Studies of calmodulin structure: laser raman spectroscopy of biomolecules

The structure of bovine brain calmodulin was probed by using laser Raman spectroscopy to elucidate cation-induced conformational changes in the protein. Local changes, most likely reflecting metal binding but not rearrangement of the peptide backbone, were observed in the presence of calcium or magnesium. A conformational change involving the peptide backbone and secondary structure content of calmodulin was observed only in the presence of calcium. The calcium-induced conformational change in the peptide backbone involves increased alpha helix and beta sheet. This was the only major calcium-specific change observed in the Raman spectrum, which suggests that the flexibility of the backbone conformation may play a critical role in the physiological activity of calmodulin.     

Characterization of lanthanide (III) ion binding to calmodulin using luminescence spectroscopy

Pulsed dye laser excitation spectroscopy of the 7F0----5D0 transition of Eu(III) reveals only a single peak as this ion is titrated into apocalmodulin. A titration based on the intensity of this transition shows that the first two Eu(III) ions bind quantitatively to two tight sites, followed by weaker binding (Kd = 2 microM) to two additional sites under conditions of high ionic strength (0.5 M KC1). This excitation experiment is also shown to be a general method for measuring contaminating levels of EDTA down to 0.2 microM in proton solutions. Experiments with Tb(III) using both direct laser excitation and indirect sensitization of Tb(III) luminescence through tyrosine residues in calmodulin also give evidence for two tight and two weaker binding sites (Kd = 2-3 microM). The indirect sensitization results primarily upon binding to the two weaker sites, implying that Tb(III) binds first to domains I and II, which are remote from tyrosine-containing domains III and IV. The 7F0----5D0 excitation signal of Eu(III) was used to measure the relative overall affinities of the tripositive lanthanide ions, Ln(III), across the series. Ln(III) ions at the end of the series are found to bind more weakly than those at the beginning and middle of the series. Eu(III) excited-state lifetime measurements in H2O and D2O reveal that two water molecules are coordinated to the Eu(III) at each of the four metal ion binding sites. Measurements of F?rster-type nonradiative energy-transfer efficiencies between Eu(III) and Nd(III) in the two tight sites were carried out by monitoring the excited-state lifetimes of Eu(III) in the presence and absence of the energy acceptor ion Nd(III).(ABSTRACT TRUNCATED AT 250 WORDS)     

Drug-protein interactions: binding of chlorpromazine to calmodulin, calmodulin fragments, and related calcium binding proteins

The quantitative binding of a phenothiazine drug to calmodulin, calmodulin fragments, and structurally related calcium binding proteins was measured under conditions of thermodynamic equilibrium by using a gel filtration method. Plant and animal calmodulins, troponin C, S100 alpha, and S100 beta bind chlorpromazine in a calcium-dependent manner with different stoichiometries and affinities for the drug. The interaction between calmodulin and chlorpromazine appears to be a complex, calcium-dependent phenomenon. Bovine brain calmodulin bound approximately 5 mol of drug per mol of protein with apparent half-maximal binding at 17 microM drug. Large fragments of calmodulin had limited ability to bind chlorpromazine. The largest fragment, containing residues 1-90, retained only 5% of the drug binding activity of the intact protein. A reinvestigation of the chlorpromazine inhibition of calmodulin stimulation of cyclic nucleotide phosphodiesterase further indicated a complex, multiple equilibrium among the reaction components and demonstrated that the order of addition of components to the reaction altered the drug concentration required for half-maximal inhibition of the activity over a 10-fold range. These results confirm previous observations using immobilized phenothiazines [Marshak, D.R., Watterson, D.M., & Van Eldik, L.J. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 6793-6797] that indicated a subclass of calcium-modulated proteins bound phenothiazines in a calcium-dependent manner, demonstrate that the interaction between phenothiazines and calmodulin is more complex than previously assumed, and suggest that extended regions of the calmodulin molecule capable of forming the appropriate conformation are required for specific, high-affinity, calcium-dependent drug binding activity.     

Thermodynamic analyses of calcium binding to troponin C, calmodulin and parvalbumins by using microcalorimetry

Results of microcalorimetric titrations of calcium-binding proteins with calcium or magnesium have been reviewed and evaluated. Results were analyzed mostly in terms of heat capacity changes, which is most closely related to the structural changes of the molecule on metal binding. Two high-affinity sites of rabbit skeletal troponin C are distinguishable in terms of their affinity to calcium and associated enthalpy changes. Heat capacity changes on calcium binding to one of the two high-affinity sites is negative and is in the range ascribed to the ligand binding. In contrast, that to the other of the high-affinity sites is large and positive, indicating that a substantial area of hydrophobic groups become exposed to the solvent. In frog skeletal troponin C, the anomalous positive heat capacity changes occur in one of the low-affinity calcium-specific sites, so that this may be involved in the regulation of contraction. Unlike skeletal troponin C, both of the two high-affinity sites of cardiac troponin C show negative heat capacity changes. In calmodulin, heat capacity changes are positive but small, indicating that calcium binding may induce clustering of the hydrophobic residues on the surface of the molecule. In parvalbumins, heat capacity changes are negative, characteristic of most ligand binding.     

Multiple calcium binding sites make calmodulin multifunctional

Protein-protein or protein-ion interactions with multisite proteins are essential to the regulation of intracellular and extracellular events. There is, however, limited understanding of how ligand-multisite protein interactions selectively regulate the activities of multiple protein targets. In this paper, we focus on the important calcium (Ca(2+)) binding protein calmodulin (CaM), which has four Ca(2+) ion binding sites and regulates the activity of over 30 other proteins. Recent progress in structural studies has led to significant improvements in the understanding of Ca(2+)-CaM-dependent regulation mechanisms. However, no quantitative model is currently available that can fully explain how the structural diversity of protein interaction surfaces leads to selective activation of protein targets. In this paper, we analyze the multisite protein-ligand binding mechanism using mathematical modelling and experimental data for Ca(2+)-CaM-dependent protein targets. Our study suggests a potential mechanism for selective and differential activation of Ca(2+)-CaM targets by the same CaM molecules, which are involved in a variety of intracellular functions. The close agreement between model predictions and experimental dose-response curves for CaM targets available in the literature suggests that such activation is due to the selective activity of CaM conformations in complexes with variable numbers of Ca(2+) ions. Although the paper focuses on the Ca(2+)-CaM pair as a particularly data rich example, the proposed model predictions are quite general and can easily be extended to other multisite proteins. The results of the study may therefore be proposed as a general explanation for multifunctional target regulation by multisite proteins.     

Estimation of pH-dependence of calcium binding with calmodulin

A methodical approach to estimating calmodulin Ca(2+)-binding properties based on its interaction with highly porous watman and consequent 45Ca2+ binding was proposed. At changing pH from 6.5 until 7.5 the affinity of Ca2+ to calmodulin increases in 4.3-fold. The article displays a model of mechanism for Ca(2+)-binding with calmodulin where the dissociation of H+ from Ca(2+)-binding sites is a limited stage of the process.     

Site-selective laser spectroscopy of lanthanide-binding sites in calmodulin

Site-selective laser spectroscopy has been used to resolve the spectral features of lanthanide fluorescence probe ions in calcium-binding proteins. The capabilities and characteristics of this technique are studied using bovine brain calmodulin where the calcium-binding sites are very similar. Two distinct spectral features are identified. These features were followed during a Eu3+ titration and were found to fill successively, showing they correspond to the high- and low-affinity sites. One set of spectral features is assigned to domains I and III, which are the high-affinity domains, while the other set is assigned to domains II and IV. Additional nonspecific binding is observed after the domains are filled. Tb3+ titrations confirmed earlier results that the tyrosine-containing domains fill second and third (R. W. Wallace, E. A. Tallant, M. E. Duckter, and W. Y. Cheung, 1982, J. Biol. Chem. 257(4), 1845-1854). Site-selective laser spectroscopy was also used to identify the presence of ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid contamination that could cause interference in titrations.     

Calcium binding to complexes of calmodulin and calmodulin binding proteins

The free energy of coupling for binding of Ca2+ and the calmodulin-sensitive phosphodiesterase to calmodulin was determined and compared to coupling energies for two other calmodulin binding proteins, troponin I and myosin light chain kinase. Free energies of coupling were determined by quantitating binding of Ca2+ to calmodulin complexed to calmodulin binding proteins with Quin 2 to monitor free Ca2+ concentrations. The geometric means of the dissociation constants (-Kd) for Ca2+ binding to calmodulin in the presence of equimolar rabbit skeletal muscle troponin I, rabbit skeletal muscle myosin light chain kinase, and bovine heart calmodulin sensitive phosphodiesterase were 2.1, 1.1, and 0.55 microM. The free-energy couplings for the binding of four Ca2+ and these proteins to calmodulin were -4.48, -6.00, and -7.64 kcal, respectively. The Ca2+-independent Kd for binding of the phosphodiesterase to calmodulin was estimated at 80 mM, indicating that complexes between calmodulin and this enzyme would not exist within the cell under low Ca2+ conditions. The large free-energy coupling values reflect the increase in Ca2+ affinity of calmodulin when it is complexed to calmodulin binding proteins and define the apparent positive cooperativity for Ca2+ binding expected for each system. These data suggest that in vitro differences in free-energy coupling for various calmodulin-regulated enzymes may lead to differing Ca2+ sensitivities of the enzymes.     

Increased covalent binding of acetaldehyde to calmodulin in the presence of calcium

The regulatory protein, calmodulin, undergoes major conformational changes in response to changes in intracellular calcium concentration. Furthermore, calmodulin has been reported to have lysine residues which markedly increase their reactivity toward electrophilic substances in the calcium-loaded state. We found that calmodulin formed two to three times more stable adducts with acetaldehyde in the calcium-loaded state as compared to the calcium-free state. Competition-binding studies showed that calmodulin could preferentially compete with albumin for acetaldehyde in the presence, but not in the absence, of calcium. When calmodulin was in the calcium-loaded state, trifluoperazine, an inhibitor of calmodulin activity, significantly decreased the stable binding of acetaldehyde to the protein, whereas in the calcium-free state, minimal effects on binding were observed. Since calmodulin is involved in regulation of multiple important processes in the cell, it is possible that acetaldehyde-calmodulin adducts could contribute to liver injury by perturbation of calcium-dependent homeostatic mechanisms within the hepatocyte.     

Defining the intramembrane binding mechanism of sarcolipin to calcium ATPase using solution NMR spectroscopy

Sarcolipin (SLN) is an integral membrane protein that is expressed in both skeletal and cardiac muscle, where it inhibits SERCA (calcium ATPase) by lowering its apparent Ca2+ affinity in a manner similar to that of its homologue phospholamban (PLN). We use solution NMR to map the structural changes occurring within SLN upon interaction with the regulatory target, SERCA, co-reconstituting the two proteins in dodecylphosphocholine (DPC) detergent micelles, a system that preserves the native structure of SLN and the activity of SERCA, with the goal of comparing these interactions with those of the previously studied PLN-SERCA complex. Our analysis of the structural dynamics of SLN in DPC micelles shows this polypeptide to be partitioned into four subdomains: a short unstructured N terminus (residues 1-6), a short dynamic helix (residues 7-14), a more rigid helix (residues 15-26), and an unstructured C terminus (residues 27-31). Upon addition of SERCA, the different domains behave according to their dynamics, molding onto the surface of the enzyme. Remarkably, each domain of SLN behaves in a manner similar to that of the corresponding domains in PLN, supporting the hypothesis that both SLN and PLN bind SERCA in the same groove and with similar mechanisms.     

Ion binding to calmodulin

Summary Over the past few years calcium has emerged as an important bioregulator. Upon external stimulation, the cell generates a transient Ca2+ increase, which is transformed into a cellular event through a molecular cascade. The first step in this cascade is the binding of calcium to proteins present in the cytosol. These proteins capable of binding Ca²⁺ under physiological conditions all belong to the same evolutionary family that evolved from a common ancestor. However, they strongly differ in the properties of their calcium binding sites. Calmodulin, the ubiquitous calcium binding protein present in all eukaryotic cells, is very close to the ancestor protein, presents four calcium binding sites which bind calcium, magnesium and monovalent ions competitively and is involved in the triggering of cellular processes. Parvalbumin, another member of the family, is more specialized and found mostly in fast-twitch skeletal muscle. It binds calcium and magnesium with high affinity and seems to be involved in muscle relaxation. On the other hand, troponin C which confers Ca²⁺ sensitivity to acto-myosin interaction exhibits both triggering and relaxing sites. The study of intracellular Ca^2– binding proteins has shown that calcium binding proteins have evolved from a simple common structure to fulfill different functions.Abbreviations CaBP calcium-binding protein - ICaBP the vitamin D-dependent intestinal Cat+binding protein - S-100 the glial S-100 protein - RLC the phosphorylatable myosin regulatory light chain - CaM calmodulin - Pa parvalbumin - TnC troponin C - TnI troponin I - Hepes N-2-hydroxyethylpipezarine, N-2-ethane-sulfonic acid - W7 N-(6-Aminohexyl)-5-chloro-l-Naphtalene sulfonamide - SDS sodium dodecyl sulfate - NMR nuclear magnetic resonance     

Helix A stabilization precedes amino-terminal lobe activation upon calcium binding to calmodulin

The structural coupling between opposing domains of CaM was investigated using the conformationally sensitive biarsenical probe 4,5-bis(1,3,2-dithioarsolan-2-yl)resorufin (ReAsH), which upon binding to an engineered tetracysteine motif near the end of helix A (Thr-5 to Phe-19) becomes highly fluorescent. Changes in conformation and dynamics are reflective of the native CaM structure, as there is no change in the (1)H- (15)N HSQC NMR spectrum in comparison to wild-type CaM. We find evidence of a conformational intermediate associated with CaM activation, where calcium occupancy of sites in the amino-terminal and carboxyl-terminal lobes of CaM differentially affect the fluorescence intensity of bound ReAsH. Insight into the structure of the conformational intermediate is possible from a consideration of calcium-dependent changes in rates of ReAsH binding and helix A mobility, which respectively distinguish secondary structural changes associated with helix A stabilization from the tertiary structural reorganization of the amino-terminal lobe of CaM necessary for high-affinity binding to target proteins. Helix A stabilization is associated with calcium occupancy of sites in the carboxyl-terminal lobe ( K d = 0.36 +/- 0.04 microM), which results in a reduction in the rate of ReAsH binding from 4900 M (-1) s (-1) to 370 M (-1) s (-1). In comparison, tertiary structural changes involving helix A and other structural elements in the amino-terminal lobe require calcium occupancy of amino-terminal sites (K d = 18 +/- 3 microM). Observed secondary and tertiary structural changes involving helix A in response to the sequential calcium occupancy of carboxyl- and amino-terminal lobe calcium binding sites suggest an important involvement of helix A in mediating the structural coupling between the opposing domains of CaM. These results are discussed in terms of a model in which carboxyl-terminal lobe calcium activation induces secondary structural changes within the interdomain linker that release helix A, thereby facilitating the formation of calcium binding sites in the amino-terminal lobe and linked tertiary structural rearrangements to form a high-affinity binding cleft that can associate with target proteins.     

Binding of calcium by calmodulin: influence of the calmodulin binding domain of the plasma membrane calcium pump.

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.     

FRET detection of calmodulin binding to the cardiac RyR2 calcium release channel

Calmodulin (CaM) binding to the type 2 ryanodine receptor (RyR2) regulates Ca release from the cardiac sarcoplasmic reticulum (SR). However, the structural basis of CaM regulation of the RyR2 is poorly defined, and the presence of other potential CaM binding partners in cardiac myocytes complicates resolution of CaM's regulatory interactions with RyR2. Here, we show that a fluorescence-resonance-energy-transfer (FRET)-based approach can effectively resolve RyR2 CaM binding, both in isolated SR membrane vesicles and in permeabilized ventricular myocytes. A small FRET donor was targeted to the RyR2 cytoplasmic assembly via fluorescent labeling of the FKBP12.6 subunit. Acceptor fluorophore was attached at discrete positions within either the N- or the C-lobe of CaM. FRET between FKBP12.6 and CaM bound to SR vesicles indicated CaM binding at a single high-affinity site within 60 Å of FKBP12.6. Micromolar Ca increased the apparent affinity of CaM binding and slowed CaM dissociation, but did not significantly affect maximal FRET efficiency at saturating CaM. FRET was strongest when the acceptor was attached at either of two positions within CaM's N-lobe versus sites in CaM's C-lobe, providing CaM orientation information. In permeabilized ventricular myocytes, FKBP12.6 and CaM colocalized to Z-lines, and the efficiency of energy transfer to both the N- and C-lobes of CaM was comparable to that observed in SR vesicle experiments. Results also indicate that both the location and orientation of CaM binding on the RyR2 are very similar to the skeletal muscle RyR1 isoform. Specific binding of CaM to functional RyR2 channels in the cardiac myocyte environment can be monitored using FKBP biosensors and FRET.     

Conformation and ion binding properties of peptides related to calcium binding domain III of bovine brain calmodulin

The conformational and ion binding properties of the sequences 93-104, 96-104, and 93-98 of domain III of bovine brain calmodulin (CaM) have been studied by CD and Tb3+-mediated fluorescence. In aqueous solution the interaction of all fragments with Ca2+ and Mg2+ ions is very weak and without any effect on the peptide conformation, which remains always random. In trifluoroethanol the interaction is very strong and the different fragments exhibit very distinct binding properties. In particular, the dodecapeptide fragment 93-104, and its N-terminal hexapeptide 98-104, bind calcium and magnesium with a very high binding constant (Kb greater than 10(5) M-1), undergoing a substantial conformational change. The structural rearrangement is particularly evident in the hexapeptide fragment, which tend to form a beta-bend. The C-terminal nonapeptide fragment 96-104 interacts with calcium and magnesium more weakly, and the binding process causes a decrease of ordered structure. These results suggest that, even in the entire dodecapeptide sequence corresponding to the loop of domain III of CaM, the calcium binding site is shifted toward the N-terminal hexapeptide segment. This interpretation is consistent with the results of crystallographic studies of CaM, which show that the calcium ions are located toward the amino terminal portion of the loop.     

Regulation of calmodulin binding to P-57. A neurospecific calmodulin binding protein

P-57 is a neural-specific calmodulin binding protein with novel calmodulin binding properties. P-57 exhibits higher affinity for calmodulin-Sepharose in the absence of free Ca2+ than in the presence of Ca2+ (Andreasen, T.J., Luetje, C.W., Heideman, W. & Storm, D.R. (1983) Biochemistry 22, 4615-4618; Cimler, B. M., Andreasen, T.J., Andreasen, K.I. & Storm, D.R. (1985) J. Biol. Chem. 260, 10784-10788). In this study, the dissociation constants for P-57 and immunopurified 5-[[(iodoacetylamino)ethyl]-amino]-1-naphthalenesulfonic acid-labeled calmodulin (AEDANS-CaM) were determined under low and high ionic strength conditions. In the absence of added KCl, the dissociation constants for the P-57 X AEDANS-CaM complex were 2.3 X 10(-7) +/- 6 X 10(-8) M and 1.0 X 10(-6) +/- 3 X 10(-7) M in the presence and absence of excess Ca2+ chelator. The addition of KCl to 150 mM increased the Ca2+-independent and -dependent dissociation constants to 3.4 X 10(-6) +/- 9 X 10(-7) M and 3.0 X 10(-6) +/- 9 X 10(-7) M, respectively. The association of P-57 with AEDANS-CaM under low Ca2+ conditions was determined as a function of KCl concentrations. By taking into account the amount of P-57 found in brain and its affinity for calmodulin, it is concluded that most or all of the CaM would be complexed to P-57 in unstimulated cells. P-57 was phosphorylated by the Ca2+-phospholipid-dependent protein kinase (protein kinase C) with a phosphate:protein molar ratio of 1.3. Phosphoamino acid analysis demonstrated phosphorylation at a serine residue. CaM decreased the rate of phosphorylation of P-57 by protein kinase C, and phosphorylation prevented P-57 binding to calmodulin-Sepharose. P-57 was not phosphorylated by the catalytic subunit of the cAMP-dependent protein kinase. It is proposed that P-57 binds and localizes calmodulin at specific sites within the cell and that free calmodulin is released locally in response to phosphorylation of P-57 by protein kinase C and/or to increases in intracellular free Ca2+. This regulatory mechanism, which appears to be specific to brain, would serve to decrease the response time for Ca2+-calmodulin-regulated processes.     

Apo calmodulin binding to the L-type voltage-gated calcium channel Cav1.2 IQ peptide

The influx of calcium through the L-type voltage-gated calcium channels (LTCCs) is the trigger for the process of calcium-induced calcium release (CICR) from the sarcoplasmic reticulum, an essential step for cardiac contraction. There are two feedback mechanisms that regulate LTCC activity: calcium-dependent inactivation (CDI) and calcium-dependent facilitation (CDF), both of which are mediated by calmodulin (CaM) binding. The IQ domain (aa 1645-1668) housed within the cytoplasmic domain of the LTCC Cav1.2 subunit has been shown to bind both calcium-loaded (Ca2+CaM ) and calcium-free CaM (apoCaM). Here, we provide new data for the structural basis for the interaction of apoCaM with the IQ peptide using NMR, revealing that the apoCaM C-lobe residues are most significantly perturbed upon complex formation. In addition, we have employed transmission electron microscopy of purified LTCC complexes which shows that both apoCaM and Ca2+CaM can bind to the intact channel.     

Ruthenium red inhibits the binding of calcium to calmodulin required for enzyme activation.

The Ca(2+)-calmodulin (CaM)-dependent activation of myosin light chain kinase is inhibited by ruthenium red competitively with respect to Ca2+, with a Ki value of 8.6 microM. The binding of Ca2+ to CaM is inhibited by micromolar concentrations of ruthenium red. In the absence of Ca2+, CaM has two binding sites for ruthenium red with the dissociation constants of 0.36 and 8.7 microM, respectively. Ca2+ antagonizes the binding of ruthenium red to the low-affinity site on CaM. Binding of ruthenium red to the high-affinity site is not affected by Ca2+. The low- and high-affinity sites for ruthenium red are shown to be located in the NH2-terminal half and the COOH-terminal half of CaM, respectively. Lower concentrations of ruthenium red are needed for enzyme inactivation than for the dissociation of enzyme-CaM-Sepharose complex, suggesting these events have different Ca2+ requirements. Moreover, ruthenium red inhibits Ca(2+)-induced contraction of depolarized vascular smooth muscle in a competitive manner with respect to Ca2+. These results suggest that ruthenium red may be a new type of CaM antagonist that inhibits the binding of Ca2+ to CaM and thereby inhibits Ca(2+)-CaM-dependent enzymes and smooth muscle contraction competitively with respect to Ca2+.