Use of site-directed mutations in the individual Ca2(+)-binding sites of calmodulin to examine Ca2(+)-induced conformational changes

Mutant versions of the calmodulin of Drosophila melanogaster have been prepared for use in the study of Ca2+ binding and Ca2(+)-induced conformational changes. In each mutant, a conserved glutamic acid residue indicated to play a critical role in Ca2+ binding has been mutated to glutamine in one of the Ca2(+)-binding sites. Thus a series of four proteins, each with an analogous mutation in one of the four binding sites, has been generated. Here the Ca2(+)-induced conformational changes in these proteins have been examined by use of the fluorescent hydrophobic reporter molecule, 9-anthroyl choline. These studies confirm earlier work which indicates that the carboxyl-terminal pair of Ca2(+)-binding sites shows cooperative Ca2+ binding to produce a major conformational change in the protein. However, these studies provide evidence that the sites of the amino-terminal pair are more independent in their Ca2+ binding properties and contribute individually to the conformational changes associated with Ca2+ binding in the amino-terminal half of the protein. This work also indicates that mutation of either of the amino-terminal Ca2(+)-binding sites can influence the conformational change produced by Ca2+ binding to the carboxyl-terminal sites.     

Low-affinity Ca(2+)-binding sites versus Zn(2+)-binding sites in histidine-rich Ca(2+)-binding protein of skeletal muscle sarcoplasmic reticulum.

Histidine-rich Ca(2+)-binding protein (HRC) is a 170 kDa protein that can be identified in the isolated sarcoplasmic reticulum from rabbit skeletal muscle by its ability to bind [125I]low-density lipoprotein on blots after SDS-PAGE and that appears to be bound to the junctional membrane through calcium bridges. Molecular cDNA cloning of this protein predicts the existence of a Ca(2+)-binding domain and of a distinct heavy-metal binding domain at the cystein-rich COOH-terminus. Here we demonstrate, using radioactive ligand blot techniques, that HRC protein binds 45Ca at low affinity, as well as being able to bind 65Zn, but at different sites, that are largely inhibitable by prior reductive alkylation of the protein. In contrast to Ca(2+)-binding protein calsequestrin not having detectable 65Zn-binding sites, HRC protein bound selectively to immobilized Zn2+ on IDA-agarose affinity columns. Our results also indicate that rabbit and human 140 kDa HRC protein have common properties.     

Kinetic studies show that Ca2+ and Tb3+ have different binding preferences toward the four Ca2+-binding sites of calmodulin

The stepwise addition of Tb3+ to calmodulin yields a large tyrosine-sensitized Tb3+ luminescence enhancement as the third and fourth ions bind to the protein [Wang, C.-L. A., Aquaron, R. R., Leavis, P. C., & Gergely, J. (1982) Eur. J. Biochem. 124, 7-12]. Since the only tyrosine residues in calmodulin are located within binding sites III and IV, these results suggest that Tb3+ binds first to sites I and II. Recent NMR studies have provided evidence that Ca2+, on the other hand, binds preferentially to sites III and IV. Kinetic studies using a stopped-flow apparatus also show that the preferential binding of Ca2+ and lanthanide ions is different. Upon rapid mixing of 2Ca-calmodulin with two Tb3+ ions, there was a small and rapid tyrosine fluorescence change, but no Tb3+ luminescence was observed, indicating that Tb3+ binds to sites I and II but not sites III and IV. When two Tb3+ ions are mixed with 2Dy-calmodulin, Tb3+ luminescence rises rapidly as Tb3+ binds to the empty sites III and IV, followed by a more gradual decrease (k = 0.4 s-1 as the ions redistribute themselves over the four sites. These results indicate that (i) both Tb3+ and Dy3+ prefer binding to sites I and II of calmodulin and (ii) the binding of Tb3+ to calmodulin is not impeded by the presence of two Ca2+ ions initially bound to the protein. Thus, the Ca2+ and lanthanide ions must exhibit opposite preferences for the four sites of calmodulin: sites III and IV are the high-affinity sites for Ca2+, whereas Tb3+ and Dy3+ prefer sites I and II.     

Kinetics of the conformational change of skeletal troponin C induced by Ca2+-Mg2+ exchange at the high-affinity Ca2+-binding sites

The rate constant of the conformational change of skeletal troponin C (TnC) induced by the Ca2+ binding reaction with the high-affinity Ca2+-binding sites was determined in the presence of Mg2+ by the fluorescence stopped-flow method in 0.1 M KCl, 50 mM Na-cacodylate-HCl pH 7.0 at 20 degrees C. The [MgCl2] dependence of the rate constants of the observed biphasic conformational change leveled off at the high [MgCl2] region: the rate constants were 60 +/- 9 s-1 and 8 +/- 2 s-1, respectively. These values are larger than the rate constants of the biphasic fluorescence intensity change of TnC induced by Mg2+ removal reaction at the high-affinity Ca2+-binding sites (37 +/- 7 s-1 and 3.0 +/- 0.6 s-1) under the same experimental conditions. These results suggest that the Ca2+-Mg2+ exchange reaction at the high-affinity Ca2+-binding sites is faster than the resultant conformational change accompanying the fluorescence intensity change. Based on these results, we also reexamine the molecular kinetic mechanism of the conformational change of the protein induced by the Mg2+ binding or removal reaction with the high affinity Ca2+-binding sites of skeletal TnC.     

Nature of the individual Ca2+ binding sites in Ca2+-regenerated bacteriorhodopsin

The binding constants, K₁ and K₂, and the number of Ca²⁺ ions in each of the two high affinity sites of Ca²⁺-regenerated bacteriorhodopsin (bR) are determined potentiometrically at different pH values in the range of pH 3.5-4.5 by using the Scatchard plot method. From the pH dependence of K₁ and K₂, it was found that two hydrogen ions are released for each Ca²⁺ bound to each of the two high affinity sites. Furthermore, we have measured by a direct spectroscopic method the association constant, K_s, for the binding of Ca²⁺ to deionized bR, which is responsible for producing the blue to purple color change. Comparing the value of K_s and its pH dependence with those of K₁ and K₂ showed that the site corresponding to K_s is to be identified with that of K₂. This is in agreement with the conclusion reached previously, using a different approach, which showed that it is the second Ca²⁺ that causes the blue to purple color change.

Our studies also show that in addition to the two distinct high affinity sites, there are about four to six sites with lower binding constants. These are attributed to the nonspecific binding in bR.

     

Thermodynamics of cation binding to rabbit skeletal muscle calsequestrin. Evidence for distinct Ca(2+)- and Mg(2+)-binding sites

Ca2+ binding to rabbit skeletal calsequestrin was studied at physiological ionic strength by equilibrium flow dialysis, Hummel-Dryer gel filtration and microcalorimetry. 31 Ca(2+)-binding sites with a mean dissociation constant (KD) of 0.79 mM were titrated in the absence, and 23 sites with a KD of 0.88 mM in the presence of 3 mM Mg2+. No cooperativity was observed. For Mg2+ binding, the combination of gel filtration and microcalorimetry yielded a stoichiometry of 26 Mg2+/protein with a KD of 2mM. 1 mM Ca2+ decreased the stoichiometry to 20 Mg2+/protein. Binding of Ca2+ in the absence and presence of 3 mM Mg2+ was accompanied by a release of 2.0 and 2.7 H+/protein, respectively. Mg2+ binding did not lead to a significant proton release suggesting a qualitative difference in the Ca(2+)- and Mg(2+)-binding sites. After correction for proton release, the enthalpy change for Ca2+ binding was very low (-1.5 kJ/protein in the absence, and -15 kJ/protein in the presence of 3 mM Mg2+). The entropy change (+59 J/K.site in the absence and +56 J/K.site in the presence of Mg2+) was therefore virtually the sole driving force for Ca2+ binding. Mg2+ binding is slightly more exothermic (-12.6 kJ/protein), but as for Ca2+, the entropy change (+50 J/K.site) constituted the major driving force of the reaction. A fluorimetric study indicates that the conformation of tryptophan in Mg(2+)-saturated calsequestrin was clearly different from that in the Ca(2+)-saturated protein, but that the (Ca2+ + Mg2+)-saturated protein was not distinct from the Ca(2+)-saturated protein. Thus, in addition to the thermodynamic characterization of the Ca2+/calsequestrin interaction, our data indicate that Ca2+ and Mg2+ do not bind to the same sites on calsequestrin. The data also predict considerable proton fluxes upon Ca(2+)-Mg2+ exchange in vivo.     

Circular dichroism studies on calcium binding to two series of Ca2+ binding site mutants of Drosophila melanogaster calmodulin.

The Ca(2+)-induced structural changes in mutant calmodulins from Drosophila melanogaster have been studied by circular dichroism. The proteins comprise eight site-specific mutants, in which a bidentate glutamic acid (at position 12 in each Ca2+ binding loop) is replaced with either glutamine (BQ series) or lysine (BK series). Previous studies of these proteins indicate that Ca2+ binding at the mutated site is effectively eliminated by each of these substitutions, with additional effects at nonmutated sites. Circular dichroism has now been used to assess Ca(2+)-induced changes in secondary and tertiary structure in these proteins. In the absence of Ca2+, the helical content of these mutant calmodulins is close to that of the wild-type protein. In excess Ca2+, calmodulins with a mutation in the N-terminal sites show Ca(2+)-induced increases in helicity (CD at 222 nm) that are similar to those of the wild-type protein. In contrast, much less additional helix is induced by Ca2+ in calmodulins with mutations in the C-terminal sites, with the two mutations to site IV showing a particularly poor response. Ca(2+)-induced changes to the environment of the single tyrosine of Drosophila calmodulin (Tyr-138 in site IV of the C-terminal domain) have been monitored via CD at 280 nm. The signal from this residue is significantly altered in the Ca(2+)-free form of almost all these mutants, including those in the N-terminal domain. This indicates significant interaction between the N- and C-terminal domains of these mutants.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of Ca(2+)-binding properties of calmodulin nitrite-anion and hydrogen peroxide

The effect of NO2- and H2O2 on Ca(2+)-binding properties of calmodulin has been studied. The decrease of Ca2+ affinity for calmodulin and limiting quantity of the sites of cation cross-linking by the protein molecule under the effect of 1 nM NO2 and 10 nM H2O2. The increase of the acting substances concentration above physiological one results in the considerable decrease of the observed effects. The investigation results indicate to possible inhibition of Ca(2+)-calmodulin-dependent cytosole reaction of the smooth-muscle cells under the effect of NO2 and H2O2, and this will result in the myometrium relaxation. At the same time the data from literature and results of the authors' experiments allow supposing the difference in the mechanisms of NO2 and H2O2 actions on these processes.     

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.     

Ca2+ and Mg2+-binding and a putative calmodulin type Ca2+-binding site in Synechococcus Photosystem II

Thylakoids and Photosystem II particles prepared from the cyanobacterium Synechococcus PCC 7942 washed with a HEPES/glycerol buffer exhibited low rates of light-induced oxygen evolution. Addition of either Ca²⁺ or Mg²⁺ to both thylakoids and Photosystem II particles increased oxygen evolution independently, maximal rates being obtained by addition of both ions. If either preparation was washed with NaCl, light induced O₂ evolution was completely inhibited, but re-activated in the same manner by Ca²⁺ and Mg²⁺ but to a lower level. In the presence of Mg²⁺, the reactivation of O₂ evolution by Ca²⁺ allowed sigmoid kinetics, implying co-operative binding. The results are interpreted as indicating that not only Ca²⁺, but also Mg²⁺, is essential for light-induced oxygen evolution in thylakoids and Photosystem II particles from Synechococcus PC 7942. The significance of the reactivation kinetics is discussed. Reactivation by Ca²⁺ was inhibited by antibodies to mammalian calmodulin, indicating that the binding site in Photosystem II may be analogous to that of this protein.Abbreviation HEPES n-2-Hydroxyethylpiperazine--2-ethane sulphonic acid     

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.     

1H-NMR studies of calmodulin. The nature of the Ca2+-dependent conformational change

The replication of M13 single-stranded DNA by the 9S DNA polymerase alpha from calf thymus has been studied in vitro. Priming conditions, the nature of the replication products and conditions for optimal elongation have been investigated. Oligonucleotides comprising only four nucleotides can serve as primers. Both ribo and deoxy oligonucleotides can be elongated. Priming by the short oligonucleotides occurs at multiple sites on the M13 genome. If replication is primed at single sites with a specific pentadecamer or with RNA in the origin of replication, specific pausing sites are observed. These pausing sites can partly be correlated with secondary structures in the template DNA. Addition of Escherichia coli single-stranded DNA binding protein leads to a weakening of pausing sites and to the synthesis of longer products. The 9S enzyme is able to proceed through most of the pausing sites resulting in the synthesis of product molecules as long as 6600 nucleotides. The 9S DNA polymerase alpha contains a potent DNA primase activity which enables it to initiate replication on a single-stranded template in the presence of the four NTPs . However, priming is also possible in the presence of ATP alone. The priming sites are not randomly distributed over the M13 DNA.     

Ca(2+)-dependent and Ca(2+)-independent calmodulin binding sites in erythrocyte protein 4.1. Implications for regulation of protein 4.1 interactions with transmembrane proteins

In vitro protein binding assays identified two distinct calmodulin (CaM) binding sites within the NH(2)-terminal 30-kDa domain of erythrocyte protein 4.1 (4.1R): a Ca(2+)-independent binding site (A(264)KKLWKVCVEHHTFFRL) and a Ca(2+)-dependent binding site (A(181)KKLSMYGVDLHKAKDL). Synthetic peptides corresponding to these sequences bound CaM in vitro; conversely, deletion of these peptides from a 30-kDa construct reduced binding to CaM. Thus, 4.1R is a unique CaM-binding protein in that it has distinct Ca(2+)-dependent and Ca(2+)-independent high affinity CaM binding sites. CaM bound to 4.1R at a stoichiometry of 1:1 both in the presence and absence of Ca(2+), implying that one CaM molecule binds to two distinct sites in the same molecule of 4.1R. Interactions of 4.1R with membrane proteins such as band 3 is regulated by Ca(2+) and CaM. While the intrinsic affinity of the 30-kDa domain for the cytoplasmic tail of erythrocyte membrane band 3 was not altered by elimination of one or both CaM binding sites, the ability of Ca(2+)/CaM to down-regulate 4. 1R-band 3 interaction was abrogated by such deletions. Thus, regulation of protein 4.1 binding to membrane proteins by Ca(2+) and CaM requires binding of CaM to both Ca(2+)-independent and Ca(2+)-dependent sites in protein 4.1.     

Mapping of three unique Ca(2+)-binding sites in human annexin II.

Site-directed mutagenesis was employed to map and characterize Ca(2+)-binding sites in annexin II, a member of the annexin family of Ca(2+)- and phospholipid-binding proteins which serves as a major cellular substrate for the tyrosine kinase encoded by the src oncogene. Several single amino acid substitutions were introduced in the human annexin II and the various mutant proteins were scored for their affinity towards Ca2+ in different assays. The data support our previous finding [Thiel, C., Weber, K. and Gerke V. (1991) J. Biol. Chem. 266, 14,732-14,739] that a Ca(2+)-binding site is present in the third of the four repeat segments which comprise the 33-kDa protein core of annexin II. In addition to Gly206 and Thr207, which are localized in the highly conserved endonexin fold of the third repeat, Glu246 is involved in the formation of this site. Thus the architecture of this Ca(2+)-binding site in solution is very similar, if not identical, to that of Ca2+ sites identified recently in annexin V crystals [Huber, R., Schneider, M., Mayr, I., R?misch, J. and Paques, E.-P. (1990) FEBS Lett. 275, 15-21]. In addition to the site in repeat 3, we have mapped sites of presumably similar architecture in repeats 2 and 4 of annexin II. Again, an acidic amino acid which is located 40 residues C-terminal to the conserved glycine at position 4 of the endonexin fold is indispensable for high-affinity Ca2+ binding: Asp161 in the second and Asp321 in the fourth repeat. In contrast, repeat 1 does not contain an acidic amino acid at a corresponding position and also shows deviations from the other repeats in the sequence surrounding the conserved glycine. These results on annexin II together with the crystallographic information on annexin V reveal that annexins can differ in the position of the Ca2+ sites. Ca(2+)-binding sites of similar structure are present in repeats 2, 3, and 4 of annexin II while in annexin V they occur in repeats 1, 2, and 4. We also synthesized an annexin II derivative with mutations in all three Ca2+ sites. This molecule shows a greatly reduced affinity for the divalent cation. However, it is still able to bind Ca2+, indicating the presence of (an) additional Ca2+ site(s) of presumably different architecture.     

Ca2+ binding sites in calmodulin and troponin C alter interhelical angle movements

Molecular dynamics analyses were performed to examine conformational changes in the C-domain of calmodulin and the N-domain of troponin C induced by binding of Ca(2+) ions. Analyses of conformational changes in calmodulin and troponin C indicated that the shortening of the distance between Ca(2+) ions and Ca(2+) binding sites of helices caused widening of the distance between Ca(2+) binding sites of helices on opposite sides, while the hydrophobic side chains in the center of helices hardly moved due to their steric hindrance. This conformational change acts as the clothespin mechanism.     

A note on Ca2+ binding to calmodulin

Binding of Ca2+ to calmodulin has been simulated on the basis of a model that assumes two classes, two sites in each class, of Ca2+ binding sites. With properly chosen values of binding constants for the two classes of sites, and with the assumption that certain degree of positive cooperativity exists between the two sites in each class, the overall binding isotherm can be generated so that it appears to be a single-transition, non-cooperative binding curve of four equivalent sites. Thus this model offers a resolution for some of the discrepancies among Ca2+ binding studies of calmodulin.     

Low affinity Ca2+-binding sites of calcineurin B mediate conformational changes in calcineurin A

Limited proteolysis of calcineurin in the presence of Ca(2+) suggested that its calmodulin-binding domain, readily degraded by proteases, was unfolded while calcineurin B was compactly folded [Hubbard, M. J., and Klee, C. B. (1989) Biochemistry 28, 1868-1874]. Moreover, in the crystal structure of calcineurin, with the four Ca(2+) sites of calcineurin B occupied, the calmodulin-binding domain is not visible in the electron density map [Kissinger, C. R., et al. (1995) Nature 378, 641-644]. Limited proteolysis of calcineurin in the presence of EGTA, shows that, when the low affinity sites of calcineurin B are not occupied, the calmodulin-binding domain is completely protected against proteolytic attack. Slow cleavages are, however, detected in the linker region between the calmodulin-binding and the autoinhibitory domains of calcineurin A. Upon prolonged exposure to the protease, selective cleavages in carboxyl-terminal end of the first helix and the central helix linker of calcineurin B and the calcineurin B-binding helix of calcineurin A are also detected. Thus, Ca(2+) binding to the low-affinity sites of calcineurin B affects the conformation of calcineurin B and induces a conformational change of the regulatory domain of calcineurin A, resulting in the exposure of the calmodulin-binding domain. This conformational change is needed for the partial activation of the enzyme in the absence of calmodulin and its full activation by calmodulin. A synthetic peptide corresponding to the calmodulin-binding domain is shown to interact with a peptide corresponding to the calcineurin B-binding domain, and this interaction is prevented by calcineurin B in the presence but not the absence of Ca(2+). These observations provide a mechanism to explain the dependence on Ca(2+) binding to calcineurin B for calmodulin activation and for the 10-20-fold increase in affinity of calcineurin for Ca(2+) upon removal of the regulatory domain by limited proteolysis [Stemmer, P. M., and Klee, C. B. (1994) Biochemistry 33, 6859-6866].     

Volume changes upon addition of Ca2+ to calmodulin: Ca2+-calmodulin conformational states

Measurement of the volume change by a rapid density method upon sequential addition of calcium ion to calmodulin showed relatively large, nonuniform increases for the first 4 moles Ca2+ per mole calmodulin. Substantially larger volume increases (approximately 15 ml/mol protein) were observed upon addition of the second and fourth moles Ca2+ relative to the first and third moles added per mole calmodulin. A total volume increase of approximately 170 ml/mol protein attended the addition of 4 moles Ca2+, as expected for multidentate carboxylate coordination to metal ion. Marginal changes in volume were observed upon further additions, the data showing a remarkably sharp transition after [Ca2+]/[calmodulin] = 4. The results are consistent with an ordered binding of Ca2+ in which pair-wise additions produce similar volume changes; the volume change behavior, however, does not indicate an absence of distinct conformational states for a Ca2+(1)-calmodulin and a Ca2+(3)-calmodulin complex as has been proposed on the basis of 1H-NMR evidences.