a mutant calmodulin that cannot bind Ca(2+) was used to evaluate the effects of Ca(2+)-free calmodulin on Ca(2+)-bound RYR1. We demonstrate that Ca(2+)-free calmodulin enhances the affinity of RYR1 for Ca(2+) while Ca(2+) binding to calmodulin converts calmodulin from an activator to an inhibitor. Furthermore, Ca(2+) binding to RYR1 enhances its affinity for both Ca(2+)-free and Ca(2+)-bound calmodulin.     

Regulation of the erythrocyte Ca(2+)-ATPase by mutant calmodulins with Glu----Ala substitutions in the Ca(2+)-binding domains.

We have used four mutant calmodulins to study the regulation of human erythrocyte Ca(2+)-ATPase by the calmodulin-dependent pathway; the conserved Glu at position 12 in each of the four Ca(2+)-binding domains of calmodulin (Glu31, Glu67, Glu104, or Glu140) was replaced by Ala. At pCa 7, where unmodified calmodulin maximally activates the erythrocyte Ca(2+)-ATPase, all four mutants stimulated Ca(2+)-ATPase activity to the same maximal velocity. However, the concentrations of mutant calmodulins required for half-maximal activation (KCaM) were significantly higher than that for unmodified calmodulin and were strongly dependent on the domain in which the mutated Glu was located; substitution in either the first or second Ca(2+)-binding domain had little effect (2-3-fold increase in KCaM), whereas substitution in either the third or fourth domain resulted in a dramatic, 25-71-fold increase in KCaM. The same order of sensitivity was observed when the Ca2+ dependence of enzyme activation was measured at a constant 100 nM concentration of mutant calmodulin. These data point to dramatic differences in the functional significance of the replacement of the Glu at position 12 in each of the four Ca(2+)-binding domains for activation of the Ca(2+)-ATPase. The 2 Glu residues located in the carboxyl-terminal half of calmodulin (particularly Glu140) are crucial for activation of the Ca(2+)-ATPase at physiologically significant Ca2+ concentrations.     

Can calmodulin function without binding calcium?

Calmodulin is a small Ca(2+)-binding protein proposed to act as the intracellular Ca2+ receptor that translates Ca2+ signals into cellular responses. We have constructed mutant yeast calmodulins in which the Ca(2+)-binding loops have been altered by site-directed mutagenesis. Each of the mutant proteins has a dramatically reduced affinity for Ca2+; one does not bind detectable levels of 45Ca2+ either during gel filtration or when bound to a solid support. Furthermore, none of the mutant proteins change conformation even in the presence of high Ca2+ concentrations. Surprisingly, yeast strains relying on any of the mutant calmodulins not only survive but grow well. In contrast, yeast strains deleted for the calmodulin gene are not viable. Thus, calmodulin is required for growth, but it can perform its essential function without the apparent ability to bind Ca2+.     

The unconventional myosin, Myo2p, is a calmodulin target at sites of cell growth in Saccharomyces cerevisiae

Myo2p is an unconventional myosin required for polarized growth in Saccharomyces cerevisiae. Four lines of evidence suggest that (a) Myo2p is a target of calmodulin at sites of cell growth, and (b) the interaction between Myo2p and calmodulin is Ca2+ independent. First, as assessed by indirect immunofluorescence, the distributions of Myo2p and calmodulin are nearly indistinguishable throughout the cell cycle. Second, a genetic analysis indicates that mutations in CMD1 show allele- specific synthetic lethality with the myo2-66 conditional mutation. Mutations that inactivate the Ca(2+)-binding sites of calmodulin have little or no effect on strains carrying myo2-66, whereas an allele with a mutation outside the Ca(2+)-binding sites dramatically increases the severity of the phenotype conferred by myo2-66. Third, Myo2p coimmunoprecipitates with calmodulin in the presence of Ca2+ or EGTA. Finally, we used a modified gel overlay assay to demonstrate direct interaction between calmodulin and fusion proteins containing portions of Myo2p. Calmodulin binds specifically to the region of Myo2p containing six tandem repeats of a motif called an IQ site. Binding occurs in either Ca2+ or EGTA, and only two sites are required to observe binding.     

Detection of Ca(2+)-binding proteins by electrophoretic migration in the presence of Ca2+ combined with 45Ca2+ overlay of protein blots.

When high affinity Ca(2+)-binding proteins like calmodulin, or proteins with a high Ca(2+)-binding capacity like calsequestrin, underwent sodium dodecyl sulfate-gel electrophoresis in Laemmli systems, their electrophoretic migration rates were much higher in gels containing 1 mM Ca2+ than in gels containing ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA). Replacement of EGTA by Ca2+ in the gel, combined with the blotting of electrophoretically separated proteins on polyvinylidene difluoride membranes and subsequent 45Ca2+ overlay, proved a very effective means of detecting Ca(2+)-binding proteins. This combined approach is important since artifacts occur in both techniques when used separately. We found that the usual procedure of adding Ca2+ to the sample before electrophoresis without including it in the gel itself (C.B. Klee, T. H. Crouch, and M. H. Krinks, 1979, Proc. Natl. Acad. Sci. USA 76, 6270-6273) permitted the detection of only very high affinity Ca(2+)-binding proteins.     

Structural analysis of wild-type and mutant yeast calmodulins by limited proteolysis and electrospray ionization mass spectrometry.

Calmodulin from Saccharomyces cerevisiae was expressed in Escherichia coli and purified. The purified protein was structurally characterized using limited proteolysis followed by ESI mass spectrometry to identify the fragments. In the presence of Ca2+, yeast calmodulin is sequentially cleaved at arginine 126, then lysine 115, and finally at lysine 77. The rapid cleavage at Arg-126 suggests that the fourth Ca(2+)-binding loop does not bind Ca2+. In the presence of EGTA, yeast calmodulin is more susceptible to proteolysis and is preferentially cleaved at Lys-106. In addition, mutant proteins carrying I100N, E104V or both mutations, which together confer temperature sensitivity to yeast, were characterized. The mutant proteins are more susceptible than wild-type calmodulin to proteolysis, suggesting that each mutation disrupts the structure of calmodulin. Furthermore, whereas wild-type calmodulin is cut at Lys-106 only in the presence of EGTA, this cleavage site is accessible in the mutants in the presence of Ca2+ as well. In these ways, the structural consequence of each mutation mimics the loss of a calcium ion in the third loop. In addition, although wild-type calmodulin binds to four proteins in a yeast crude extract in the presence of Ca2+, the mutants bind only to a subset of these. Thus, the inability to adopt the stable Ca(2+)-bound conformation in the third Ca(2+)-binding loop alters the ability of calmodulin to interact with yeast proteins in a Ca(2+)-dependent manner.     

The plasma membrane calcium pump: regulation by a soluble Ca2+ binding protein

The Ca2+ pump of the plasma membrane of human red blood cells is associated with the activity of a (Ca2+ + Mg2+)-ATPase. Both the ATPase and the pump are stimulated above basal activities by calmodulin, an ubiquitous Ca2+-binding protein. Calmodulin isolated from human red blood cells was shown to be equipotent and equieffective with that isolated from beef brain. Half-maximal activation of ATPase (isolated red blood cell membranes, 37 C) and transport (inside-out red blood cell membrane vesicles, 25 C) were obtained with 2.5 and 4.4 nM calmodulin, respectively. Ca2+ dependence of Ca2+ transport was measured in the absence and in the presence of 50 nM calmodulin. At all Ca2+ concentrations above 2 X 10(-7) M Ca2+, the rate of transport was greater in the presence of calmodulin. The results implicate calmodulin in the regulation of the plasma membrane Ca2+ pump, but the mechanism(s) remain to be elucidated.     

Binding shift assay of parvalbumin, calmodulin and carbonic anhydrase by high-performance capillary electrophoresis

Shifts in mobility caused by binding of Ca2+ to calmodulin and parvalbumin were studied using high-performance capillary electrophoresis in a Tris-glycine buffer, rather than conventional polyacrylamide gel electrophoresis which requires larger amounts of sample and longer assay time. A Zn(2+)-binding protein, carbonic anhydrase, also showed a partial shift in mobility following Zn(2+)-binding.     

Specific conformation and Ca(2+)-binding mode of yeast calmodulin: insight into evolutionary development

The vertebrate calmodulin is configured with two structurally independent globular lobes in N- and C-terminus, and a flexible central linker. Distinctly, two lobes of calmodulin from Saccharomyces cerevisiae (yCaM) interact and influence the Ca(2+)-binding profile of each other. We explored this further using the mutant proteins with eliminated Ca(2+)-binding ability in one of the lobes and found that the Ca(2+)-bound N-lobe associates with the Ca(2+)-free C-lobe to gain the Ca(2+) affinity of a wild-type level. Next, analysing series of C-terminal residue truncation mutant, we found that the truncation of C-terminal three residues induce the hyper Ca(2+) affinity. These residues are also important for the general structural behaviour of calmodulin, such as Ca(2+)-induced slow mobility shift in polyacrylamide gel electrophoresis and for the ability to activate Cmk1p (yeast calmodulin kinase). These suggest: (i) when Ca(2+) occupies only N-lobe, two lobes interact and form the stable intermediate leading to a proper level of Ca(2+) affinity; (ii) the C-terminal three residues are required to prohibit abnormal stabilization of the intermediate promoting abnormally high Ca(2+) affinity and for recognition of target enzymes. A model for Ca(2+) and target bindings of yCaM is proposed. Evolutional aspect concerning the biological significance of this model was discussed.     

Construction and molecular dynamics simulation of calmodulin in the extended and in a bent conformation.

Analysis of sequence similarity and comparison of the three-dimensional (3D) structures of troponin C and calmodulin have revealed a sequence in the central helix of calmodulin with a high probability for bending. The three amino acids known to form a bend in the N-terminal portion of troponin C are also found in the central helix of calmodulin. The modelling of a bent calmodulin structure, using the dihedral angles of the three residues in the bend of troponin C as a 3D template, results in a conformation of calmodulin where the N- and C-terminal domains are able to form contacts. Dynamics simulations starting from the X-ray structure of calmodulin and from the modelled bent calmodulin were carried out to compare flexibility and correlated movements of Ca2+ in the binding loops. Both conformations of calmodulin remained stable during the period of simulation. In the simulation of calmodulin in the extended form, the motions of the Ca2+ atoms in the two domains (Ca2+1 and Ca2+2 in one domain, and Ca2+3 and Ca2+4 in the other) are correlated. In the simulation of the bent form, an additional correlation between the Ca atoms in the two different domains is observed. The results are compatible with the occurrence of a bent conformation of calmodulin in the presence of targets, and with increased Ca2+ affinity and cooperativity of the Ca(2+)-binding loops in the calmodulin-peptide complexes.     

Monoclonal antibodies to the calcium-binding region peptide of human C-reactive protein alter its conformation.

Five mouse mAb were generated against a synthetic peptide corresponding to the proposed Ca(2+)-binding region of human C-reactive protein (CRP). The peptide consists of amino acids 134 to 148 and possesses a calmodulin Ca(2+)-binding sequence. The mAb reacted with a surface epitope(s) on native, intact CRP as well as the closely related pentraxin protein, serum amyloid P-component. Three of the 5 mAb inhibited the Ca(2+)-dependent phosphorylcholine-(PC) binding activity of CRP, but did not bind to the PC-binding region itself. Four of the five mAb also inhibited the recognition of an epitope in the PC-binding site of CRP. Four of the mAb partially, or completely, protected CRP from selective cleavage by pronase between residues 146 and 147. The findings suggest that the Ca(2+)-binding region is on the surface of CRP, has substantial flexibility, and is probably responsible for the allosteric effects of Ca2+ ions on CRP.     

Ca(2+) binding and energy coupling in the calmodulin-myosin light chain kinase complex

We have previously shown that 3 Ca(2+) ions are released cooperatively and 1 independently from the complex between (Ca(2+))4-calmodulin and skeletal muscle myosin light chain kinase or a peptide containing its core calmodulin-binding sequence. We now have found that three Ca(2+)-binding sites also function cooperatively in equilibrium Ca(2+) binding to these complexes. Replacement of sites I and II in calmodulin by a copy of sites III and IV abolishes these cooperative effects. Energy coupling-dependent increases in Ca(2+)-binding affinity in the mutant and native calmodulin complexes with enzyme are considerably less than in the peptide complexes, although the complexes have similar affinities. Ca(2+) binding to three sites in the native calmodulin-enzyme complex is enhanced; the affinity of the remaining site is slightly reduced. In the mutant enzyme complex Ca(2+) binding to one pair of sites is enhanced; the other pair is unaffected. In this complex reversal of enzyme activation occurs when Ca(2+) dissociates from the pair of sites with enhanced affinity; more rapid dissociation from the other pair has no effect, although both pairs participate in activation. Ca(2+)-independent interactions with calmodulin clearly play a major role in the enzyme complex, and appear to weaken Ca(2+)-dependent interactions with the core calmodulin-binding sequence.     

Calcium binding properties of recombinant calcium binding protein 40, a major calcium binding protein of lower eukaryote Physarum polycephalum

Calcium binding protein 40 (CBP40) is a Ca(2+)-binding protein abundant in the plasmodia of Physarum polycephalum. CBP40 consists four EF-hand domains in the COOH-terminal half and a putative alpha-helix domain in the NH(2)-terminal half. We expressed recombinant proteins of CBP40 in Escherichia coli to investigate its Ca(2+)-binding properties. Recombinant proteins of CBP40 bound 4 mol of Ca(2+) with much higher affinity (pCa(1/2) = 6.5) than that of calmodulin. When residues 1-196 of the alpha-helix domain were deleted, the affinity for Ca(2+) decreased to pCa(1/2) = 4.6. A chimeric calmodulin was generated by conjugating the alpha-helix domain of CBP40 with calmodulin. The affinity of Ca(2+) for the chimeric calmodulin was higher than that for calmodulin, suggesting that the alpha-helix domain is responsible for the high affinity of CBP40 for Ca(2+). CBP40 forms large aggregates reversibly in a Ca(2+)-dependent manner. A mutant protein with a deletion of NH(2)-terminal 32 residues, however, could not aggregate, indicating the importance of these residues for the aggregation. The aggregation occurs above micromolar levels of Ca(2+) concentration, so it may only occur when CBP40 is secreted out of the plasmodial cells.     

The Ayerst Award Lecture 1990. Calcium-dependent mechanisms of regulation of smooth muscle contraction.

The contractile state of smooth muscle is regulated primarily by the sarcoplasmic (cytosolic) free Ca2+ concentration. A variety of stimuli that induce smooth muscle contraction (e.g., membrane depolarization, alpha-adrenergic and muscarinic agonists) trigger an increase in sarcoplasmic free [Ca2+] from resting levels of 120-270 to 500-700 nM. At the elevated [Ca2+], Ca2+ binds to calmodulin, the ubiquitous and multifunctional Ca(2+)-binding protein. The interaction of Ca2+ with CaM induces a conformational change in the Ca(2+)-binding protein with exposure of a site(s) of interaction with target proteins, the most important of which in the context of smooth muscle contraction is the enzyme myosin light chain kinase. The interaction of calmodulin with myosin light chain kinase results in activation of the kinase that catalyzes phosphorylation of myosin at serine-19 of each of the two 20-kDa light chains (native myosin is a hexamer composed of two heavy chains (230 kDa each) and two pairs of light chains (one pair of 20 kDa each and the other pair of 17 kDa each)). This simple phosphorylation reaction triggers cycling of myosin cross-bridges along actin filaments and the development of force. Relaxation of the muscle follows removal of Ca2+ from the sarcoplasm, whereupon calmodulin dissociates from myosin light chain kinase regenerating the inactive kinase; myosin is dephosphorylated by myosin light chain phosphatase(s), whereupon it dissociates and remains detached from the actin filament and the muscle relaxes. A substantial body of evidence has been accumulated in support of this central role of myosin phosphorylation-dephosphorylation in the regulation of smooth muscle contraction. However, a wide range of physiological and biochemical studies supports the existence of additional, secondary Ca(2+)-dependent mechanisms that can modulate or fine-tune the contractile state of the smooth muscle cell. Three such mechanisms have emerged: (i) the actin-, tropomyosin-, and calmodulin-binding protein, calponin; (ii) the actin-, myosin-, tropomyosin-, and calmodulin-binding protein, caldesmon; and (iii) the Ca(2+)- and phospholipid-dependent protein kinase (protein kinase C).     

Acid pairs increase the N-terminal Ca2+ affinity of CaM by increasing the rate of Ca2+ association

A series of N-terminal calmodulin (CaM) mutants was generated to probe the relationship between the N-terminal Ca(2+) affinity and the number of paired, negatively charged Ca(2+) chelating residues in the N-terminal Ca(2+)-binding sites of CaM. When the number of acid pairs [negatively charged residues at positions +x and -x (X-axis), +y and -y (Y-axis), and +z and -z (Z-axis)] was increased from zero to one and then to two, a progressive increase was seen in the N-terminal Ca(2+) affinities. The maximal ranges of the increases observed in the N-terminal Ca(2+) affinity were approximately 8-8.5-fold for site I, approximately 4.5-5-fold for site II, and approximately 11-fold for both sites, in comparison to the mutants containing no acid pairs. The maximal values of N-terminal Ca(2+) affinity were bestowed by the presence of five acidic chelating residues in site I or II, individually. Addition of the sixth acidic chelating residue (third acid pair) to both N-terminal Ca(2+)-binding sites reduced the N-terminal Ca(2+) affinity. The increases in Ca(2+) affinity observed were caused by an increase in the Ca(2+) association rates for the Y- and Z-axis acid pairs, while the X-axis acid pair caused a reduction in the Ca(2+) dissociation rates.     

The whole is not the simple sum of its parts in calmodulin from S. cerevisiae

Calmodulin is an essential Ca(2+)-binding protein involved in a multitude of cellular processes. The calmodulin sequence is highly conserved among all eukaryotic species; calmodulin from the yeast S. cerevisiae (yCaM) is the most divergent form, while still sharing 60% sequence identity with vertebrate calmodulin (vCaM). Although yCaM can be functionally substituted by vCaM in vivo, the two calmodulin proteins possess significantly different Ca(2+)-binding properties as well as abilities to activate vertebrate target enzymes in vitro. In addition, it has been observed that certain properties of the N-terminal and C-terminal domains of Ca(2+)-yCaM differ depending on whether they are in the context of the whole protein or isolated as half-molecule fragments. To investigate the structural basis for these differing properties, we have undertaken nuclear magnetic resonance (NMR) studies on yCaM and the two half-molecule fragments representing its two individual domains, yTr1(residues 1-76) and yTr2 (residues 75-146). We present direct evidence that the two domains of Ca(2+)-yCaM interact via their exposed hydrophobic surfaces. Thus, the Ca(2+)-bound form of yCaM exists in a novel compact structure in direct contrast to the well-established structure of Ca(2+)-vCaM comprised of two independent globular domains.     

Interactions of calcineurin A, calcineurin B, and Ca2+.

Calcineurin B (CN-B) is the Ca(2+)-binding, regulatory subunit of the phosphatase calcineurin. Point mutations to Ca(2+)-binding sites in CN-B were generated to disable individual Ca(2+)-binding sites and evaluate contributions from each site to calcineurin heterodimer formation. Ca(2+)-binding properties of four CN-B mutants and wild-type CN-B were analyzed by flow dialysis confirming that each CN-B mutant binds three Ca2+ and that wild-type CN-B binds four Ca2+. Macroscopic dissociation constants indicate that N-terminal Ca(2+)-binding sites have lower affinity for Ca2+ than the C-terminal sites. Each CN-B mutant was coexpressed with the catalytic subunit of calcineurin, CN-A, to produce heterodimers with specific disruption of one Ca(2+)-binding site. Enzymes containing CN-B with a mutation in Ca(2+)-binding sites 1 or 2 have a lower ratio of CN-B to CN-A and a lower phosphatase activity than those containing wild-type CN-B or mutants in sites 3 or 4. Effects of heterodimer formation on Ca2+ binding were assessed by monitoring (45)Ca2+ exchange by flow dialysis. Enzymes containing wild-type CN-B and mutants in sites 1 and 2 exchange (45)Ca2+ slowly from two sites whereas mutants in sites 3 and 4 exchange (45)Ca2+ slowly from a single site. These data indicate that the Ca2+ bound to sites 1 and 2 is likely to vary with Ca2+ concentration and may act in dynamic modulation of enzyme function, whereas Ca(2+)-binding sites 3 and 4 are saturated at all times and that Ca2+ bound to these sites is structural.     

Calcium-induced refolding of the calmodulin V136G mutant studied by NMR spectroscopy: evidence for interaction between the two globular domains

The Ca(2+) titration of the (15)N-labeled mutant V136G calmodulin has been monitored using (1)H-(15)N HSQC NMR spectra. Up to a [Ca(2+)]/[CaM] ratio of 2, the Ca(2+) ions bind predominantly to sites I and II on the N-domain in contrast with the behavior of the wild-type calmodulin where the C-terminal domain has the higher affinity for Ca(2+). Surprisingly, the Ca(2+)-binding affinity for the N-domain in the mutant calmodulin is greater than that for the N-domain in the wild-type protein. The mutated C-domain is observed as a mixture of unfolded, partially folded (site III occupied), and native-like folded (sites III and IV occupied) conformations, with relative populations dependent on the [Ca(2+)]/[CaM] ratio. The occupancy of site III independently of site IV in this mutant shows that the cooperativity of Ca(2+) binding in the C-domain is mediated by the integrity of the domain structure. Several NH signals from residues in the Ca(2+)-bound N-domain appear as two signals during the Ca(2+) titration indicating separate species in slow exchange, and it can be deduced that these result from the presence and absence of interdomain interactions in the mutant. It is proposed that an unfolded part of the mutated C-domain interacts with sites on the N-domain that normally bind to target proteins. This would also account for the increase in the Ca(2+) affinity for the N-domain in the mutant compared with the wild-type calmodulin. The results therefore show the wide-ranging effects of a point mutation in a single Ca(2+)-binding site, providing details of the involvement of individual residues in the calcium-induced folding reactions.     

A calcium-binding protein from rabbit lung cytosol identified as the product of growth-regulated gene (2A9) and its binding proteins

Using Ca(2+)-dependent affinity chromatography on a synthetic compound (W-77)-coupled Sepharose 4B column, we purified two different Ca(2+)-binding proteins from rabbit lung extracts. The molecular weights of these proteins were estimated to be 17 kDa (calmodulin) and 10 kDa, respectively. The partial amino acid sequence of the 10-kDa protein revealed that it has two EF-hand structures. In addition, the 10-kDa protein was highly homologous (91%) to the product of growth-regulated gene, 2A9 (calcyclin). The Ca(2+)-binding property of the 10-kDa protein was observed by a change in the uv difference spectrum. Equilibrium dialysis showed that 1 mol of the 10-kDa protein bound to 2.04 +/- 0.05 mol of Ca2+ in the presence of 10(-4) M Ca2+. However, the protein failed to activate calmodulin-dependent enzymes such as Ca2+/CaM kinase II, myosin light chain kinase, and phosphodiesterase. We found that a 50-kDa cytosolic protein of the rabbit lung, intestine, and spleen bound to the 10-kDa protein, in a Ca(2+)-dependent manner. The distribution of calcyclin and calcyclin binding proteins was unique and seems to differ from that of calmodulin and calmodulin-binding proteins. Thus, calcyclin probably plays a physiological role through its binding proteins for the Ca(2+)-dependent cellular response.     

Three-dimensional structure of a sarcoplasmic calcium-binding protein from Nereis diversicolor

The three-dimensional structure of a sarcoplasmic Ca2(+)-binding protein from the sandworm Nereis diversicolor has been determined at 3.0 A resolution using multiple isomorphous replacement techniques. The NH2-terminal half of the molecule contains one variant Ca2(+)-binding domain with a novel helix-loop-helix conformation and one Ca2(+)-binding domain that is no longer functional because of amino acid changes. The overall conformation of this pair of domains is different from any previously described Ca2(+)-binding protein. The COOH-terminal half of the protein contains two Ca2(+)-binding domains with the usual helix-loop-helix configuration and is similar to calmodulin and troponin C. Unlike calmodulin or troponin C, there is no exposed alpha-helix connecting the two halves of the molecule, so the overall structure is much more compact.