Calcium-induced folding of a fragment of calmodulin composed of EF-hands 2 and 3

Calmodulin (CaM) is an EF-hand protein composed of two calcium (Ca(2+))-binding EF-hand motifs in its N-domain (EF-1 and EF-2) and two in its C-domain (EF-3 and EF-4). In this study, we examined the structure, dynamics, and Ca(2+)-binding properties of a fragment of CaM containing only EF-2 and EF-3 and the intervening linker sequence (CaM2/3). Based on NMR spectroscopic analyses, Ca(2+)-free CaM2/3 is predominantly unfolded, but upon binding Ca(2+), adopts a monomeric structure composed of two EF-hand motifs bridged by a short antiparallel beta-sheet. Despite having an "even-odd" pairing of EF-hands, the tertiary structure of CaM2/3 is similar to both the "odd-even" paired N- and C-domains of Ca(2+)-ligated CaM, with the conformationally flexible linker sequence adopting the role of an inter-EF-hand loop. However, unlike either CaM domain, CaM2/3 exhibits stepwise Ca(2+) binding with a K (d1) = 30 +/- 5 microM to EF-3, and a K (d2) > 1000 microM to EF-2. Binding of the first equivalent of Ca(2+) induces the cooperative folding of CaM2/3. In the case of native CaM, stacking interactions between four conserved aromatic residues help to hold the first and fourth helices of each EF-hand domain together, while the loop between EF-hands covalently tethers the second and third helices. In contrast, these aromatic residues lie along the second and third helices of CaM2/3, and thus are positioned adjacent to the loop between its "even-odd" paired EF-hands. This nonnative hydrophobic core packing may contribute to the weak Ca(2+) affinity exhibited by EF-2 in the context of CaM2/3.     

Apoptosis-linked gene product ALG-2 is a new member of the calpain small subunit subfamily of Ca2+-binding proteins.

ALG-2 is a newly discovered Ca2+-binding protein which has been demonstrated to be directly linked to apoptosis. Structurally, ALG-2 is expressed as a single polypeptide chain corresponding to a 22 kDa protein containing five putative EF-hand Ca2+-binding sites. In this work, we have developed an efficient expression and purification scheme for recombinant ALG-2. Utilizing this protocol, we can routinely obtain purified recombinant protein with a yield of approximately 100 mg per liter of bacterial cell cultures. Gel filtration and chemical cross-linking experiments have shown that Ca2+-free ALG-2 forms a weak homodimer in solution. Biochemical and spectroscopic studies of truncated and point mutants of ALG-2 demonstrated that the fifth EF-hand Ca2+-binding motif is likely to participate in the formation of the dimer complex. Experimentally, both the amino- and carboxyl-terminal truncated mutants of ALG-2 have shown their ability to retain the structural, as well as, Ca2+-binding integrity when individually expressed in bacteria. In this respect, the N-terminal domain encompasses the first two EF-hands, and the C-terminal domain contains the remaining three EF-hands. Combining mutagenesis and spectroscopic studies, we showed that ALG-2 possesses two strong Ca2+-binding sites. Employing fluorescence spectroscopy and circular dichroism, we showed that the binding of Ca2+ to ALG-2 induced significant conformational changes in both the N-terminal and C-terminal domains of the protein. Furthermore, our studies demonstrated that Ca2+ binding to both strong Ca2+-binding sites of ALG-2 is required for ion-induced aggregation of the protein. We also report here the expression, purification, and partial characterization of a Ca2+-binding-deficient ALG-2 mutant (Glu47Ala/Glu114Ala). In light of its much decreased affinity for Ca2+, this mutant could prove to be instrumental in elucidating the Ca2+-mediated function of ALG-2 within the context of its cellular environment.     

Structures and metal-ion-binding properties of the Ca2+-binding helix-loop-helix EF-hand motifs

The 'EF-hand' Ca2+-binding motif plays an essential role in eukaryotic cellular signalling, and the proteins containing this motif constitute a large and functionally diverse family. The EF-hand is defined by its helix-loop-helix secondary structure as well as the ligands presented by the loop to bind the Ca2+ ion. The identity of these ligands is semi-conserved in the most common (the 'canonical') EF-hand; however, several non-canonical EF-hands exist that bind Ca2+ by a different co-ordination mechanism. EF-hands tend to occur in pairs, which form a discrete domain so that most family members have two, four or six EF-hands. This pairing also enables communication, and many EF-hands display positive co-operativity, thereby minimizing the Ca2+ signal required to reach protein saturation. The conformational effects of Ca2+ binding are varied, function-dependent and, in some cases, minimal, but can lead to the creation of a protein target interaction site or structure formation from a molten-globule apo state. EF-hand proteins exhibit various sensitivities to Ca2+, reflecting the intrinsic binding ability of the EF-hand as well as the degree of co-operativity in Ca2+ binding to paired EF-hands. Two additional factors can influence the ability of an EF-hand to bind Ca2+: selectivity over Mg2+ (a cation with very similar chemical properties to Ca2+ and with a cytoplasmic concentration several orders of magnitude higher) and interaction with a protein target. A structural approach is used in this review to examine the diversity of family members, and a biophysical perspective provides insight into the ability of the EF-hand motif to bind Ca2+ with a wide range of affinities.     

The primary structure of a new Mr 18,000 calcium vector protein from amphioxus

The amino acid sequence of a new Ca2+-binding protein (CaVP) from Amphioxus muscle (Cox, J. A., J. Biol. Chem. 261, 13173-13178) has been determined. The protein contains 161 amino acid residues and has a molecular weight of 18,267. The N terminus is blocked by an acetyl group. The two functional Ca2+-binding sites have been localized based on homology with known Ca2+-binding domains, on internal homology and on secondary structure prediction, and appear to be the domains III and IV. The C-terminal half of CaVP, which contains the two Ca2+-binding sites, shows a remarkable similarity with human brain calmodulin (45%) and with rabbit skeletal troponin C (40%). Functional domain III contains 2 epsilon-N-trimethyllysine residues in the alpha-helices flanking the Ca2+-binding loop. Sequence determination revealed two abortive Ca2+-binding domains in the N-terminal half of CaVP with a similarity of 24 and 30% as compared with calmodulin and troponin C, respectively. This half is also characterized by the presence of a disulfide bridge linking the N-terminal helix of domain I to the C-terminal helix of domain II. This disulfide bond is very resistant to reduction in the native state, but not in denatured CaVP. The optically interesting aromatic chromophores (2 tryptophan and 1 tyrosine residues) are all located in the nonfunctional domain II.     

Structural changes in the C-terminus of Ca2+-bound rat S100B (beta beta) upon binding to a peptide derived from the C-terminal regulatory domain of p53.

S100B(beta beta) is a dimeric Ca2+-binding protein that interacts with p53, inhibits its phosphorylation by protein kinase C (PKC) and promotes disassembly of the p53 tetramer. Likewise, a 22 residue peptide derived from the C-terminal regulatory domain of p53 has been shown to interact with S100B(beta beta) in a Ca2+-dependent manner and inhibits its phosphorylation by PKC. Hence, structural studies of Ca2+-loaded S100B(beta beta) bound to the p53 peptide were initiated to characterize this interaction. Analysis of nuclear Overhauser effect (NOE) correlations, amide proton exchange rates, 3J(NH-H alpha) coupling constants, and chemical shift index data show that, like apo- and Ca2+-bound S100B(beta beta), S100B remains a dimer in the p53 peptide complex, and each subunit has four helices (helix 1, Glu2-Arg20; helix 2, Lys29-Asn38; helix 3, Gln50-Asp61; helix 4, Phe70-Phe87), four loops (loop 1, Glu21-His25; loop 2, Glu39-Glu49; loop 3, Glu62-Gly66; loop 4, Phe88-Glu91), and two beta-strands (beta-strand 1, Lys26-Lys28; beta-strand 2, Glu67-Asp69), which forms a short antiparallel beta-sheet. However, in the presence of the p53 peptide helix 4 is longer by five residues than in apo- or Ca2+-bound S100B(beta beta). Furthermore, the amide proton exchange rates in helix 3 (K55, V56, E58, T59, L60, D61) are significantly slower than those of Ca2+-bound S100B(beta beta). Together, these observations plus intermolecular NOE correlations between the p53 peptide and S100B(beta beta) support the notion that the p53 peptide binds in a region of S100B(beta beta), which includes residues in helix 2, helix 3, loop 2, and the C-terminal loop, and that binding of the p53 peptide interacts with and induces the extension of helix 4.     

Calcium-binding by p26olf, an S100-like protein in the frog olfactory epithelium.

Frog p26olf is a novel S100-like Ca2+-binding protein found in olfactory cilia. It consists of two S100-like domains aligned sequentially, and has a total of four Ca2+-binding sites (known as EF-hands). In this study, to elucidate the mechanism of Ca2+-binding to each EF-hand (named EF-A, -B, -C and -D from the N-terminus of p26olf), we examined Ca2+-binding in wild-type p26olf and also in its mutants in which a glutamate at the -z coordinate position within each Ca2+-binding loop was substituted for a glutamine. Flow dialysis experiments showed that the wild-type binds nearly four Ca2+ per molecule maximally, while all the mutants bind approximately three Ca2+. Although EF-B and -D are p26olf-specific EF-hands and their role in Ca2+-binding is not known, the result unequivocally showed that they actually bind Ca2+. The overall Ca2+-binding affinity decreased in the three mutants. The decrease was very large in the mutants of EF-A and -B, which suggested that the Ca2+-affinities are high in EF-A and -B in the wild-type. Assuming the presence of four steps of Ca2+-binding, we determined the dissociation constant of each step in wild-type p26olf. To assign which step takes place at which EF-hand, we measured the antagonistic effect of K+ on each step, as the effect of K+ is thought to be a function of the number of the carboxyl groups in an EF-hand. Although the actual Ca2+-binding mechanism may not be so simple, this study together with the mutation study suggested a tentative Ca2+-binding model of p26olf: the order of Ca2+-binding to p26olf is EF-B, EF-A, EF-C and EF-D. Based on these results, we speculate that similar Ca2+-binding takes place in an S100 dimer.     

A novel calmodulin-Ca2+ target recognition activates the Bcl-2 regulator FKBP38

The FK506-binding protein 38 (FKBP38) affects neuronal apoptosis control by suppressing the anti-apoptotic function of Bcl-2. The direct interaction between FKBP38 and Bcl-2, however, requires a prior activation of FKBP38 by the Ca2+ sensor calmodulin (CaM). Here we demonstrate for the first time that the formation of a complex between FKBP38 and CaM-Ca2+ involves two separate interaction sites, thus revealing a novel scenario of target protein regulation by CaM-Ca2+. The C-terminal FKBP38 residues Ser290-Asn313 bind to the target protein-binding cleft of the Ca2+-coordinated C-terminal CaM domain, thereby enabling the N-terminal CaM domain to interact with the catalytic domain of FKBP38 in a Ca2+-independent manner. Only the latter interaction between the catalytic FKBP38 domain and the N-terminal CaM domain activates FKBP38 and, as a consequence, also regulates Bcl-2.     

Molecular modeling of single polypeptide chain of calcium-binding protein p26olf from dimeric S100B(betabeta).

P26olf from olfactory tissue of frog, which may be involved in olfactory transduction or adaptation, is a Ca2+-binding protein with 217 amino acids. The p26olf molecule contains two homologous parts consisting of the N-terminal half with amino acids 1-109 and the C-terminal half with amino acids 110-217. Each half resembles S100 protein with about 100 amino acids and contains two helix-loop-helix Ca2+-binding structural motifs known as EF-hands: a normal EF-hand at the C-terminus and a pseudo EF-hand at the N-terminus. Multiple alignment of the two S100-like domains of p26olf with 18 S100 proteins indicated that the C-terminal putative EF-hand of each domain contains a four-residue insertion when compared with the typical EF-hand motifs in the S100 protein, while the N-terminal EF-hand is homologous to its pseudo EF-hand. We constructed a three-dimensional model of the p26olf molecule based on results of the multiple alignment and NMR structures of dimeric S100B(betabeta) in the Ca2+-free state. The predicted structure of the p26olf single polypeptide chain satisfactorily adopts a folding pattern remarkably similar to dimeric S100B(betabeta). Each domain of p26olf consists of a unicornate-type four-helix bundle and they interact with each other in an antiparallel manner forming an X-type four-helix bundle between the two domains. The two S100-like domains of p26olf are linked by a loop with no steric hindrance, suggesting that this loop might play an important role in the function of p26olf. The circular dichroism spectral data support the predicted structure of p26olf and indicate that Ca2+-dependent conformational changes occur. Since the C-terminal putative EF-hand of each domain fully keeps the helix-loop-helix motif having a longer Ca2+-binding loop, regardless of the four-residue insertion, we propose that it is a new, novel EF-hand, although it is unclear whether this EF-hand binds Ca2+. P26olf is a new member of the S100 protein family.     

Apo-calmodulin binds with its C-terminal domain to the N-methyl-D-aspartate receptor NR1 C0 region

Calmodulin (CaM) is the major Ca2+ sensor in eukaryotic cells. It consists of four EF-hand Ca2+ binding motifs, two in its N-terminal domain and two in its C-terminal domain. Through a negative feedback loop, CaM inhibits Ca2+ influx through N-methyl-D-aspartate-type glutamate receptors in neurons by binding to the C0 region in the cytosolic tail of the NR1 subunit. Ca2+ -depleted (apo)CaM is pre-associated with a variety of ion channels for fast and effective regulation of channel activities upon Ca2+ influx. Using the NR1 C0 region for fluorescence and circular dichroism spectroscopy studies we found that not only Ca2+ -saturated CaM but also apoCaM bound to NR1 C0. In vitro interaction assays showed that apoCaM also binds specifically to full-length NR1 solubilized from rat brain and to the complete C terminus of the NR1 splice form that contains the C0 plus C2' domain. The Ca2+ -independent interaction of CaM was also observed with the isolated C-but not N-terminal fragment of calmodulin in the independent spectroscopic assays. Fluorescence polarization studies indicated that apoCaM associated via its C-terminal domain with NR1 C0 in an extended conformation and collapsed to adopt a more compact conformation of faster rotational mobility in its complex with NR1 C0 upon addition of Ca2+. Our results indicate that apoCaM is associated with NR1 and that the complex of CaM bound to NR1 C0 undergoes a dramatic conformational change when Ca2+ binds to CaM.     

X-ray Structures of Magnesium and Manganese Complexes with the N-Terminal Domain of Calmodulin: Insights into the Mechanism and Specificity of Metal Ion Binding to an EF-Hand

Calmodulin (CaM), a member of the EF-hand superfamily, regulates many aspects of cell function by responding specifically to micromolar concentrations of Ca(2+) in the presence of an ～1000-fold higher concentration of cellular Mg(2+). To explain the structural basis of metal ion binding specificity, we have determined the X-ray structures of the N-terminal domain of calmodulin (N-CaM) in complexes with Mg(2+), Mn(2+), and Zn(2+). In contrast to Ca(2+), which induces domain opening in CaM, octahedrally coordinated Mg(2+) and Mn(2+) stabilize the closed-domain, apo-like conformation, while tetrahedrally coordinated Zn(2+) ions bind at the protein surface and do not compete with Ca(2+). The relative positions of bound Mg(2+) and Mn(2+) within the EF-hand loops are similar to those of Ca(2+); however, the Glu side chain at position 12 of the loop, whose bidentate interaction with Ca(2+) is critical for domain opening, does not bind directly to either Mn(2+) or Mg(2+), and the vacant ligand position is occupied by a water molecule. We conclude that this critical interaction is prevented by specific stereochemical constraints imposed on the ligands by the EF-hand β-scaffold. The structures suggest that Mg(2+) contributes to the switching off of calmodulin activity and possibly other EF-hand proteins at the resting levels of Ca(2+). The Mg(2+)-bound N-CaM structure also provides a unique view of a transiently bound hydrated metal ion and suggests a role for the hydration water in the metal-induced conformational change.     

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

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

Peptide binding by a fragment of calmodulin composed of EF-hands 2 and 3

Calmodulin (CaM) is composed of two EF-hand domains tethered by a flexible linker. Upon Ca2+-binding, a fragment of CaM encompassing EF-hands 2 and 3 (CaM2/3; residues 46-113) folds into a structure remarkably similar to the N- and C-domains of CaM. In this study, we demonstrate that Ca2+-ligated CaM2/3 can also bind to a peptide representing the CaM-recognition sequence of skeletal muscle myosin light chain kinase (M13) with an equimolar stoichiometry and a dissociation constant of 0.40 +/- 0.05 microM. On the basis of an analytical ultracentrifugation measurement, the resulting complex exists as an equilibrium mixture of 2:2 heterotetrameric and 1:1 heterodimeric species. Chemical shift perturbation mapping indicates that, similar to CaM, the peptide associates with a hydrophobic groove crossing both EF-hands in CaM2/3. However, upon binding the M13 peptide, many residues in CaM2/3 yielded two equal intensity NMR signals with the same 15N relaxation properties. Thus, the 2:2 CaM2/3-M13 tetramer, which predominates under the conditions used for these studies, is asymmetric with each component adopting spectroscopically distinguishable conformations within the complex. CaM2/3 also weakly stimulates the phosphatase activity of calcineurin and inhibits stimulation by native CaM. These studies highlight the remarkable plasticity of EF-hand association and expand the diverse repertoire of mechanisms possible for CaM-target protein interactions.     

The N-terminal portion of the main cytosolic loop mediates K+ sensitivity in the retinal rod Na+/Ca2+-K+-exchanger.

Two types of Na+/Ca2+-exchangers have been characterized in the literature: The first is the cardiac, skeletal muscle and brain type, which exchanges 1 Ca2+ for 3 Na+, the second, found in retinal photosensor cells, transports 1 Ca2+ and 1 K+ in exchange for 4 Na+. The present work describes the properties of chimeric constructs of the two exchanger types. Ca2+ gel overlay experiments have identified a high affinity (Kd in the 1 microM range) Ca2+-binding domain between Glu601 and Asp733 in the main cytosolic loop of the retinal protein, just after transmembrane domain 5. Insertion of the retinal Ca2+-binding domain in the cytosolic loop of the cardiac exchanger conferred K+-dependence to the Ca2+ uptake activity of the chimeric constructs expressed in HeLa cells. The apparent Km of the K+ effect was about 1 mM. Experiments with C-terminally truncated versions of the retinal insert indicated that the sequence between Leu643 and Asp733 was critical in mediating K+ sensitivity of the recombinant chimeras. Thus, the high affinity Ca2+-binding domain in the main cytosolic loop of the retinal exchanger may regulate the activity of the retinal protein by binding Ca2+, and by conferring to it K+ sensitivity.     

The two IQ-motifs and Ca2+/calmodulin regulate the rat myosin 1d ATPase activity

The light chain binding domain of rat myosin 1d consists of two IQ-motifs, both of which bind the light chain calmodulin (CaM). To analyze the Myo1d ATPase activity as a function of the IQ-motifs and Ca2+/CaM binding, we expressed and affinity purified the Myo1d constructs Myo1d-head, Myo1d-IQ1, Myo1d-IQ1.2, Myo1d-IQ2 and Myo1dDeltaLV-IQ2. IQ1 exhibited a high affinity for CaM both in the absence and presence of free Ca2+. IQ2 had a lower affinity for CaM in the absence of Ca2+ than in the presence of Ca2+. The actin-activated ATPase activity of Myo1d was approximately 75% inhibited by Ca2+-binding to CaM. This inhibition was observed irrespective of whether IQ1, IQ2 or both IQ1 and IQ2 were fused to the head. Based on the measured Ca2+-dependence, we propose that Ca2+-binding to the C-terminal pair of high affinity sites in CaM inhibits the Myo1d actin-activated ATPase activity. This inhibition was due to a conformational change of the C-terminal lobe of CaM remaining bound to the IQ-motif(s). Interestingly, a similar but Ca2+-independent inhibition of Myo1d actin-activated ATPase activity was observed when IQ2, fused directly to the Myo1d-head, was rotated through 200 degrees by the deletion of two amino acids in the lever arm alpha-helix N-terminal to the IQ-motif.     

The kinetics of Ca(2+)-dependent switching in a calmodulin-IQ domain complex

We have performed a kinetic analysis of Ca2+-dependent switching in the complex between calmodulin (CaM) and the IQ domain from neuromodulin, and have developed detailed kinetic models for this process. Our results indicate that the affinity of the C-ter Ca2+-binding sites in bound CaM is reduced due to a approximately 10-fold decrease in the Ca2+ association rate, while the affinity of the N-ter Ca2+-binding sites is increased due to a approximately 3-fold decrease in the Ca2+ dissociation rate. Although the Ca2+-free and Ca2+-saturated forms of the CaM-IQ domain complex have identical affinities, CaM dissociates approximately 100 times faster in the presence of Ca2+. Furthermore, under these conditions CaM can be transferred to the CaM-binding domain from CaM kinase II via a ternary complex. These properties are consistent with the hypothesis that CaM bound to neuromodulin comprises a localized store that can be efficiently delivered to neuronal proteins in its Ca2+-bound form in response to a Ca2+ signal.     

Target-induced conformational adaptation of calmodulin revealed by the crystal structure of a complex with nematode Ca(2+)/calmodulin-dependent kinase kinase peptide.

Calmodulin (CaM) is a ubiquitous calcium (Ca(2+)) sensor which binds and regulates protein serine/threonine kinases along with many other proteins in a Ca(2+)-dependent manner. For this multi-functionality, conformational plasticity is essential; however, the nature and magnitude of CaM's plasticity still remains largely undetermined. Here, we present the 1.8 A resolution crystal structure of Ca(2+)/CaM, complexed with the 27-residue synthetic peptide corresponding to the CaM-binding domain of the nematode Caenorhabditis elegans Ca(2+)/CaM-dependent kinase kinase (CaMKK). The peptide bound in this crystal structure is a homologue of the previously NMR-derived complex with rat CaMKK, but benefits from improved structural resolution. Careful comparison of the present structure to previous crystal structures of CaM complexed with unrelated peptides derived from myosin light chain kinase and CaM kinase II, allow a quantitative analysis of the differences in the relative orientation of the N and C-terminal domains of CaM, defined as a screw axis rotation angle ranging from 156 degrees to 196 degrees. The principal differences in CaM interaction with various peptides are associated with the N-terminal domain of CaM. Unlike the C-terminal domain, which remains unchanged internally, the N-terminal domain of CaM displays significant differences in the EF-hand helix orientation between this and other CaM structures. Three hydrogen bonds between CaM and the peptide (E87-R336, E87-T339 and K75-T339) along with two salt bridges (E11-R349 and E114-K334) are the most probable determinants for the binding direction of the CaMKK peptide to CaM.     

Coupling of ligand binding and dimerization of helix-loop-helix peptides: spectroscopic and sedimentation analyses of calbindin D9k EF-hands

Isolated Ca2+-binding EF-hand peptides have a tendency to dimerize. This study is an attempt to account for the coupled equilibria of Ca2+-binding and peptide association for two EF-hands with strikingly different loop sequence and net charge. We have studied each of the two separate EF-hand fragments from calbindin D9k. A series of Ca2+-titrations at different peptide concentrations were monitored by CD and fluorescence spectroscopy. All data were fitted simultaneously to both a complete model of all possible equilibrium intermediates and a reduced model not including dimerization in the absence of Ca2+. Analytical ultracentrifugation shows that the peptides may occur as monomers or dimers depending on the solution conditions. Our results show strikingly different behavior for the two EF-hands. The fragment containing the N-terminal EF-hand shows a strong tendency to dimerize in the Ca2+-bound state. The average Ca2+-affinity is 3.5 orders of magnitude lower than for the intact protein. We observe a large apparent cooperativity of Ca2+ binding for the overall process from Ca2+-free monomer to fully loaded dimer, showing that a Ca2+-free EF-hand folds upon dimerization to a Ca2+-bound EF-hand, thereby presenting a preformed binding site to the second Ca2+-ion. The C-terminal EF-hand shows a much smaller tendency to dimerize, which may be related to its larger net negative charge. In spite of the differences in dimerization behavior, the Ca2+ affinities of both EF-hand fragments are similar and in the range lgK = 4.6-5.3.     

Refined crystal structure of troponin C from turkey skeletal muscle at 2.0 A resolution

The crystal structure of troponin C from turkey skeletal muscle has been refined at 2.0 A resolution (1 A = 0.1 nm). The resulting crystallographic R factor (R = sigma[[Fo[-[Fc[[/sigma[Fo[, where [Fo[ and [Fc[ are the observed and calculated structure factor amplitudes) is 0.155 for the 8054 reflections with intensities I greater than or equal to 2 sigma(I) within the 10 A to 2.0 A resolution range. With 66% of the residues in helical conformation, troponin C provides a good sample for helix analysis. The mean alpha-helix dihedral angles (phi, psi = -62 degrees, -42 degrees) agree with values observed for helical regions in other proteins. The helices are all curved and/or kinked. In particular, the 31 amino acid long inter-domain helix is smoothly curved, with a rather large radius of curvature of 137 A. Helix packing is different in the Ca2+-free domain (N-terminal) and the Ca2+-bound domain (C-terminal). The inter-helix angles for the two helix-loop-helix motifs in the regulatory domain are 133 degrees and 151 degrees, whereas the value for the two motifs in the C-terminal domain is 110 degrees, as observed in the EF-hands of parvalbumin. These differences affect the packing of the respective hydrophobic cores of each domain, in particular the disposition of aromatic rings. Pairwise arrangement of Ca2+-binding loops is common to both states, but the conformation is markedly different. Conversion of one to the other can be achieved by small cumulative changes of main-chain dihedral angles. The integrity of loop structure is maintained by numerous electrostatic interactions. Both salt bridges and carboxyl-carboxylate interactions are observed in TnC. There are more intramolecular (9) than intermolecular (1) salt bridges. Carboxyl-carboxylate interactions occur because the pH of the crystals is 5.0 and there is a multitude of aspartate and glutamate residues. One is intramolecular and four are intermolecular. Polar side-chain interactions occur more commonly with main-chain carbonyls and amides than with other polar side-chains. These interactions are mostly short range, and are similar to those observed in other proteins with one exception: negatively charged side-chains interact more frequently with main-chain carbonyl oxygen atoms. However, out of 19 such interactions, 10 involve oxygen atoms of the Ca2+ ligands. These unfavorable interactions are compensated by the favorable interactions with the Ca2+ ions and with main-chain amides. They are a trivial consequence of the tight fold of the Ca2+-binding loops.     

Structural basis for diversity of the EF-hand calcium-binding proteins

The calcium binding proteins of the EF-hand super-family are involved in the regulation of all aspects of cell function. These proteins exhibit a great diversity of composition, structure, Ca2+-binding and target interaction properties. Here, our current understanding of the Ca2+-binding mechanism is assessed. The structures of the EF-hand motifs containing 11-14 amino acid residues in the Ca2+-binding loop are analyzed within the framework of the recently proposed two-step Ca2+-binding mechanism. A hypothesis is put forward that in all EF-hand proteins the Ca2+-binding and the resultant conformational responses are governed by the central structure connecting the Ca2+-binding loops in the two-EF-hand domain. This structure, named EFbeta-scaffold, defines the position of the bound Ca2+, and coordinates the function of the N-terminal (variable and flexible) with the C-terminal (invariable and rigid) parts of the Ca2+-binding loop. It is proposed that the nature of the first ligand of the Ca2+-binding loop is an important determinant of the conformational change. Additional factors, including the interhelical contacts, the length, structure and flexibility of the linker connecting the EF-hand motifs, and the overall energy balance provide the fine-tuning of the Ca2+-induced conformational change in the EF-hand proteins.     

Protein Ser/Thr phosphatases PPEF interact with calmodulin

Regulation of protein dephosphorylation by cytoplasmic Ca(2+) levels and calmodulin (CaM) is well established and considered to be mediated solely by calcineurin. Yet, recent identification of protein phosphatases with EF-hand domains (PPEF/rdgC) point to the existence of another group of Ca(2+)-dependent protein phosphatases. We have recently hypothesised that PPEF/rdgC phosphatases might possess CaM-binding sites of the IQ-type in their N-terminal domains. We now employed yeast two-hybrid system and surface plasmon resonance (SPR) to test this hypothesis. We found that entire human PPEF2 interacts with CaM in the in vivo tests and that its N-terminal domain binds to CaM in a Ca(2+)-dependent manner with nanomolar affinity in vitro. The fragments corresponding to the second exons of PPEF1 and PPEF2, containing the IQ motifs, are sufficient for specific Ca(2+)-dependent interaction with CaM both in vivo and in vitro. These findings demonstrate the existence of mammalian CaM-binding protein Ser/Thr phosphatases distinct from calcineurin and suggest that the activity of PPEF phosphatases may be controlled by Ca(2+) in a dual way: via C-terminal Ca(2+)-binding domain and via interaction of the N-terminal domain with CaM.