The W105G and W99G sorcin mutants demonstrate the role of the D helix in the Ca(2+)-dependent interaction with annexin VII and the cardiac ryanodine receptor

Sorcin, a 21.6 kDa two-domain penta-EF-hand (PEF) protein, when activated by Ca(2+) binding, interacts with target proteins in a largely uncharacterized process. The two physiological EF-hands EF3 and EF2 do not belong to a structural pair but are connected by the D helix. To establish whether this helix is instrumental in sorcin activation, two D helix residues were mutated: W105, located near EF3 and involved in a network of interactions, and W99, located near EF2 and facing solvent, were substituted with glycine. Neither mutation alters calcium affinity. The interaction of the W105G and W99G mutants with annexin VII and the cardiac ryanodine receptor (RyR2), requiring the sorcin N-terminal and C-terminal domain, respectively, was studied. Surface plasmon resonance experiments show that binding of annexin VII to W99G occurs at the same Ca(2+) concentration as that of the wild type, whereas W105G requires a significantly higher Ca(2+) concentration. Ca(2+) spark activity of isolated heart cells monitors the sorcin-RyR2 interaction and is unaltered by W105G but is reduced equally by W99G and the wild type. Thus, substitution of W105, via disruption of the network of D helix interactions, affects the capacity of sorcin to recognize and interact with either target at physiological Ca(2+) concentrations, while mutation of solvent-facing W99 has little effect. The D helix appears to amplify the localized structural changes that occur at EF3 upon Ca(2+) binding and thereby trigger a structural rearrangement that enables interaction of sorcin with its molecular targets. The same activation process may apply to other PEF proteins in view of the D helix conservation.     

Crystal structure of calcium-free human sorcin: A member of the penta-EF-hand protein family

Sorcin is a 22 kD calcium-binding protein that is found in a wide variety of cell types, such as heart, muscle, brain and adrenal medulla. It belongs to the penta-EF-hand (PEF) protein family, which contains five EF-hand motifs that associate with membranes in a calcium-dependent manner. Prototypic members of this family are the calcium-binding domains of calpain, such as calpain dVI. Full-length human sorcin has been crystallized in the absence of calcium and the structure determined at 2.2 A resolution. Apart from an extended N-terminal portion, the sorcin molecule has a globular shape. The C-terminal domain is predominantly alpha-helical, containing eight alpha-helices and connecting loops incorporating five EF hands. Sorcin forms dimers through the association of the unpaired EF5, confirming this as the mode of association in the dimerization of PEF proteins. Comparison with calpain dVI reveals that the general folds of the individual EF-hand motifs are conserved, especially that of EF1, the novel EF-hand motif characteristic of the family. Detailed structural comparisons of sorcin with other members of PEF indicate that the EF-hand pair EF1-EF2 is likely to correspond to the two physiologically relevant calcium-binding sites and that the calcium-induced conformational change may be modest and localized within this pair of EF-hands. Overall, the results derived from the structural observations support the view that, in sorcin, calcium signaling takes place through the first pair of EF-hands.     

Calcium-binding properties of wild-type and EF-hand mutants of S100B in the presence and absence of a peptide derived from the C-terminal negative regulatory domain of p53

S100B is a dimeric Ca(2+)-binding protein that undergoes a 90 +/- 3 degrees rotation of helix 3 in the typical EF-hand domain (EF2) upon the addition of calcium. The large reorientation of this helix is a prerequisite for the interaction between each subunit of S100B and target proteins such as the tumor suppressor protein, p53. In this study, Tb(3+) was used as a probe to examine how binding of a 22-residue peptide derived from the C-terminal regulatory domain of p53 affects the rate of Ca(2+) ion dissociation. In competition studies with Tb(3+), the dissociation rates of Ca(2+) (k(off)) from the EF2 domains of S100B in the absence and presence of the p53 peptide was determined to be 60 and 7 s(-)(1), respectively. These data are consistent with a previously reported result, which showed that that target peptide binding to S100B enhances its calcium-binding affinity [Rustandi et al. (1998) Biochemistry 37, 1951-1960]. The corresponding Ca(2+) association rate constants for S100B, k(on), for the EF2 domains in the absence and presence of the p53 peptide are 1.1 x 10(6) and 3.5 x 10(5) M(-)(1) s(-)(1), respectively. These two association rate constants are significantly below the diffusion control ( approximately 10(9) M(-)(1) s(-)(1)) and likely involve both Ca(2+) ion association and a Ca(2+)-dependent structural rearrangement, which is slightly different when the target peptide is present. EF-hand calcium-binding mutants of S100B were engineered at the -Z position (EF-hand 1, E31A; EF-hand 2, E72A; both EF-hands, E31A + E72A) and examined to further understand how specific residues contribute to calcium binding in S100B in the absence and presence of the p53 peptide.     

Calcium and chlorpromazine binding to the EF-hand peptides of neuronal calcium sensor-1

Neuronal calcium sensor-1, a protein of calcium sensor family, is known to have four structural EF-hands. We have synthesised peptides corresponding to all the four EF-hands and studied their conformation and calcium-binding. Our data confirm that the first putative site, a non-canonical one (EF1), does not bind calcium. We have investigated if this lack of binding is due to the presence of non-favoured residues (particularly at +x and -z co-ordinating positions) of the loop. We have mutated these residues and found that after modification the peptides bound calcium. However, these mutated peptides (EF1 and its functional mutants) do not show any Ca(2+) induced changes in far-UV CD. EF2, EF3, and EF4 peptides bind Ca(2+), EF3 being the strongest binder, followed by EF4. Our data of Ca(2+)-binding to individual EF peptides show that there are three active Ca(2+)-binding sites in NCS-1. We have also studied the binding of a neuroleptic drug, chlorpromazine, with the protein as well as with its EF-hands. CPZ binds myristoylated as well as non-myristoylated NCS-1 in Ca(2+)-dependent manner, with dynamic interaction to myristoylated protein. CPZ does not bind to EF1, but binds to functional EF-hand peptides and induces changes in far-UV CD. Our results suggest that NCS-1 could be a target of such antipsychotic and neuroleptic drugs.     

The crystal structure of the sorcin calcium binding domain provides a model of Ca2+-dependent processes in the full-length protein

Sorcin is a 21.6 kDa calcium binding protein, expressed in a number of mammalian tissues that belongs to the small, recently identified penta-EF-hand (PEF) family. Like all members of this family, sorcin undergoes a Ca2+-dependent translocation from cytosol to membranes where it binds to target proteins. For sorcin, the targets differ in different tissues, indicating that it takes part in a number of Ca2+-regulated processes. The sorcin monomer is organized in two domains like in all PEF proteins: a flexible, hydrophobic, glycine-rich N-terminal region and a calcium binding C-terminal domain. In vitro, the PEF proteins are dimeric in their Ca2+-free form, but have a marked tendency to precipitate when bound to calcium. Stabilization of the dimeric structure is achieved by pairing of the uneven EF-hand, EF5. Sorcin can also form tetramers at acid pH.The sorcin calcium binding domain (SCBD, residues 33-198) expressed in Escherichia coli was crystallized in the Ca2+-free form. The structure was solved by molecular replacement and was refined to 2.2 A with a crystallographic R-factor of 22.4 %. Interestingly, the asymmetric unit contains two dimers.The structure of the SCBD leads to a model that explains the solution properties and describes the Ca2+-induced conformational changes. Phosphorylation studies show that the N-terminal domain hinders phosphorylation of SCBD, i.e. the rate of phosphorylation increased twofold in the absence of the N-terminal region. In addition, previous fluorescence studies indicated that hydrophobic residues are exposed to solvent upon Ca2+ binding to full-length sorcin. The model accounts for these data by proposing that Ca2+ binding weakens the interactions between the two domains and leads to their reorientation, which exposes hydrophobic regions facilitating the Ca2+-dependent binding to target proteins at or near membranes.     

Structure of apoptosis-linked protein ALG-2: insights into Ca2+-induced changes in penta-EF-hand proteins

BACKGROUND: The Ca2+ binding apoptosis-linked gene-2 (ALG-2) protein acts as a proapoptotic factor in a variety of cell lines and is required either downstream or independently of caspases for apoptosis to occur. ALG-2 belongs to the penta-EF-hand (PEF) protein family and has two high-affinity and one low-affinity Ca2+ binding sites. Like other PEF proteins, its N terminus contains a Gly/Pro-rich segment. Ca2+ binding is required for the interaction with the target protein, ALG-2 interacting protein 1 (AIP1). RESULTS: We present the 2.3 A resolution crystal structure of Ca2+-Ioaded des1-20ALG-2 (aa 21-191), which was obtained by limited proteolysis of recombinant ALG-2 with elastase. The molecule contains eight alpha helices that fold into five EF-hands, and, similar to other members of this protein family, the molecule forms dimers. Ca2+ ions bind to EF1, EF3, and, surprisingly, to EF5. In the related proteins calpain and grancalcin, the EF5 does not bind Ca2+ and is thought to primarily facilitate dimerization. Most importantly, the conformation of des1-20ALG-2 is significantly different from that of calpain and grancalcin. This difference can be described as a rigid body rotation of EF1-2 relative to EF4-5 and the dimer interface, with a hinge within the EF3 loop. An electron density, which is interpreted as a hydrophobic Gly/Pro-rich decapeptide that is possibly derived from the cleaved N terminus, was found in a hydrophobic cleft between these two halves of the molecule. CONCLUSIONS: A different relative orientation of the N- and C-terminal halves of des1-20ALG-2 in the presence of Ca2+ and the peptide as compared to other Ca2+loaded PEF proteins changes substantially the shape of the molecule, exposing a hydrophobic patch on the surface for peptide binding and a large cleft near the dimer interface. We postulate that the binding of a Gly/ Pro-rich peptide in the presence of Ca2+ induces a conformational rearrangement in ALG-2, and that this mechanism is common to other PEF proteins.     

Calbindin D28k EF-hand ligand binding and oligomerization: four high-affinity sites--three modes of action

Calbindin D28k, a highly conserved protein with Ca2+-sensing and Ca2+-buffering capabilities, is abundant in brain and sensory neurons. This protein contains six EF-hand subdomains, four of which bind Ca2+ with high affinity. Calbindin D28k can be reconstituted from six synthetic peptides corresponding to the six EF-hands, indicating a single-domain structure with multiple interactions between the EF-hand subdomains. In this study, we have undertaken a detailed characterization of the Ca2+-binding and oligomerization properties of each individual EF-hand peptide using CD spectroscopy and analytical ultracentrifugation. Under the conditions tested, EF2 is monomeric and does not bind Ca2+, whereas EF6, which binds Ca2+ weakly, aggregates severely. We have therefore focused this study on the high-affinity binding sites, EF-hands 1, 3, 4, and 5. Our sedimentation equilibrium data show that, in the presence of Ca2+, EF-hands 1, 3, 4, and 5 all form dimers in solution in which the distribution between the monomer, dimer, and higher order oligomers differs. The processes of Ca2+ binding and oligomerization are linked to different degrees, and three main mechanisms emerge. For EF-hands 1 and 5, the dimer binds Ca2+ more strongly than the monomer and Ca2+ binding drives dimerization. For EF-hand 4, dimer formation requires only one of the monomers to be Ca2+-bound. In this case, the Ca2+ affinity is independent of dimerization. For EF-hand 3, dimerization occurs both in the absence and presence of Ca2+, while oligomerization increases in the presence of Ca2+.     

Ca2+-dependent interaction of the inhibitory region of troponin I with acidic residues in the N-terminal domain of troponin C

Ca2+ regulation of vertebrate striated muscle contraction is initiated by conformational changes in the N-terminal, regulatory domain of the Ca2+-binding protein troponin C (TnC), altering the interaction of TnC with the other subunits of troponin complex, TnI and TnT. We have investigated the role of acidic amino acid residues in the N-terminal, regulatory domain of TnC in binding to the inhibitory region (residues 96-116) of TnI. We constructed three double mutants of TnC (E53A/E54A, E60A/E61A and E85A/D86A), in which pairs of acidic amino acid residues were replaced by neutral alanines, and measured their affinities for synthetic inhibitory peptides. These peptides had the same amino acid sequence as TnI segments 95-116, 95-119 or 95-124, except that the natural Phe-100 of TnI was replaced by a tryptophan residue. Significant Ca2+-dependent increases in the affinities of the two longer peptides, but not the shortest one, to TnC could be detected by changes in Trp fluorescence. In the presence of Ca2+, all the mutant TnCs showed about the same affinity as wild-type TnC for the inhibitory peptides. In the presence of Mg2+ and EGTA, the N-terminal, regulatory Ca2+-binding sites of TnC are unoccupied. Under these conditions, the affinity of TnC(E85A/D86A) for inhibitory peptides was about half that of wild-type TnC, while the other two mutants had about the same affinity. These results imply a Ca2+-dependent change in the interaction of TnC Glu-85 and/or Asp-86 with residues (117-124) on the C-terminal side of the inhibitory region of TnI. Since Glu-85 and/or Asp-86 of TnC have also been demonstrated to be involved in Ca2+-dependent regulation through interaction with TnT, this region of TnC must be critical for troponin function.     

Identification of pore residues engaged in determining divalent cationic permeation in transient receptor potential melastatin subtype channel 2

The molecular basis for divalent cationic permeability in transient receptor potential melastatin subtype (TRPM) channels is not fully understood. Here we studied the roles of all eight acidic residues, glutamate or aspartate, and also the glutamine residue between pore helix and selectivity filter in the pore of TRPM2 channel. Mutants with alanine substitution in each of the acidic residues, except Glu-960 and Asp-987, formed functional channels. These channels exhibited similar Ca(2+) and Mg(2+) permeability to wild type channel, with the exception of the E1022A mutant, which displayed increased Mg(2+) permeability. More conservative E960Q, E960D, and D987N mutations also led to loss of function. The D987E mutant was functional and showed greater Ca(2+) permeability along with concentration-dependent inhibition of Na(+)-carrying currents by Ca(2+). Incorporation of negative charge in place of Gln-981 between the pore helix and selectivity filter by changing it to glutamate, which is present in the more Ca(2+)-permeable TRPM channels, substantially increased Ca(2+) permeability. Expression of concatemers linking wild type and E960D mutant subunits resulted in functional channels that exhibited reduced Ca(2+) permeability. These data taken together suggest that Glu-960, Gln-981, Asp-987, and Glu-1022 residues are engaged in determining divalent cationic permeation properties of the TRPM2 channel.     

Identification of catalytic residues of Ca2+-independent 1,2-alpha-D-mannosidase from Aspergillus saitoi by site-directed mutagenesis

The roles of six conserved active carboxylic acids in the catalytic mechanism of Aspergillus saitoi 1,2-alpha-d-mannosidase were studied by site-directed mutagenesis and kinetic analyses. We estimate that Glu-124 is a catalytic residue based on the drastic decrease of kcat values of the E124Q and E124D mutant enzyme. Glu-124 may work as an acid catalyst, since the pH dependence of its mutants affected the basic limb. D269N and E411Q were catalytically inactive, while D269E and E411D showed considerable activity. This indicated that the negative charges at these points are essential for the enzymatic activity and that none of these residues can be a base catalyst in the normal sense. Km values of E273D, E414D, and E474D mutants were greatly increased to 17-31-fold wild type enzyme, and the kcat values were decreased, suggesting that each of them is a binding site of the substrate. Ca2+, essential for the mammalian and yeast enzymes, is not required for the enzymatic activity of A. saitoi 1,2-alpha-d-mannosidase. EDTA inhibits the Ca2+-free 1,2-alpha-d-mannosidase as a competitive inhibitor, not as a chelator. We deduce that the Glu-124 residue of A. saitoi 1,2-alpha-d-mannosidase is directly involved in the catalytic mechanism as an acid catalyst, whereas no usual catalytic base is directly involved. Ca2+ is not essential for the activity. The catalytic mechanism of 1,2-alpha-d-mannosidase may deviate from that typical glycosyl hydrolase.     

Structure of a trapped intermediate of calmodulin: calcium regulation of EF-hand proteins from a new perspective

Calmodulin (CaM) is a multifunctional Ca2+-binding protein that regulates the activity of many enzymes in response to changes in the intracellular Ca2+ concentration. There are two globular domains in CaM, each containing a pair of helix-loop-helix Ca2+-binding motifs called EF-hands. Ca2+-binding induces the opening of both domains thereby exposing hydrophobic pockets that provide binding sites for the target enzymes. Here, I present a 2.4 A resolution structure of a calmodulin mutant (CaM41/75) in which the N-terminal domain is locked in the closed conformation by a disulfide bond. CaM41/75 crystallized in a tetragonal lattice with the Ca2+ bound in all four EF-hands. In the closed N-terminal domain Ca ions are coordinated by the four protein ligands in positions 1, 3, 5 and 7 of the loop, and by two solvent ligands. The glutamate side-chain in the 12th position of the loop (Glu31 in site I and Glu67 in site II), which in the wild-type protein provides a bidentate Ca2+ ligand, remains in a distal position. Based on a comparison of CaM41/75 with other CaM and troponin C structures a detailed two-step mechanism of the Ca2+-binding process is proposed. Initially, the Ca2+ binds to the N-terminal part of the loop, thus generating a rigid link between the incoming helix (helix A, or helix C) and the central beta structure involving the residues in the sixth, seventh and eighth position of the loop. Then, the exiting helix (helix B or helix D) rotates causing the glutamate ligand in the 12th position to move into the vicinity of the immobilized Ca2+. An adjustment of the phi, psi backbone dihedral angles of the Ile residue in the eighth position is necessary and sufficient for the helix rotation and functions as a hinge. The model allows for a significant independence of the Ca2+-binding sites in a two-EF-hand domain.     

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.     

Crystal structure of the D94S/G98E variant of rat alpha-parvalbumin. An explanation for the reduced divalent ion affinity

Simultaneous replacement of Asp-94 with serine and Gly-98 with glutamate in rat alpha-parvalbumin creates a CD-site ligand array in the context of the EF-site binding loop. Previous work has shown that, relative to the wild-type CD site, this engineered site has markedly reduced Ca(2+) affinity. Seeking an explanation for this phenomenon, we have obtained the crystal structure of the alpha D94S/G98E variant. The Ca(2+) coordination within the engineered EF site of the 94/98E variant is nearly identical to that within the CD site, suggesting that the attenuated affinity of the EF site in 94/98E is not a consequence of suboptimal coordination geometry. We have also examined the divalent ion binding properties of the alpha 94/98E variant in both Na(+)- and K(+)-containing buffers. Although the Ca(2+) and Mg(2+) affinities are higher in K(+) solution, the increases are comparable to those observed for wild-type alpha. Consistent with that finding, the apparent Na(+) stoichiometry, estimated from stability studies conducted as a function of Na(+) concentration, is 1.0 +/- 0.1, identical to that of wild-type alpha. Thus, the reduced affinity for divalent ions is evidently not the result of heightened monovalent ion competition. The thermodynamic analysis indicates that the less favorable Gibbs free energy of binding reflects a substantial enthalpic penalty. Significantly, the crystal structure reveals a steric clash between Phe-57 and the C(gamma) atom of Glu-98. The consequent displacement of Phe-57 also produces a close contact with Ser-55. Thus, steric interference may be the source of the enthalpic penalty.     

An EF-hand phage display study of calmodulin subdomain pairing

The interaction between the two EF-hands, EF3 and EF4, in the C-terminal domain of vertebrate calmodulin is addressed using an EF-hand phage display library. Significant specificity is observed in the presence of Ca(2+), as EF3-EF4 heterodimers are favored over EF3-EF3 and EF4-EF4 homodimers. Primarily EF4-type (and not EF3-type) amino acids are selected when an EF3 peptide is used as the target and vice versa. The results show that this specificity is promoted by several factors. There are three positions, corresponding to Phe89, Ala102, and Leu105, that are strongly selected as EF3-type hydrophobic residues with an EF4 target. When EF3 is the target peptide, EF4-type residues, Ile125, Tyr138 and Phe141, are selected. Remarkably, this subset consists of the same three residue positions in EF3 or EF4 and seems to be involved in specifying the heterodimer preference in both cases. In addition, electrostatic repulsion between the acidic monomers in an EF4 homodimer may further influence the preferred stability of heterodimers. This hypothesis is based on the observation that positively charged residues are strongly selected at four positions when EF4 is the target. A survey of EF-hand pairs suggests that charge separation is a common way to achieve efficient attraction of Ca(2+) without causing electrostatic repulsion between the subdomains. No significant specificity of binding is observed in the ion free state or in the presence of magnesium as no sequence is preferentially selected. The residues at the interface between the two EF-hands are thus highly optimized for the Ca(2+) bound state. At some residue positions, EF3-type amino acids are chosen with EF3-target in the presence of Ca(2+). These residues are not involved in the preference for heterodimer over homodimer formation, but represent key positions to mutate in the intact domain to stabilize its Ca(2+)-bound state.     

Structural Insights into Activation of the Retinal L-type Ca2+ Channel (Cav1.4) by Ca2+-binding Protein 4 (CaBP4)

CaBP4 modulates Ca²⁺-dependent activity of L-type voltage-gated Ca²⁺ channels (Cav1.4) in retinal photoreceptor cells. Mg²⁺ binds to the first and third EF-hands (EF1 and EF3), and Ca²⁺ binds to EF1, EF3, and EF4 of CaBP4. Here we present NMR structures of CaBP4 in both Mg²⁺-bound and Ca²⁺-bound states and model the CaBP4 structural interaction with Cav1.4. CaBP4 contains an unstructured N-terminal region (residues 1–99) and four EF-hands in two separate lobes. The N-lobe consists of EF1 and EF2 in a closed conformation with either Mg²⁺ or Ca²⁺ bound at EF1. The C-lobe binds Ca²⁺ at EF3 and EF4 and exhibits a Ca²⁺-induced closed-to-open transition like that of calmodulin. Exposed residues in Ca²⁺-bound CaBP4 (Phe¹³⁷, Glu¹⁶⁸, Leu²⁰⁷, Phe²¹⁴, Met²⁵¹, Phe²⁶⁴, and Leu²⁶⁸) make contacts with the IQ motif in Cav1.4, and the Cav1.4 mutant Y1595E strongly impairs binding to CaBP4. We conclude that CaBP4 forms a collapsed structure around the IQ motif in Cav1.4 that we suggest may promote channel activation by disrupting an interaction between IQ and the inhibitor of Ca²⁺-dependent inactivation domain.     

Presenilin 2 interacts with sorcin, a modulator of the ryanodine receptor

Perturbed Ca(2+) homeostasis is a common molecular consequence of familial Alzheimer's disease-linked presenilin mutations. We report here the molecular interaction of the large hydrophilic loop region of presenilin 2 (PS2) with sorcin, a penta-EF-hand Ca(2+)-binding protein that serves as a modulator of the ryanodine receptor intracellular Ca(2+) channel. The association of endogenous sorcin and PS2 was demonstrated in cultured cells and human brain tissues. Membrane-associated sorcin and a subset of the functional PS2 complexes were co-localized to a novel subcellular fraction that is distinctively positive for calcineurin B. Sorcin was found to interact with PS2 endoproteolytic fragments but not full-length PS2, and the sorcin/PS2 interaction was greatly enhanced by treatment with the Ca(2+) ionophore A23187. Our findings reveal a molecular link between PS2 and intracellular Ca(2+) channels (i.e. ryanodine receptor) and substantiate normal and/or pathological roles of PS2 in intracellular Ca(2+) homeostasis.     

Ca2+ and Mg2+ binding properties of GCAP-1. Evidence that Mg2+-bound form is the physiological activator of photoreceptor guanylyl cyclase

Guanylyl cyclase-activating protein 1 (GCAP-1) is an EF-hand protein that activates retinal guanylyl cyclase (RetGC) in photoreceptors at low free Ca2+ in the light and inhibits it in the dark when Ca2+ concentrations rise. We present the first direct evidence that Mg2+-bound form of GCAP-1, not its cation-free form, is the true activator of RetGC-1 under physiological conditions. Of four EF-hand structures in GCAP-1, three bound Ca2+ ions and could exchange Ca2+ for Mg2+. At concentrations of free Ca2+ and Mg2+ typical for the light-adapted photoreceptors, all three metal-binding EF-hands were predominantly occupied by Mg2, and the presence of bound Mg2+ in GCAP-1 was essential for its ability to stimulate RetGC-1. In the Mg2+-bound form of GCAP-1 all three Trp residues became more exposed to the polar environment compared with its apo form. The replacement of Mg2+ by Ca2+ in the EF-hands 2 and 3 further exposed Trp-21 to the solution in a non-metal-binding EF-hand domain 1 that interacts with RetGC. Contrary to that, replacement of Mg2+ by Ca2+ in the EF-hand 4 moved Trp-94 in the entering alpha-helix of the EF-hand 3 back to the non-polar environment. Our results demonstrate that Mg2+ regulates GCAP-1 not only by adjusting its Ca2+ sensitivity to the physiological conditions in photoreceptors but also by creating the conformation required for RetGC stimulation.     

Solution structure and calcium-binding properties of EF-hands 3 and 4 of calsenilin

Calsenilin is a member of the recoverin branch of the EF-hand superfamily that is reported to interact with presenilins, regulate prodynorphin gene expression, modulate voltage-gated Kv4 potassium channel function, and bind to neurotoxins. Calsenilin is a Ca+2-binding protein and plays an important role in calcium signaling. Despite its importance in numerous neurological functions, the structure of this protein has not been reported. In the absence of Ca+2, the protein has limited spectral resolution that increases upon the addition of Ca+2. Here, we describe the three-dimensional solution structure of EF-hands 3 and 4 of calsenilin in the Ca+2-bound form. The Ca+2-bound structure consists of five alpha-helices and one two-stranded antiparallel beta-sheet. The long loop that connects EF hands 3 and 4 is highly disordered in solution. In addition to its structural effects, Ca+2 binding also increases the protein's propensity to dimerize. These changes in structure and oligomerization state induced upon Ca+2 binding may play important roles in molecular recognition during calcium signaling.     

NMR-driven secondary and tertiary structure model of Ca2+-loaded calexcitin

Calexcitin (CE) is a Ca2+-binding protein which is expressed in neuronal cells and is a member of the sarcoplasmic Ca2+-binding protein subfamily. The peptide backbone of Ca2+-CE has been assigned by NMR and it shows that CE is composed of nine alpha-helices-forming four EF-hands and an additional helix near the C-terminus. A structural model of CE suggests the presence of a putative recessed hydrophobic pocket that may be involved in Ca2+-mediated protein-ligand interactions. This feature is unique to CE and is absent in other SCPs, such as those from Branchiostoma and Nereis, and from calerythrin.     

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.