Calcium binding to calmodulin mutants having domain-specific effects on the regulation of ion channels

Calmodulin (CaM) is an essential, eukaryotic protein comprised of two highly homologous domains (N and C). CaM binds four calcium ions cooperatively, regulating a wide array of target proteins. A genetic screen of Paramecia by Kung [Kung, C. et al. (1992) Cell Calcium 13, 413-425] demonstrated that the domains of CaM have separable physiological roles: "under-reactive" mutations affecting calcium-dependent sodium currents mapped to the N-domain, while "over-reactive" mutations affecting calcium-dependent potassium currents localized to the C-domain of CaM. To determine whether and how these mutations affected intrinsic calcium-binding properties of CaM domains, phenylalanine fluorescence was used to monitor calcium binding to sites I and II (N-domain) and tyrosine fluorescence was used to monitor sites III and IV (C-domain). To explore interdomain interactions, binding properties of each full-length mutant were compared to those of its corresponding domain fragments. The calcium-binding properties of six under-reactive mutants (V35I/D50N, G40E, G40E/D50N, D50G, E54K, and G59S) and one over-reactive mutant (M145V) were indistinguishable from those of wild-type CaM, despite their deleterious physiological effects on ion-channel regulation. Four over-reactive mutants (D95G, S101F, E104K, and H135R) significantly decreased the calcium affinity of the C-domain. Of these, one (E104K) also increased the calcium affinity of the N-domain, demonstrating that the magnitude and direction of wild-type interdomain coupling had been perturbed. This suggests that, while some of these mutations alter calcium-binding directly, others probably alter CaM-channel association or calcium-triggered conformational change in the context of a ternary complex with the affected ion channel.     

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.     

Molecular dynamics of calmodulin as monitored by fluorescence anisotropy

Static and dynamic measurements of fluorescence anisotropy have been made for calmodulin, employing both the intrinsic fluorescence of Tyr-99 and Tyr-138 and the fluorescence of dansyl and fluorescein groups attached to Tyr-99, as well as AEDANS groups attached to methionines. All approaches indicate the presence of a significant internal mobility involving the probe for calmodulin in the absence of Ca²⁺. This is diminished in the presence of Ca²⁺.²     

Calcium binding to tryptic fragments of calmodulin

Fragments of scallop testis calmodulin were prepared by tryptic digestion. One peptide consisted of 75 amino acid residues from N-acetylalanine to lysine at position 75 (F12) and the other of 71 residues from aspartic acid at position 78 to C-terminal lysine (F34). Flow dialysis and equilibrium dialysis experiments revealed the existence of two Ca2+ binding sites in each fragment. Half-saturating concentrations of the Ca2+ titration curves were 11 microM for F12 and 3.2 microM for F34, and Hill coefficients were obtained as 1.14 and 1.84, respectively. The results indicate that the high-affinity sites for Ca2+ are located on the C-terminal region of the calmodulin. The sum of the two Ca2+ titration curves of F12 and F34 fits well to the curves of Ca2+ binding to intact calmodulin. This shows that the characteristic of Ca2+ bindings in intact calmodulin did not change after separation of the whole molecule into two domains, F12 and F34. The domains corresponding to F12 and F34 may exist independently from each other in the intact calmodulin molecule.     

Calcium binding to calmodulin and its globular domains

The macroscopic Ca(2+)-binding constants of bovine calmodulin have been determined from titrations with Ca2+ in the presence of the chromophoric chelator 5,5'-Br2BAPTA in 0, 10, 25, 50, 100, and 150 mM KCl. Identical experiments have also been performed for tryptic fragments comprising the N-terminal and C-terminal domains of calmodulin. These measurements indicate that the separated globular domains retain the Ca2+ binding properties that they have in the intact molecule. The Ca2+ affinity is 6-fold higher for the C-terminal domain than for the N-terminal domain. The salt effect on the free energy of binding two Ca2+ ions is 20 and 21 kJ. mol-1 for the N- and C-terminal domain, respectively, comparing 0 and 150 mM KCl. Positive cooperativity of Ca2+ binding is observed within each globular domain at all ionic strengths. No interaction is observed between the globular domains. In the N-terminal domain, the cooperativity amounts to 3 kJ.mol-1 at low ionic strength and greater than or equal to 10 kJ.mol-1 at 0.15 M KCl. For the C-terminal domain, the corresponding figures are 9 +/- 2 kJ.mol-1 and greater than or equal to 10 kJ.mol-1. Two-dimensional 1H NMR studies of the fragments show that potassium binding does not alter the protein conformation.     

Metal ion binding to calmodulin: NMR and fluorescence studies

Calmodulin is an important second messenger protein which is involved in a large variety of cellular path-ways.Calmodulin is sensitive to fluctuations in the intracellular Ca levels and is activated by the bindingof four Ca ions. In spite of the important role it plays in signal transduction pathways, it shows a surpris-inglybroad specificity for binding metal ions. Using 15N-Gly biosynthetically-labelled calmodulin, we havestudied the binding of different metal ions to calmodulin, including K+, Na+, Ca, Mg, Zn, Cd, Pb, Hg, Sr, La and Lu, by 1H, 15N HMQC NMR experiments. The effects of these ions on the substrate-bindingability of calmodulin have also been studied by fluorescence spectroscopy of the single tryptophan residue in a 22-residue synthetic peptide encompassing the skeletal muscle myosin light chain kinase calmod-ulin-binding domain. Most of these metal ions can activate a calmodulin target enzyme to some extent,though they bind to calmodulin in a different manner. Mg, which is of direct physiological interest, has adistinct site-preference for calmodulin, as it shows the highest affinity for site I in the N-terminal domain,while the C-terminal sites III and IV are the high affinity binding sites for Ca (as well as for Cd ). At ahigh concentration of Mg and a low concentration of Ca, calmodulin can bind Mg in its N-terminallobe while the C-terminal domain is occupied by Ca; this species could exist in resting cells in which the Mg level significantly exceeds that of Ca. Moreover, our data suggest that the toxicity of Pb-which,like Sr, binds with an equal and high affinity to all four sites-may be related to its capacity to tightlybind and improperly activate calmodulin.     

Calcium binding to calmodulin. Cooperativity of the calcium-binding sites

The effects of Mg2+ ion, pH, and KCl concentration on Ca2+ binding to calmodulin were studied by using a Ca2+ ion-sensitive electrode. The Ca2+ ion affinity of calmodulin increased with increasing pH or decreasing KCl concentration. Cooperativity between the Ca2+-binding sites was observed, and increased with decreasing pH or increasing KCl concentration. Free Ca2+ ion concentration was decreased by adding MgCl2 ion at low Mg2+ concentration and increased at higher concentrations in the presence of small amounts of Ca2+ ion. The decrease of free Ca2+ ion concentration by Mg2+ ion strongly suggests cooperativity between the Ca2+-binding sites, and it is difficult to explain the decrease in terms of the ordered binding models previously proposed. These results can be explained by a simple model which has four equivalent binding sites that bind Ca2+ and Mg2+ competitively, and showing cooperativity when either Ca2+ or Mg2+ is bound. Mg2+ ion binding to calmodulin was measured in the presence or absence of Ca2+ to confirm the validity of this model, and no Mg2+-specific site was observed.     

Sodium and potassium binding to parvalbumins measured by means of intrinsic protein fluorescence

The binding of Na+ and K+ to whiting parvalbumin (pI 4.4) and pike parvalbumins (pI 4.2 and 5.0) results in a shift of the tryptophan fluorescence spectrum towards shorter wavelengths by 2-4 nm for the whiting protein and in a rise of the tyrosine and phenylalanine fluorescence quantum yield for the pike proteins. The effective binding constants of Na+ and K+ to parvalbumins are within the range of 10 M-1 to 100 M-1. Physiological concentrations of Na+ and K+ lower the affinity of whiting parvalbumin for Ca2+ and Mg2+ by almost an order of magnitude.     

Sugar binding to recombinant wild-type and mutant glucokinase monitored by kinetic measurement and tryptophan fluorescence

Tryptophan fluorescence was used to study GK (glucokinase), an enzyme that plays a prominent role in glucose homoeostasis which, when inactivated or activated by mutations, causes diabetes mellitus or hypoglycaemia in humans. GK has three tryptophan residues, and binding of D-glucose increases their fluorescence. To assess the contribution of individual tryptophan residues to this effect, we generated GST-GK [GK conjugated to GST (glutathione transferase)] and also pure GK with one, two or three of the tryptophan residues of GK replaced with other amino acids (i.e. W99C, W99R, W167A, W167F, W257F, W99R/W167F, W99R/W257F, W167F/W257F and W99R/W167F/W257F). Enzyme kinetics, binding constants for glucose and several other sugars and fluorescence quantum yields (varphi) were determined and compared with those of wild-type GK retaining its three tryptophan residues. Replacement of all three tryptophan residues resulted in an enzyme that retained all characteristic features of GK, thereby demonstrating the unique usefulness of tryptophan fluorescence as an indicator of GK conformation. Curves of glucose binding to wild-type and mutant GK or GST-GK were hyperbolic, whereas catalysis of wild-type and most mutants exhibited co-operativity with D-glucose. Binding studies showed the following order of affinities for the enzyme variants: N-acetyl-D-glucosamine>D-glucose>D-mannose>D-mannoheptulose>2-deoxy-D-glucose>L-glucose. GK activators increased sugar binding of most enzymes, but not of the mutants Y214A/V452A and C252Y. Contributions to the fluorescence increase from Trp(99) and Trp(167) were large compared with that from Trp(257) and are probably based on distinct mechanisms. The average quantum efficiency of tryptophan fluorescence in the basal and glucose-bound state was modified by activating (Y214A/V452A) or inactivating (C213R and C252Y) mutations and was interpreted as a manifestation of distinct conformational states.     

Calcium/calmodulin inhibits direct binding of spectrin to synaptosomal membranes

Brain spectrin, through its beta subunit, binds with high affinity to protein-binding sites on brain membranes quantitatively depleted of ankyrin (Steiner, J., and Bennett, V. (1988) J. Biol. Chem. 263, 14417-14425). In this study, calmodulin is demonstrated to inhibit binding of brain spectrin to synaptosomal membranes. Submicromolar concentrations of calcium are required for inhibition of binding, with half-maximal effects at pCa = 6.5. Calmodulin competitively inhibits binding of spectrin to protein(s) in stripped synaptosomal membranes, with Ki = 1.3 microM in the presence of 10 microM calcium. A reversible receptor-mediated process, and not proteolysis, is responsible for inhibition since the effect of calcium/calmodulin is reversed by the calmodulin antagonist trifluoperazine and by chelation of calcium with sodium [ethylenebis(oxyethylenenitrilo)]tetraacetic acid. The target of calmodulin is most likely the spectrin attachment protein(s) rather than spectrin itself since: (a) membrane binding of the brain spectrin beta subunit, which does not associate with calmodulin, is inhibited by calcium/calmodulin, and (b) red cell spectrin which binds calmodulin very weakly, is inhibited from interacting with membrane receptors in the presence of calcium/calmodulin. Ca2+/calmodulin inhibited association of erythrocyte spectrin with synaptosomal membranes but had no effect on binding of erythrocyte or brain spectrin to ankyrin in erythrocyte membranes. These experiments demonstrate the potential for differential regulation of spectrin-membrane protein interactions, with the consequence that Ca2+/calmodulin can dissociate direct spectrin-membrane interactions locally or regionally without disassembly of the areas of the membrane skeleton stabilized by linkage of spectrin to ankyrin. A membrane protein of Mr = 88,000 has been identified that is dissociated from spectrin affinity columns by calcium/calmodulin and is a candidate for the calmodulin-sensitive spectrin-binding site in brain.     

Rapid kinetic studies of calmodulin interactions with calcium and troponin I as monitored by anthroylcholine fluorescence

Anthroylcholine was utilized as an extrinsic fluorescent probe in rapid kinetic studies of calcium dissociation from calmodulin (k_off = 10 S^?1) and the calmodulin-troponin I complex (k_off = 6 S^?1). At concentrations lower than 70 μM, the mechanism of dye binding agreed with the simple kinetic scheme in which the dye binds exclusively to the respective calcium complexes of calmodulin and calmodulin-troponin I. The sensitivity of anthroylcholine also made possible the estimation of values for the association (1.0 ± 0.8) × 10⁸$\text{M}{}^{\mathrm{?1}}$ S^?1) and dissociation rate constants (2 ± 170 S^?1) for troponin I binding to the calcium₄-calmodulin complex.     

Calcium release from calmodulin and its C-terminal or N-terminal halves in the presence of the calmodulin antagonists phenoxybenzamine and melittin measured by stopped-flow fluorescence with Quin 2 and intrinsic tyrosine. Inhibition of calmodulin-dependent protein kinase of cardiac sarcoplasmic reticulum

Calcium dissociation from the C-terminal and N-terminal halves of calmodulin, intact bovine brain calmodulin and the respective phenoxybenzamine complexes or melittin complexes was measured directly by stopped-flow fluorescence with the calcium chelator Quin 2 and, when possible, also by protein fluorescence using endogenous tyrosine fluorescence by mixing with EGTA. Calcium dissociation from the C-terminal half of calmodulin, which contains only the two high-affinity calcium-binding sites, and from intact calmodulin was monophasic, with good correlation of the rates of calcium dissociation obtained by the two methods. The apparent rates with Quin 2 and endogenous tyrosine fluorescence were 13.4 s-1 and 12.8 s-1, respectively, in the C-terminal half and 10.5 s-1 and 10.8 s-1, respectively, in intact calmodulin (pH 7.0, 25 degrees C, 100 mM KCl). Alkylation of the C-terminal half resulted in a biphasic calcium dissociation (Quin 2: kobs 1.90 s-1 and 0.73 s-1 respectively; tyrosine: kobs 1.65 s-1 and 0.61 s-1 respectively). Alkylation of intact calmodulin resulted in a four-phase calcium dissociation measured with Quin 2 (kobs 85.3 s-1, 11.1 s-1, 1.92 s-1 and 0.59 s-1); the latter two phases are assumed to represent calcium release from high-affinity sites since they correspond to the biphasic tyrosine fluorescence change in intact alkylated calmodulin (kobs 2.04 s-1 and 0.53 s-1 respectively) and the rate parameters determined in the C-terminal half. Evidently perturbation of the calcium-binding sites by alkylation reduces the rate of calcium dissociation and allows a distinction to be made between dissociation from each of the two high-affinity sites as well as the distinct conformational change on dissociation of each calcium. Alkylation of the N-terminal half resulted in biphasic calcium release with rates (kobs 153 s-1 and 10.9 s-1 respectively) similar to those observed in intact alkylated calmodulin. The rates of calcium dissociation from calmodulin-melittin or fragment-melittin complexes, measured with Quin 2, were slower and monophasic in the C-terminal half (kobs 1.12 s-1), biphasic in the N-terminal half (kobs 140 s-1 and 26.8 s-1 respectively) and triphasic in intact calmodulin (kobs 126 s-1, 12.1 s-1 and 1.38 s-1). Calmodulin antagonists thus increase the apparent calcium affinity of high and low-affinity sites mainly due to a reduced calcium 'off rate', presumably because of conformation restrictions.(ABSTRACT TRUNCATED AT 400 WORDS)     

Phenylalanine fluorescence studies of calcium binding to N-domain fragments of Paramecium calmodulin mutants show increased calcium affinity correlates with increased disorder

Calmodulin (CaM) is a ubiquitous, essential calcium-binding protein that regulates diverse protein targets in response to physiological calcium fluctuations. Most high-resolution structures of CaM-target complexes indicate that the two homologous domains of CaM are equivalent partners in target recognition. However, mutations between calcium-binding sites I and II in the N-domain of Paramecium calmodulin (PCaM) selectively affect calcium-dependent sodium currents. To understand these domain-specific effects, N-domain fragments (PCaM(1-75)) of six of these mutants were examined to determine whether energetics of calcium binding to sites I and II or conformational properties had been perturbed. These PCaM((1-75)) sequences naturally contain 5 Phe residues but no Tyr or Trp; calcium binding was monitored by observing the reduction in intrinsic phenylalanine fluorescence at 280 nm. To assess mutation-induced conformational changes, thermal denaturation of the apo PCaM((1-75)) sequences, and calcium-dependent changes in Stokes radii were determined. The free energy of calcium binding to each mutant was within 1 kcal/mole of the value for wild type and calcium reduced the R(s) of all of them. A striking trend was observed whereby mutants showing an increase in calcium affinity and R(s) had a concomitant decrease in thermal stability (by as much as 18 degrees C). Thus, mutations between the binding sites that increased disorder and reduced tertiary constraints in the apo state promoted calcium coordination. This finding underscores the complexity of the linkage between calcium binding and conformational change and the difficulty in predicting mutational effects.     

Calcium binding to calmodulin leads to an N-terminal shift in its binding site on the ryanodine Receptor

The skeletal muscle calcium release channel, ryanodine receptor, is activated by calcium-free calmodulin and inhibited by calcium-bound calmodulin. Previous biochemical studies from our laboratory have shown that calcium-free calmodulin and calcium bound calmodulin protect sites at amino acids 3630 and 3637 from trypsin cleavage (Moore, C. P., Rodney, G., Zhang, J. Z., Santacruz-Toloza, L., Strasburg, G., and Hamilton, S. L. (1999) Biochemistry 38, 8532-8537). We now demonstrate that both calcium-free calmodulin and calcium-bound calmodulin bind with nanomolar affinity to a synthetic peptide matching amino acids 3614-3643 of the ryanodine receptor. Deletion of the last nine amino acids (3635-3643) destroys the ability of the peptide to bind calcium-free calmodulin, but not calcium-bound calmodulin. We propose a novel mechanism for calmodulin's interaction with a target protein. Our data suggest that the binding sites for calcium-free calmodulin and calcium-bound calmodulin are overlapping and, when calcium binds to calmodulin, the calmodulin molecule shifts to a more N-terminal location on the ryanodine receptor converting it from an activator to an inhibitor of the channel. This region of the ryanodine receptor has previously been identified as a site of intersubunit contact, suggesting the possibility that calmodulin regulates ryanodine receptor activity by regulating subunit-subunit interactions.     

Time-resolved fluorescence anisotropy studies show domain-specific interactions of calmodulin with IQ target sequences of myosin V

Single cysteine mutants of calmodulin, Cam(S38C) and Cam(N111C), have been specifically labelled with Alexa488 maleimide to study the effects of calcium on the structural dynamics of calmodulin complexed with IQ3, IQ4 and IQ34 target peptide motifs of mouse unconventional myosin-V. Using phase fluorometry, the time-resolved anisotropy shows well-separated global and segmental correlation times. The calcium-sensitive global motion of either calmodulin domain can be independently monitored in domain-specific interactions of either apo- or Ca(4).calmodulin with IQ3 or IQ4 peptides. C-domain interactions predominate, and apo-N-domain interactions are unexpectedly weak. The 1:1 complex of Ca(4).calmodulin with IQ34 behaves as a compact globular species. The results demonstrate novel dynamic aspects of calmodulin-IQ interactions relating to the calcium regulation of motility of unconventional myosin.     

Nucleotide-induced conformational changes in P-glycoprotein and in nucleotide binding site mutants monitored by trypsin sensitivity

Limited trypsin digestion was used to monitor nucleotide-induced conformational changes in wild-type P-glycoprotein (Pgp) as well as in nucleotide binding domain (NBD) Pgp mutants. Purified and reconstituted wild-type or mutant mouse Mdr3 Pgps were preincubated with different hydrolyzable or nonhydrolyzable nucleotides, followed by limited proteolytic cleavage at different trypsin:protein ratios. The Pgp tryptic digestion products were separated by SDS-PAGE followed by immunodetection with the mouse monoclonal anti-Pgp antibody C219, which recognizes a conserved epitope (VVQE/AALD) in each half of the protein. Different trypsin digestion patterns were observed for wild-type Pgp incubated with MgCl(2) alone, MgADP, MgAMP.PNP, MgATP, and MgATP + vanadate. A unique trypsin digestion profile suggestive of enhanced resistance to trypsin was observed under conditions of vanadate-induced trapping of nucleotides (MgATP + vanadate). The trypsin sensitivity profiles of Pgp mutants bearing either single or double mutations in Walker A (K429R, K1072R) and Walker B (D551N, D1196N) sequence signatures of NBD1 and NBD2 were analyzed under conditions of vanadate-induced trapping of nucleotides. The proteolytic cleavage pattern observed for the double mutants K429R/K1072R and D551N/D1196N, and for the single mutants K429R, K1072R, and D1196N were similar and clearly distinct from wild-type Pgp under the same conditions. This is consistent with the absence of ATP hydrolysis and of vanadate-induced trapping of 8-azido-ADP previously reported for these mutants [Urbatsch et al. (1998) Biochemistry 37, 4592-4602]. Interestingly, the trypsin digestion profiles observed under vanadate-induced trapping for the D551N and D1196N mutants were quite different, with the D551N mutant showing a profile resembling that seen for wild-type Pgp. The different sensitivity profiles of Pgp mutants bearing mutations at the homologous residue in NBD1 (D551N) and NBD2 (D1196N) suggest possible structural and functional differences between the two sites.