Backbone dynamic properties of the central linker region of calcium-calmodulin in 35% trifluoroethanol

The backbone dynamic properties of uniformly (15)N-labeled calcium-saturated calmodulin (Ca(2+)-CaM) in 35% 2,2,2-trifluoroethanol (TFE) have been examined by (15)N NMR relaxation methods. This particular solvent was chosen in order to mimic the conditions in which CaM was crystallized, which included the presence of alcohols. Special attention was paid to the central linker region of Ca(2+)-CaM, which is a long, solvent-exposed alpha-helix in the crystal structure but is known to be partially unwound and flexible in solution. (15)N T(1), T(2), and (15)N-[(1)H] NOE values were determined for both Ca(2+)-CaM in H(2)O and Ca(2+)-CaM in 35% TFE, and the results indicated that the presence of 35% TFE did indeed induce a more ordered conformation in the central linker, with order parameters for Asp78-Glu80 of 0.29, 0.17, and 0.27 in H(2)O and 0.82, 0.66, and 0.64 in 35% TFE. However, (15)N-[(1)H] NOE values showed that these residues were still slightly more flexible than the rest of the molecule in 35% TFE (Asp78-Glu80 (15)N-[(1)H] NOE=0.46, 0.46, and 0.51). Furthermore, there is still independent motion of the two lobes of Ca(2+)-CaM in 35% TFE, with motional correlation times of approximately 10 and approximately 9 ns for the N- and C-lobes, respectively, indicating that 35% TFE was not sufficient to force Ca(2+)-CaM into a rigid dumbbell-shaped molecule as seen in the crystal structure. Additional factors that could further stabilize the structure of CaM in the crystal include pH, temperature, and crystal packing.     

Solution structure and fluctuation of the Mg(2+)-bound form of calmodulin C-terminal domain

Calmodulin (CaM) is a Ca(2+)-binding protein that functions as a ubiquitous Ca(2+)-signaling molecule, through conformational changes from the "closed" apo conformation to the "open" Ca(2+)-bound conformation. Mg(2+) also binds to CaM and stabilizes its folded structure, but the NMR signals are broadened by slow conformational fluctuations. Using the E104D/E140D mutant, designed to decrease the signal broadening in the presence of Mg(2+) with minimal perturbations of the overall structure, the solution structure of the Mg(2+)-bound form of the CaM C-terminal domain was determined by multidimensional NMR spectroscopy. The Mg(2+)-induced conformational change mainly occurred in EF hand IV, while EF-hand III retained the apo structure. The helix G and helix H sides of the binding sequence undergo conformational changes needed for the Mg(2+) coordination, and thus the helices tilt slightly. The aromatic rings on helix H move to form a new cluster of aromatic rings in the hydrophobic core. Although helix G tilts slightly to the open orientation, the closed conformation is maintained. The fact that the Mg(2+)-induced conformational changes in EF-hand IV and the hydrophobic core are also seen upon Ca(2+) binding suggests that the Ca(2+)-induced conformational changes can be divided into two categories, those specific to Ca(2+) and those common to Ca(2+) and Mg(2+).     

The 1.0 A crystal structure of Ca(2+)-bound calmodulin: an analysis of disorder and implications for functionally relevant plasticity

Calmodulin (CaM) is a highly conserved 17 kDa eukaryotic protein that can bind specifically to over 100 protein targets in response to a Ca(2+) signal. Ca(2+)-CaM requires a considerable degree of structural plasticity to accomplish this physiological role; however, the nature and extent of this plasticity remain poorly characterized. Here, we present the 1.0 A crystal structure of Paramecium tetraurelia Ca(2+)-CaM, including 36 discretely disordered residues and a fifth Ca(2+) that mediates a crystal contact. The 36 discretely disordered residues are located primarily in the central helix and the two hydrophobic binding pockets, and reveal correlated side-chain disorder that may assist target-specific deformation of the binding pockets. Evidence of domain displacements and discrete backbone disorder is provided by translation-libration-screw (TLS) analysis and multiconformer models of protein disorder, respectively. In total, the evidence for disorder at every accessible length-scale in Ca(2+)-CaM suggests that the protein occupies a large number of hierarchically arranged conformational substates in the crystalline environment and may sample a quasi-continuous spectrum of conformations in solution. Therefore, we propose that the functionally distinct forms of CaM are less structurally distinct than previously believed, and that the different activities of CaM in response to Ca(2+) may result primarily from Ca(2+)-mediated alterations in the dynamics of the protein.     

Differential codes for free Ca(2+)-calmodulin signals in nucleus and cytosol

BACKGROUND: Many targets of calcium signaling pathways are activated or inhibited by binding the Ca(2+)-liganded form of calmodulin (Ca(2+)-CaM). Here, we test the hypothesis that local Ca(2+)-CaM-regulated signaling processes can be selectively activated by local intracellular differences in free Ca(2+)-CaM concentration. RESULTS: Energy-transfer confocal microscopy of a fluorescent biosensor was used to measure the difference in the concentration of free Ca(2+)-CaM between nucleus and cytoplasm. Strikingly, short receptor-induced calcium spikes produced transient increases in free Ca(2+)-CaM concentration that were of markedly higher amplitude in the cytosol than in the nucleus. In contrast, prolonged increases in calcium led to equalization of the nuclear and cytosolic free Ca(2+)-CaM concentrations over a period of minutes. Photobleaching recovery and translocation measurements with fluorescently labeled CaM showed that equalization is likely to be the result of a diffusion-mediated net translocation of CaM into the nucleus. The driving force for equalization is a higher Ca(2+)-CaM-buffering capacity in the nucleus compared with the cytosol, as the direction of the free Ca(2+)-CaM concentration gradient and of CaM translocation could be reversed by expressing a Ca(2+)-CaM-binding protein at high concentration in the cytosol. CONCLUSIONS: Subcellular differences in the distribution of Ca(2+)-CaM-binding proteins can produce gradients of free Ca(2+)-CaM concentration that result in a net translocation of CaM. This provides a mechanism for dynamically regulating local free Ca(2+)-CaM concentrations, and thus the local activity of Ca(2+)-CaM targets. Free Ca(2+)-CaM signals in the nucleus remain low during brief or low-frequency calcium spikes, whereas high-frequency spikes or persistent increases in calcium cause translocation of CaM from the cytoplasm to the nucleus, resulting in similar concentrations of nuclear and cytosolic free Ca(2+)-CaM.     

Crystal structures of apo- and holo-bovine alpha-lactalbumin at 2. 2-A resolution reveal an effect of calcium on inter-lobe interactions

High affinity binding of Ca(2+) to alpha-lactalbumin (LA) stabilizes the native structure and is required for the efficient generation of native protein with correct disulfide bonds from the reduced denatured state. A progressive increase in affinity of LA conformers for Ca(2+) as they develop increasingly native structures can account for the tendency of the apo form to assume a molten globule state and the large acceleration of folding by Ca(2+). To investigate the effect of calcium on structure of bovine LA, x-ray structures have been determined for crystals of the apo and holo forms at 2.2-A resolution. In both crystal forms, which were grown at high ionic strength, the protein is in a similar global native conformation consisting of alpha-helical and beta-subdomains separated by a cleft. Even though alternative cations and Ca(2+) liganding solvent molecules are absent, removal of Ca(2+) has only minor effects on the structure of the metal-binding site and a structural change was observed in the cleft on the opposite face of the molecule adjoining Tyr(103) of the helical lobe and Gln(54) of the beta-lobe. Changes include increased separation of the lobes, loss of a buried solvent molecule near the Ca(2+)-binding site, and the replacement of inter- and intra-lobe H-bonds of Tyr(103) by interactions with new immobilized water molecules. The more open cleft structure in the apo protein appears to be an effect of calcium binding transmitted via a change in orientation of helix H3 relative to the beta-lobe to the inter-lobe interface. Calcium is well known to promote the folding of LA. The results from the comparison of apo and holo structures of LA provide high resolution structural evidence that the acceleration of folding by Ca(2+) is mediated by an effect on interactions between the two subdomains.     

The calmodulin-binding domain from a plant kinesin functions as a modular domain in conferring Ca2+-calmodulin regulation to animal plus- and minus-end kinesins

Plant kinesin-like calmodulin-binding protein (KCBP) is a novel member of the kinesin superfamily that interacts with calmodulin (CaM) via its CaM-binding domain (CBD). Activated CaM (Ca(2+)-CaM) has been shown to inhibit KCBP interaction with microtubules (MTs) thereby abolishing its motor- and MT-dependent ATPase activities. To test whether the fusion of CBD to non-CaM-binding kinesins confers Ca(2+)-CaM regulation, we fused the CBD of KCBP to the N or C terminus of a minus-end (non-claret disjunction) or C terminus of a plus-end (Drosophila kinesin) motor. Purified chimeric kinesins bound CaM in a Ca(2+)-dependent manner whereas non-claret disjunction, Drosophila kinesin, and KCBP that lack a CBD did not. As in the case of KCBP with CBD, the interaction of chimeric motors with MTs, as well as their MT-stimulated ATPase activity, was inhibited by Ca(2+)-CaM. The presence of a spacer between the motor and CBD did not alter Ca(2+)-CaM regulation. However, KCBP interaction with MTs and its MT-stimulated ATPase activity were not inhibited when the motor domain and CBD were added separately, suggesting that Ca(2+)-CaM regulation of CaM-binding motors occurs only when the CBD is attached to the motor domain. These results show that the fusion of the CBD to animal motors confers Ca(2+)-CaM regulation and suggest that the CBD functions as a modular domain in disrupting motor-MT interaction. Our data also support the hypothesis that CaM-binding kinesins may have evolved by addition of a CBD to a kinesin motor domain.     

Human calcium-calmodulin dependent protein kinase I: cDNA cloning, domain structure and activation by phosphorylation at threonine-177 by calcium-calmodulin dependent protein kinase I kinase.

Human Ca(2+)-calmodulin (CaM) dependent protein kinase I (CaMKI) encodes a 370 amino acid protein with a calculated M(r) of 41,337. The 1.5 kb CaMKI mRNA is expressed in many different human tissues and is the product of a single gene located on human chromosome 3. CaMKI 1-306, was unable to bind Ca(2+)-CaM and was completely inactive thereby defining an essential component of the CaM-binding domain to residues C-terminal to 306. CaMKI 1-294 did not bind CaM but was fully active in the absence of Ca(2+)-CaM, indicating that residues 295-306 are sufficient to maintain CaMKI in an auto-inhibited state. CaMKI was phosphorylated on Thr177 and its activity enhanced approximately 25-fold by CaMKI kinase in a Ca(2+)-CaM dependent manner. Replacement of Thr177 with Ala or Asp prevented both phosphorylation and activation by CaMKI kinase and the latter replacement also led to partial activation in the absence of CaMKI kinase. Whereas CaMKI 1-306 was unresponsive to CaMKI kinase, the 1-294 mutant was phosphorylated and activated by CaMKI kinase in both the presence and absence of Ca(2+)-CaM although at a faster rate in its presence. These results indicate that the auto-inhibitory domain in CaMKI gates, in a Ca(2+)-CaM dependent fashion, accessibility of both substrates to the substrate binding cleft and CaMKI kinase to Thr177. Additionally, CaMKI kinase responds directly to Ca(2+)-CaM with increased activity.     

Structured functional domains of myelin basic protein: cross talk between actin polymerization and Ca(2+)-dependent calmodulin interaction

The 18.5-kDa myelin basic protein (MBP), the most abundant isoform in human adult myelin, is a multifunctional, intrinsically disordered protein that maintains compact assembly of the sheath. Solution NMR spectroscopy and a hydrophobic moment analysis of MBP's amino-acid sequence have previously revealed three regions with high propensity to form strongly amphipathic α-helices. These regions, located in the central, N- and C-terminal parts of the protein, have been shown to play a role in the interactions of MBP with cytoskeletal proteins, Src homology 3-domain-containing proteins, Ca(2+)-activated calmodulin (Ca(2+)-CaM), and myelin-mimetic membrane bilayers. Here, we have further characterized the structure-function relationship of these three domains. We constructed three recombinant peptides derived from the 18.5-kDa murine MBP: (A22-K56), (S72-S107), and (S133-S159) (which are denoted α1, α2, and α3, respectively). We used a variety of biophysical methods (circular dichroism spectroscopy, isothermal titration calorimetry, transmission electron microscopy, fluorimetry, and solution NMR spectroscopy and chemical shift index analysis) to characterize the interactions of these peptides with actin and Ca(2+)-CaM. Our results show that all three peptides can adopt α-helical structure inherently even in aqueous solution. Both α1- and α3-peptides showed strong binding with Ca(2+)-CaM, and both adopted an α-helical conformation upon interaction, but the binding of the α3-peptide appeared to be more dynamic. Only the α1-peptide exhibited actin polymerization and bundling activity, and the addition of Ca(2+)-CaM resulted in depolymerization of actin that had been polymerized by α1. The results of this study proved that there is an N-terminal binding domain in MBP for Ca(2+)-CaM (in addition to the primary site located in the C-terminus), and that it is sufficient for CaM-induced actin depolymerization. These three domains of MBP represent molecular recognition fragments with multiple roles in both membrane- and protein-association.     

Ca2+-calmodulin inhibits tail-anchored protein insertion into the mammalian endoplasmic reticulum membrane

Cytosolic components and pathways have been identified that are involved in inserting tail-anchored (TA) membrane proteins into the yeast or mammalian endoplasmic reticulum (ER) membrane. Searching for regulatory mechanisms of TA protein biogenesis, we found that Ca(2+)-calmodulin (CaM) inhibits the insertion of TA proteins into mammalian ER membranes and that this inhibition is prevented by trifluoperazine, a CaM antagonist that interferes with substrate binding of Ca(2+)-CaM. The effects of Ca(2+)-CaM on cytochrome b(5) and Synaptobrevin 2 suggest a direct interaction between Ca(2+)-CaM and TA proteins. Thus, CaM appears to regulate TA insertion into the ER membrane in a Ca(2+) dependent manner.     

A molecular dynamics study of the effect of Ca2+ removal on calmodulin structure

Calmodulin is a small (148 residues), ubiquitous, highly-conserved Ca(2+) binding protein serving as a modulator of many calcium-dependent processes. In this study, we followed, by means of molecular dynamics, the structural stability of the protein when one of its four bound Ca(2+) ions is removed, and compared it to a simulation of the fully Ca(2+) bound protein. We found that the removal of a single Ca(2+) ion from the N-lobe of the protein, which has a lower affinity for the ion, is sufficient to initiate a considerable structural rearrangement. Although the overall structure of the fully 4 Ca(2+) bound protein remained intact in the extended conformation, the Ca(2+)-removed protein changed its conformation into a compact state. The observation that the 3 Ca(2+) loaded protein assumes a compacted solution state is in accord with experimental observation that the NSCP protein, which binds only three Ca(2+) ions, is natively in a compact state. Examination of the folding dynamics reveals a cooperation between the C-lobe, N-lobe, and the interdomain helix that enable the conformation change. The forces driving this conformational change are discussed.     

Distribution of activated calmodulin in the chloronema tip cells of the moss Funaria hygrometrica

Auxin (indole-3-acetic acid) regulates caulonema differentiation as a result of gradual transitional events in the chloronema tip cells in moss protonema. This auxin action in the moss Funaria hygrometrica involves a rapid influx of calcium ions from the extracellular medium. This investigation demonstrates spatial and temporal changes in calmodulin (CaM) activation (formation of Ca(2+)-CaM complex) in the chloronema tip cells subjected to auxin treatment. Photomicroscopic localisation of the fluorescence (excitation at 365 nm and emission of 397 nm) from the tricomplex of Ca(2+)-CaM with trifluoperazine (TFP, a blocker of Ca(2+)-CaM action) shows a tip to base (tip high) gradient of Ca(2+)-CaM in the chloronema tip cells. Comparison of Ca(2+)-CaM-TFP fluorescence over time in the chloronema tip cells of wild type Funaria with the response in an auxin overproducer mutant (86.1) and an auxin deficient mutant (87.13) reveals the involvement of auxin in calmodulin activation as a rapid response prior to cell differentiation.     

Multicolor imaging of Ca(2+) and protein kinase C signals using novel epifluorescence microscopy.

Dynamic changes in intracellular free Ca(2+) concentrations ([Ca(2+)](i)s) control many important cellular events, including binding of Ca(2+)-calmodulin (Ca(2+)-CaM) and phosphorylation by protein kinase C (PKC). The two signals compete for the same domains in certain substrates, such as myristoylated alanine-rich PKC-substrate (MARCKS). To observe the convergence and relative time of arrival of CaM and PKC signals at their shared domain of MARCKS, we need to image cells that are loaded with more than two fluorescent dyes at a reasonable speed. We have developed a simple and powerful multicolor imaging system using conventional fluorescence microscopy. The epifluorescence configuration uses a glass reflector and rotating filter wheels for excitation and emission paths. As it is free of dichroic (multichroic) mirrors, multiple fluorescence images can be acquired rapidly regardless of the colors of fluorophores. We visualized Ca(2+)-CaM and PKC together with the dynamics of their common target, MARCKS, in single live cells. Receptor-activation resulted in translocation of MARCKS from the plasma membrane to cytosol through its phosphorylation by PKC. By observing fluorescence resonance energy transfer, we also obtained direct evidence that Ca(2+)-CaM binds MARCKS to drag it away from the membrane in circumstances when Ca(2+)-mobilization predominates over PKC activation.     

Calmodulin oxidation and methionine to glutamine substitutions reveal methionine residues critical for functional interaction with ryanodine receptor-1

Calmodulin (CaM) binds to the skeletal muscle ryanodine receptor Ca(2+) release channel (RyR1) with high affinity, and it may act as a Ca(2+)-sensing subunit of the channel. Apo-CaM increases RyR1 channel activity, but Ca(2+)-CaM is inhibitory. Here we examine the functional effects of CaM oxidation on RyR1 regulation by both apo-CaM and Ca(2+)-CaM, as assessed via determinations of [(3)H]ryanodine and [(35)S]CaM binding to skeletal muscle sarcoplasmic reticulum vesicles. Oxidation of all nine CaM Met residues abolished functional interactions of CaM with RyR1. Incomplete CaM oxidation, affecting 5-8 Met residues, increased the CaM concentration required to modulate RyR1, having a greater effect on the apo-CaM species. Mutating individual CaM Met residues to Gln demonstrated that Met-109 was required for apo-CaM activation of RyR1 but not for Ca(2+)-CaM inhibition of the channel. Furthermore, substitution of Gln for Met-124 increased the apo- and Ca(2+)-CaM concentrations required to regulate RyR1. These results thus identify Met residues critical for the productive association of CaM with RyR1 channels and suggest that oxidation of CaM may contribute to altered regulation of sarcoplasmic reticulum Ca(2+) release during oxidative stress.     

Energetics of target peptide binding by calmodulin reveals different modes of binding

Thermodynamic parameters of interactions of calcium-saturated calmodulin (Ca(2+)-CaM) with melittin, C-terminal fragment of melittin, or peptides derived from the CaM binding regions of constitutive (cerebellar) nitric-oxide synthase, cyclic nucleotide phosphodiesterase, calmodulin-dependent protein kinase I, and caldesmon (CaD-A, CaD-A*) have been measured using isothermal titration calorimetry. The peptides could be separated into two groups according to the change in heat capacity upon complex formation, DeltaC(p). The calmodulin-dependent protein kinase I, constitutive (cerebellar) nitric-oxide synthase, and melittin peptides have DeltaC(p) values clustered around -3.2 kJ.mol(-1).K(-1), consistent with the formation of a globular CaM-peptide complex in the canonical fashion. In contrast, phosphodiesterase, the C-terminal fragment of melittin, CaD-A, and CaD-A* have DeltaC(p) values clustered around -1.6 kJ.mol(-1).K(-1), indicative of interactions between the peptide and mostly one lobe of CaM, probably the C-terminal lobe. It is also shown that the interactions for different peptides with Ca(2+)-CaM can be either enthalpically or entropically driven. The difference in the energetics of peptide/Ca(2+)-CaM complex formation appears to be due to the coupling of peptide/Ca(2+)-CaM complex formation to the coil-helix transition of the peptide. The binding of a helical peptide to Ca(2+)-CaM is dominated by favorable entropic effects, which are probably mostly due to hydrophobic interactions between nonpolar groups of the peptide and Ca(2+)-CaM. Applications of these findings to the design of potential CaM inhibitors are discussed.     

Calcium binding decreases the stokes radius of calmodulin and mutants R74A, R90A, and R90G.

Calmodulin (CaM) is an intracellular cooperative calcium-binding protein essential for activating many diverse target proteins. Biophysical studies of the calcium-induced conformational changes of CaM disagree on the structure of the linker between domains and possible orientations of the domains. Molecular dynamics studies have predicted that Ca4(2+)CaM is in equilibrium between an extended and compact conformation and that Arg74 and Arg90 are critical to the compaction process. In this study gel permeation chromatography was used to resolve calcium-induced changes in the hydrated shape of CaM at pH 7.4 and 5.6. Results showed that mutation of Arg 74 to Ala increases the R(s) as predicted; however, the average separation of domains in Ca4(2+)-CaM was larger than predicted by molecular dynamics. Mutation of Arg90 to Ala or Gly affected the dimensions of apo-CaM more than those of Ca4(2+)-CaM. Calcium binding to CaM and mutants (R74A-CaM, R90A-CaM, and R90G-CaM) lowered the Stokes radius (R(s)). Differences between R(s) values reported here and Rg values determined by small-angle x-ray scattering studies illustrate the importance of using multiple techniques to explore the solution properties of a flexible protein such as CaM.     

Intermolecular tuning of calmodulin by target peptides and proteins: differential effects on Ca2+ binding and implications for kinase activation.

Ca(2+)-activated calmodulin (CaM) regulates many target enzymes by docking to an amphiphilic target helix of variable sequence. This study compares the equilibrium Ca2+ binding and Ca2+ dissociation kinetics of CaM complexed to target peptides derived from five different CaM-regulated proteins: phosphorylase kinase. CaM-dependent protein kinase II, skeletal and smooth myosin light chain kinases, and the plasma membrane Ca(2+)-ATPase. The results reveal that different target peptides can tune the Ca2+ binding affinities and kinetics of the two CaM domains over a wide range of Ca2+ concentrations and time scales. The five peptides increase the Ca2+ affinity of the N-terminal regulatory domain from 14- to 350-fold and slow its Ca2+ dissociation kinetics from 60- to 140-fold. Smaller effects are observed for the C-terminal domain, where peptides increase the apparent Ca2+ affinity 8- to 100-fold and slow dissociation kinetics 13- to 132-fold. In full-length skeletal myosin light chain kinase the inter-molecular tuning provided by the isolated target peptide is further modulated by other tuning interactions, resulting in a CaM-protein complex that has a 10-fold lower Ca2+ affinity than the analogous CaM-peptide complex. Unlike the CaM-peptide complexes, Ca2+ dissociation from the protein complex follows monoexponential kinetics in which all four Ca2+ ions dissociate at a rate comparable to the slow rate observed in the peptide complex. The two Ca2+ ions bound to the CaM N-terminal domain are substantially occluded in the CaM-protein complex. Overall, the results indicate that the cellular activation of myosin light chain kinase is likely to be triggered by the binding of free Ca2(2+)-CaM or Ca4(2+)-CaM after a Ca2+ signal has begun and that inactivation of the complex is initiated by a single rate-limiting event, which is proposed to be either the direct dissociation of Ca2+ ions from the bound C-terminal domain or the dissociation of Ca2+ loaded C-terminal domain from skMLCK. The observed target-induced variations in Ca2+ affinities and dissociation rates could serve to tune CaM activation and inactivation for different cellular pathways, and also must counterbalance the variable energetic costs of driving the activating conformational change in different target enzymes.     

Locating phospholamban in co-crystals with Ca(2+)-ATPase by cryoelectron microscopy.

Phospholamban (PLB) is responsible for regulating Ca(2+) transport by Ca(2+)-ATPase across the sarcoplasmic reticulum of cardiac and smooth muscle. This regulation is coupled to beta-adrenergic stimulation, and dysfunction has been associated with end-stage heart failure. PLB appears to directly bind to Ca(2+)-ATPase, thus slowing certain steps in the Ca(2+) transport cycle. We have determined 3D structures from co-crystals of PLB with Ca(2+)-ATPase by cryoelectron microscopy of tubular co-crystals at 8--10 A resolution. Specifically, we have used wild-type PLB, a monomeric PLB mutant (L37A), and a pentameric PLB mutant (N27A) for co-reconstitution and have compared resulting structures with three control structures of Ca(2+)-ATPase alone. The overall molecular shape of Ca(2+)-ATPase was indistinguishable in the various reconstructions, indicating that PLB did not have any global effects on Ca(2+)-ATPase conformation. Difference maps reveal densities which we attributed to the cytoplasmic domain of PLB, though no difference densities were seen for PLB's transmembrane helix. Based on these difference maps, we propose that a single PLB molecule interacts with two Ca(2+)-ATPase molecules. Our model suggests that PLB may resist the large domain movements associated with the catalytic cycle, thus inhibiting turnover.     

Calcium activates erythrocyte AMP deaminase [isoform E (AMPD3)] through a protein-protein interaction between calmodulin and the N-terminal domain of the AMPD3 polypeptide

Erythrocyte AMP deaminase [isoform E (AMPD3)] is activated in response to increased intracellular calcium levels in Tarui's disease, following exposure of ionophore-treated cells to extracellular calcium, and by the addition of calcium to freshly prepared hemolysates. However, the assumption that Ca(2+) is a positive effector of isoform E is inconsistent with the loss of sensitivity to this divalent cation following dilution of erythrocyte lysates or enzyme purification. Ca(2+) regulation of isoform E was studied by examining in vitro effects of calmodulin (CaM) on this enzyme and by monitoring the influence of CaM antagonists on purine catabolic flow in freshly prepared erythrocytes under various conditions of energy imbalance. Erythrocyte and recombinant isoform E both adsorb to immobilized Ca(2+)-CaM, and relative adsorption across a series of N-truncated recombinant enzymes localizes CaM binding determinants to within residues 65-89 of the AMPD3 polypeptide. Ca(2+)-CaM directly stimulates isoform E catalytic activity through a K(mapp) effect and also antagonizes the protein-lipid interaction between this enzyme and intracellular membranes that inhibits catalytic activity. AMP is the predominant purine catabolite in erythrocytes deprived of glucose or exposed to A23187 ionophore alone, whereas IMP accumulates when Ca(2+) is included under the latter conditions and also during autoincubation at 37 degrees C. Preincubation with a CaM antagonist significantly slows the accumulation of erythrocyte IMP under both conditions. The combined results reveal a protein-protein interaction between Ca(2+)-CaM and isoform E and identify a mechanism that advances our understanding of erythrocyte purine metabolism. Ca(2+)-CaM overcomes potent isoform E inhibitory mechanisms that function to maintain the total adenine nucleotide pool in mature erythrocytes, which are unable to synthesize AMP from IMP because of a developmental loss of adenylosuccinate synthetase. This may also explain why Tarui's disease erythrocytes exhibit accelerated adenine nucleotide depletion in response to an increase in intracellular Ca(2+) concentration. This regulatory mechanism could also play an important role in purine metabolism in other human tissues and cells where the AMPD3 gene is expressed.     

A coupled equilibrium shift mechanism in calmodulin-mediated signal transduction

We used nuclear magnetic resonance data to determine ensembles of conformations representing the structure and dynamics of calmodulin (CaM) in the calcium-bound state (Ca(2+)-CaM) and in the state bound to myosin light chain kinase (CaM-MLCK). These ensembles reveal that the Ca(2+)-CaM state includes a range of structures similar to those present when CaM is bound to MLCK. Detailed analysis of the ensembles demonstrates that correlated motions within the Ca(2+)-CaM state direct the structural fluctuations toward complex-like substates. This phenomenon enables initial ligation of MLCK at the C-terminal domain of CaM and induces a population shift among the substates accessible to the N-terminal domain, thus giving rise to the cooperativity associated with binding. Based on these results and the combination of modern free energy landscape theory with classical allostery models, we suggest that a coupled equilibrium shift mechanism controls the efficient binding of CaM to a wide range of ligands.