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.     

Calmodulin association with connexin32-derived peptides suggests trans-domain interaction in chemical gating of gap junction channels

Calmodulin plays a key role in the chemical gating of gap junction channels. Two calmodulin-binding regions have previously been identified in connexin32 gap junction protein, one in the N-terminal and another in the C-terminal cytoplasmic tail of the molecule. The aim of this study was to better understand how calmodulin interacts with the connexin32-binding domains. Lobe-specific interactions of calmodulin with connexin32 peptides were studied by stopped flow kinetics, using Ca(2+) binding-deficient mutants. Peptides corresponding to the N-terminal tail (residues 1-22) of connexin32 engaged both the N- and C-terminal lobes (N- and C-lobes) of calmodulin, binding with higher affinity to the C-lobe of calmodulin (Ca(2+) dissociation rate constants k(3,4), 1.7+/-0.5 s(-1)) than to the N-lobe (k(1,2), 10.8+/-1.3 s(-1)). In contrast, peptides representing the C-terminal tail domain (residues 208-227) of connexin32 bound either the C- or the N-lobe but only one calmodulin lobe at a time (k(3,4), 2.6+/-0.1 s(-1) or k(1), 13.8+/-0.5 s(-1) and k(2), 1000 s(-1)). The calmodulin-binding domains of the N- and C-terminal tails of connexin32 were best defined as residues 1-21 and 216-227, respectively. Our data, showing separate functions of the N- and C-lobes of calmodulin in the interactions with connexin32, suggest trans-domain or trans-subunit bridging by calmodulin as a possible mechanism of gap junction gating.     

The carboxy-terminal calcium binding sites of calmodulin control calmodulin's switch from an activator to an inhibitor of RYR1

Calcium and calmodulin both regulate the skeletal muscle calcium release channel, also known as the ryanodine receptor, RYR1. Ca(2+)-free calmodulin (apocalmodulin) activates and Ca(2+)-calmodulin inhibits the ryanodine receptor. The conversion of calmodulin from an activator to an inhibitor is due to Ca(2+) binding to calmodulin. We have previously shown that the binding sites for apocalmodulin and Ca(2+)-calmodulin on RYR1 are overlapping with the Ca(2+)-calmodulin site located slightly N-terminal to the apocalmodulin binding site. We now show that mutations of the calcium binding sites in either the N-terminal or the C-terminal lobes of calmodulin decrease the affinity of calmodulin for the ryanodine receptor, suggesting that both lobes interact with RYR1. Mutation of the two C-terminal Ca(2+) binding sites of calmodulin destroys calmodulin's ability to inhibit ryanodine receptor activity at high calcium concentrations. The mutated calmodulin, however, can still bind to RYR1 at both nanomolar and micromolar Ca(2+) concentrations. Mutating the two N-terminal calcium binding sites of calmodulin does not significantly alter calmodulin's ability to inhibit ryanodine receptor activity. These data suggest that calcium binding to the two C-terminal calcium binding sites within calmodulin is responsible for the switching of calmodulin from an activator to an inhibitor of the ryanodine receptor.     

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.     

The solution structure of apocalmodulin from Saccharomyces cerevisiae implies a mechanism for its unique Ca2+ binding property

We have determined the solution structure of calmodulin (CaM) from yeast (Saccharomyces cerevisiae) (yCaM) in the apo state by using NMR spectroscopy. yCaM is 60% identical in its amino acid sequence with other CaMs, and exhibits its unique biological features. yCaM consists of two similar globular domains (N- and C-domain) containing three Ca(2+)-binding motifs, EF-hands, in accordance with the observed 3 mol of Ca(2+) binding. In the solution structure of yCaM, the conformation of the N-domain conforms well to the one of the expressed N-terminal half-domains of yCaM [Ishida, H., et al. (2000) Biochemistry 39, 13660-13668]. The conformation of the C-domain basically consists of a pair of helix-loop-helix motifs, though a segment corresponding to the forth Ca(2+)-binding site of CaM deviates in its primary structure from a typical EF-hand motif and loses the ability to bind Ca(2+). Thus, the resulting conformation of each domain is essentially identical to the corresponding domain of CaM in the apo state. A flexible linker connects the two domains as observed for CaM. Any evidence for the previously reported interdomain interaction in yCaM was not observed in the solution structure of the apo state. Hence, the interdomain interaction possibly occurs in the course of Ca(2+) binding and generates a cooperative Ca(2+) binding among all three sites. Preliminary studies on a mutant protein of yCaM, E104Q, revealed that the Ca(2+)-bound N-domain interacts with the apo C-domain and induces a large conformational change in the C-domain.     

Conformational dynamics of yeast calmodulin in the Ca(2+)-bound state probed using NMR relaxation dispersion

Most calmodulin (CaM) in apo and Ca(2+)-bound states show a dumb-bell-like structure, involving the N- and C-terminal domains, connected with a flexible linker. However, Ca(2+)-bound yeast calmodulin (yCaM) takes on a unique globular structure; the target-binding site of this protein is autoinhibited. We applied NMR relaxation dispersion experiments to yCaM in the Ca(2+)-bound state. The amide (15)N and (1)H(N) relaxation dispersion profiles indicated the presence of conformational dynamics for specific residues at the interface between the N- and C-terminal domains. We conclude that these conformational dynamics were derived from the mobility of the C-terminal domain.     

In calmodulin-IQ domain complexes, the Ca(2+)-free and Ca(2+)-bound forms of the calmodulin C-lobe direct the N-lobe to different binding sites

We have investigated the roles played by the calmodulin (CaM) N- and C-lobes in establishing the conformations of CaM-IQ domain complexes in different Ca(2+)-free and Ca(2+)-bound states. Our results indicate a dominant role for the C-lobe in these complexes. When the C-lobe is Ca(2+)-free, it directs the N-lobe to a binding site within the IQ domain consensus sequence. It appears that the N-lobe must be Ca(2+)-free to interact productively with this site. When the C-lobe is Ca(2+)-bound, it directs the N-lobe to a site upstream of the consensus sequence, and it appears that the N-lobe must be Ca(2+)-bound to interact productively with this site. A model for switching in CaM-IQ domain complexes is presented in which the N-lobe adopts bound and extended positions that depend on the status of the Ca(2+)-binding sites in each CaM lobe and the compositions of the two N-lobe binding sites. Ca(2+)-dependent changes in the conformation of the bound C-lobe that appear to be responsible for directed N-lobe binding are also identified. Changes in the equilibria between extended and bound N-lobe positions may control bridging interactions in which the extended N-lobe is bound to another CaM-binding domain. Ca(2+)-dependent control of bridging interactions with CaM has been implicated in the regulation of ion channel and unconventional myosin activities.     

RC3/Neurogranin and Ca2+/calmodulin-dependent protein kinase II produce opposing effects on the affinity of calmodulin for calcium

The interaction of calmodulin with its target proteins is known to affect the kinetics and affinity of Ca(2+) binding to calmodulin. Based on thermodynamic principles, proteins that bind to Ca(2+)-calmodulin should increase the affinity of calmodulin for Ca(2+), while proteins that bind to apo-calmodulin should decrease its affinity for Ca(2+). We quantified the effects on Ca(2+)-calmodulin interaction of two neuronal calmodulin targets: RC3, which binds both Ca(2+)- and apo-calmodulin, and alphaCaM kinase II, which binds selectively to Ca(2+)-calmodulin. RC3 was found to decrease the affinity of calmodulin for Ca(2+), whereas CaM kinase II increases the calmodulin affinity for Ca(2+). Specifically, RC3 increases the rate of Ca(2+) dissociation from the C-terminal sites of calmodulin up to 60-fold while having little effect on the rate of Ca(2+) association. Conversely, CaM kinase II decreases the rates of dissociation of Ca(2+) from both lobes of calmodulin and autophosphorylation of CaM kinase II at Thr(286) induces a further decrease in the rates of Ca(2+) dissociation. RC3 dampens the effects of CaM kinase II on Ca(2+) dissociation by increasing the rate of dissociation from the C-terminal lobe of calmodulin when in the presence of CaM kinase II. This effect is not seen with phosphorylated CaM kinase II. The results are interpreted according to a kinetic scheme in which there are competing pathways for dissociation of the Ca(2+)-calmodulin target complex. This work indicates that the Ca(2+) binding properties of calmodulin are highly regulated and reveals a role for RC3 in accelerating the dissociation of Ca(2+)-calmodulin target complexes at the end of a Ca(2+) signal.     

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.     

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

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

Calcium-deficient calmodulin binding and activation of neuronal and inducible nitric oxide synthases

The nitric oxide synthase (NOS) enzymes are bound and activated by the Ca(2+)-binding protein, calmodulin (CaM). We have utilized CaM mutants deficient in binding Ca(2+) with mutations in the N-lobe (CaM(12)), the C-lobe (CaM(34)), or both lobes of CaM (CaM(1234)) to determine their effect on the binding and activation of the Ca(2+)-dependent neuronal (nNOS) and Ca(2+)-independent inducible NOS (iNOS) isoforms. Four different kinetic assays were employed to monitor the effect of these CaM mutants on electron transfer rates in NOS. Protein-protein interactions between CaM and NOS were studied using steady-state fluorescence and spectropolarimetry to monitor the binding of these CaM mutants to nNOS and iNOS CaM-binding domain peptides. The CaM mutants were unable to activate nNOS, however, our CD results show that the C-terminal lobe of CaM is capable of binding to nNOS peptide in the presence of Ca(2+). Our results prove for the first time without the use of chelators that apo-CaM is capable of binding to iNOS peptides and holoenzymes.     

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+.     

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.     

Role of the N- and C-lobes of calmodulin in the activation of Ca(2+)/calmodulin-dependent protein kinase II

Understanding the principles of calmodulin (CaM) activation of target enzymes will help delineate how this seemingly simple molecule can play such a complex role in transducing Ca (2+)-signals to a variety of downstream pathways. In the work reported here, we use biochemical and biophysical tools and a panel of CaM constructs to examine the lobe specific interactions between CaM and CaMKII necessary for the activation and autophosphorylation of the enzyme. Interestingly, the N-terminal lobe of CaM by itself was able to partially activate and allow autophosphorylation of CaMKII while the C-terminal lobe was inactive. When used together, CaMN and CaMC produced maximal CaMKII activation and autophosphorylation. Moreover, CaMNN and CaMCC (chimeras of the two N- or C-terminal lobes) both activated the kinase but with greater K act than for wtCaM. Isothermal titration calorimetry experiments showed the same rank order of affinities of wtCaM > CaMNN > CaMCC as those determined in the activity assay and that the CaM to CaMKII subunit binding ratio was 1:1. Together, our results lead to a proposed sequential mechanism to describe the activation pathway of CaMKII led by binding of the N-lobe followed by the C-lobe. This mechanism contrasts the typical sequential binding mode of CaM with other CaM-dependent enzymes, where the C-lobe of CaM binds first. The consequence of such lobe specific binding mechanisms is discussed in relation to the differential rates of Ca (2+)-binding to each lobe of CaM during intracellular Ca (2+) oscillations.     

Apocalmodulin and Ca2+ calmodulin-binding sites on the CaV1.2 channel

The cardiac L-type voltage-dependent calcium channel is responsible for initiating excitation-contraction coupling. Three sequences (amino acids 1609-1628, 1627-1652, and 1665-1685, designated A, C, and IQ, respectively) of its alpha(1) subunit contribute to calmodulin (CaM) binding and Ca(2+)-dependent inactivation. Peptides matching the A, C, and IQ sequences all bind Ca(2+)CaM. Longer peptides representing A plus C (A-C) or C plus IQ (C-IQ) bind only a single molecule of Ca(2+)CaM. Apocalmodulin (ApoCaM) binds with low affinity to the IQ peptide and with higher affinity to the C-IQ peptide. Binding to the IQ and C peptides increases the Ca(2+) affinity of the C-lobe of CaM, but only the IQ peptide alters the Ca(2+) affinity of the N-lobe. Conversion of the isoleucine and glutamine residues of the IQ motif to alanines in the channel destroys inactivation (Zühlke et al., 2000). The double mutation in the peptide reduces the interaction with apoCaM. A mutant CaM unable to bind Ca(2+) at sites 3 and 4 (which abolishes the ability of CaM to inactivate the channel) binds to the IQ, but not to the C or A peptide. Our data are consistent with a model in which apoCaM binding to the region around the IQ motif is necessary for the rapid binding of Ca(2+) to the C-lobe of CaM. Upon Ca(2+) binding, this lobe is likely to engage the A-C region.     

A series of point mutations reveal interactions between the calcium-binding sites of calmodulin.

Calmodulin is a member of the "EF-hand" family of Ca(2+)-binding proteins. It consists of two homologous globular domains, each containing two helix-loop-helix Ca(2+)-binding sites. To examine the contribution of individual Ca(2+)-binding sites to the Ca(2+)-binding properties of CaM, a series of four site-directed mutants has been studied. In each, the glutamic acid at position 12 in one of the four Ca(2+)-binding loops has been changed to a glutamine. One-dimensional 1H-NMR has been used to monitor Ca(2+)-induced changes in the mutant proteins, and the spectral changes observed for each mutant have been compared to those for wild-type CaM. In this way, the effect of each mutation on both the mutated site and the other Ca(2+)-binding sites has been examined. The mutation of glutamate to glutamine at position 12 in any of the EF-hand Ca(2+)-binding loops greatly decreases the Ca(2+)-binding affinity at that site, yet differs in the overall effects on Ca2+ binding depending on which of the four sites is mutated. When the mutation is in site I, there is only a small decrease in the apparent Ca(2+)-binding affinity of site II, and vice versa. Mutation in either site III or IV results in a large decrease in the apparent Ca(2+)-binding affinities of the partner C-terminal site. In both the N- and C-terminal domains, evidence for altered conformational effects in the partners of mutated sites is presented. In the C-terminus, the conformational consequences of mutating site III or site IV are strikingly different.     

Interaction of S100 proteins with the antiallergic drugs, olopatadine, amlexanox, and cromolyn: identification of putative drug binding sites on S100A1 protein

S100 proteins are a multigenic family of low-molecular-weight Ca(2+)-binding proteins comprising 19 members. These proteins undergo a conformational change by Ca(2+)-binding and consequently interact with their target proteins. Recently, we reported that two antiallergic drugs, Amlexanox and Cromolyn, bind to S100A12 and S100A13 of the S100 protein family. In the present study, we used a newly developed antiallergic drug, Olopatadine, as a ligand for affinity chromatography and examined binding specificity of the drug to S100 protein family. Olopatadine binds specifically to S100 proteins, such as S100A1, S100B, S100L, S100A12, and S100A13, in a Ca(2+)-dependent manner but not to calmodulin. Mutagenesis study showed that amino acid residues 76-85 in S100A1 are necessary for its binding to Olopatadine. In contrast, residues 89-94 were identified as an Amlexanox-binding site in S100A1. Moreover, Olopatadine did not competitively inhibit S100A1-binding site of Amlexanox. Furthermore, we showed that Olopatadine inhibited the binding of S100A1 target protein's binding site peptides to S100A1. These results indicate that C-terminal region of S100A1 is important for antiallergic drug binding, although the drug binding sites are different according to each antiallergic drug. Differences in the binding sites of S100A1 to antiallergic drugs suggest that the regulatory functions of S100 proteins may exist in several regions. Therefore, these drugs may serve as useful tools for evaluating the physiological significance of S100 protein family.     

C-terminal half of human centrin 2 behaves like a regulatory EF-hand domain

Human centrin 2 (HsCen2) is an EF-hand protein that plays a critical role in the centrosome duplication and separation during cell division. We studied the structural and Ca(2+)-binding properties of two C-terminal fragments of this protein: SC-HsCen2 (T94-Y172), covering two EF-hands, and LC-HsCen2 (M84-Y172), having 10 additional residues. Both fragments are highly disordered in the apo state but become better structured (although not conformationally homogeneous) in the presence of Ca(2+) and depending on the nature of the cations (K(+) or Na(+)) in the buffer. Only the longer C-terminal domain, in the Ca(2+)-saturated state and in the presence of Na(+) ions, was amenable to structure determination by nuclear magnetic resonance. The solution structure of LC-HsCen2 reveals an open two EF-hand structure, similar to the conformation of related Ca(2+)-saturated regulatory domains. Unexpectedly, the N-terminal helix segment (F86-T94) lies over the exposed hydrophobic cavity. This unusual intramolecular interaction increases considerably the Ca(2+) affinity and constitutes a useful model for the target binding.     

Tuning of a neuronal calcium sensor

Recoverin is a Ca(2+)-regulated signal transduction modulator expressed in the vertebrate retina that has been implicated in visual adaptation. An intriguing feature of recoverin is a cluster of charged residues at its C terminus, the functional significance of which is largely unclear. To elucidate the impact of this segment on recoverin structure and function, we have investigated a mutant lacking the C-terminal 12 amino acids. Whereas in myristoylated recoverin the truncation causes an overall decrease in Ca(2+) sensitivity, results for the non-myristoylated mutant indicate that the truncation primarily affects the high affinity EF-hand 3. The three-dimensional structure of the mutant has been determined by x-ray crystallography. In addition to significant changes in average coordinates compared with wild-type recoverin, the structure provides strong indication of increased conformational flexibility, particularly in the C-terminal domain. Based on these observations, we propose a novel role of the C-terminal segment of recoverin as an internal modulator of Ca(2+) sensitivity.     

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.