An extracellular calcium-binding domain in bacteria with a distant relationship to EF-hands

Extracellular Ca(2+)-dependent nuclease YokF from Bacillus subtilis and several other surface-exposed proteins from diverse bacteria are encoded in the genomes in two paralogous forms that differ by a approximately 45 amino acid fragment, which comprises a novel conserved domain. Sequence analysis of this domain revealed a conserved DxDxDGxxCE motif, which is strikingly similar to the Ca(2+)-binding loop of the calmodulin-like EF-hand domains, suggesting an evolutionary relationship between them. Functions of many of the other proteins in which the novel domain, named Excalibur (extracellular calcium-binding region), is found, as well as a structural model of its conserved motif are consistent with the notion that the Excalibur domain binds calcium. This domain is but one more example of the diversity of structural contexts surrounding the EF-hand-like calcium-binding loop in bacteria. This loop is thus more widespread than hitherto recognized and the evolution of EF-hand-like domains is probably more complex than previously appreciated.     

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.     

Cloning, characterization, and expression of calcyphosine 2, a novel human gene encoding an EF-hand Ca(2+)-binding protein

Calcyphosine is a calcium-binding protein containing four EF-hand domains, initially identified as thyroid protein p24. It was first cloned and its counterparts in rabbit, human, and mouse, crayfish and lobster of invertebrate were also cloned. Here we describe the cloning and characterization of a novel human calcyphosine gene. The 3829-bp cDNA encodes a EF-hand Ca(2+)-binding protein homologous to the dog calcyphosine. It also contains two EF-hand Ca(2+)-binding motif. It is abundantly expressed in many tissues including by RT-PCR analysis and believed to play important role in calcium signaling. It was mapped to human genome 12q15.     

Metal-ion affinity and specificity in EF-hand proteins: coordination geometry and domain plasticity in parvalbumin.

BACKGROUND: The EF-hand family is a large set of Ca(2+)-binding proteins that contain characteristic helix-loop-helix binding motifs that are highly conserved in sequence. Members of this family include parvalbumin and many prominent regulatory proteins such as calmodulin and troponin C. EF-hand proteins are involved in a variety of physiological processes including cell-cycle regulation, second messenger production, muscle contraction, microtubule organization and vision. RESULTS: We have determined the structures of parvalbumin mutants designed to explore the role of the last coordinating residue of the Ca(2+)-binding loop. An E101D substitution has been made in the parvalbumin EF site. The substitution decreases the Ca(2+)-binding affinity 100-fold and increases the Mg(2+)-binding affinity 10-fold. Both the Ca(2+)- and Mg(2+)-bound structures have been determined, and a structural basis has been proposed for the metal-ion-binding properties. CONCLUSIONS: The E101D mutation does not affect the Mg(2+) coordination geometry of the binding loop, but it does pull the F helix 1.1 A towards the loop. The E101D-Ca(2+) structure reveals that this mutant cannot obtain the sevenfold coordination preferred by Ca(2+), presumably because of strain limits imposed by tertiary structure. Analysis of these results relative to previously reported structural information supports a model wherein the characteristics of the last coordinating residue and the plasticity of the Ca(2+)-binding loop delimit the allowable geometries for the coordinating sphere.     

Prediction of EF-hand calcium-binding proteins and analysis of bacterial EF-hand proteins

The EF-hand protein with a helix-loop-helix Ca(2+) binding motif constitutes one of the largest protein families and is involved in numerous biological processes. To facilitate the understanding of the role of Ca(2+) in biological systems using genomic information, we report, herein, our improvement on the pattern search method for the identification of EF-hand and EF-like Ca(2+)-binding proteins. The canonical EF-hand patterns are modified to cater to different flanking structural elements. In addition, on the basis of the conserved sequence of both the N- and C-terminal EF-hands within S100 and S100-like proteins, a new signature profile has been established to allow for the identification of pseudo EF-hand and S100 proteins from genomic information. The new patterns have a positive predictive value of 99% and a sensitivity of 96% for pseudo EF-hands. Furthermore, using the developed patterns, we have identified zero pseudo EF-hand motif and 467 canonical EF-hand Ca(2+) binding motifs with diverse cellular functions in the bacteria genome. The prediction results imply that pseudo EF-hand motifs are phylogenetically younger than canonical EF-hand motifs. Our prediction of Ca(2+) binding motifs provides not only an insight into the role of Ca(2+) and Ca(2+)-binding proteins in bacterial systems, but also a way to explore and define the role of Ca(2+) in other biological systems (calciomics).     

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.     

Effects of mutations in the calcium-binding sites of recoverin on its calcium affinity: evidence for successive filling of the calcium binding sites

A molecule of the photoreceptor Ca(2+)-binding protein recoverin contains four potential EF-hand Ca(2+)-binding sites, of which only two, the second and the third, are capable of binding calcium ions. We have studied the effects of substitutions in the second, third and fourth EF-hand sites of recoverin on its Ca(2+)-binding properties and some other characteristics, using intrinsic fluorescence, circular dichroism spectroscopy and differential scanning microcalorimetry. The interaction of the two operating binding sites of wild-type recoverin with calcium increases the protein's thermal stability, but makes the environment around the tryptophan residues more flexible. The amino acid substitution in the EF-hand 3 (E121Q) totally abolishes the high calcium affinity of recoverin, while the mutation in the EF-hand 2 (E85Q) causes only a moderate decrease in calcium binding. Based on this evidence, we suggest that the binding of calcium ions to recoverin is a sequential process with the EF-hand 3 being filled first. Estimation of Ca(2+)-binding constants according to the sequential binding scheme gave the values 3.7 x 10(6) and 3.1 x 10(5) M(-1) for third and second EF-hands, respectively. The substitutions in the EF-hand 2 or 3 (or in both the sites simultaneously) do not disturb significantly either tertiary or secondary structure of the apo-protein. Amino acid substitutions, which have been designed to restore the calcium affinity of the EF-hand 4 (G160D, K161E, K162N, D165G and K166Q), increase the calcium capacity and affinity of recoverin but also perturb the protein structure and decrease the thermostability of its apo-form.     

Potential influence of Asp in the Ca2+ coordination position 5 of parvalbumin on the calcium-binding affinity: a computational study

Parvalbumins (PV) are calcium-binding proteins, all sharing the common helix-loop-helix (EF-hand) motif. This motif contains a central twelve-residue Ca(2+)-binding loop with the flanking helices positioned roughly perpendicular to each other. The precise role of these coordination residues has been the subject of intense studies. In this work, we focus on the coordination position 5 in the CD Ca(2+)-binding site of silver hake parvalbumin isoform B (SHPV-B). The most common residue at site 5 of calcium-binding loop in canonical EF-hands is Asp [B.J. Marsden, G.S. Shaw, B.D. Sykes, Biochem. Cell Biol. 68 (1990) 587-601], but in the CD site of PV, this position is almost always serine (Ser). The substitution of Ser with Asp will add the 5th carboxylate residue in the CD coordination sphere. However, as predicted by the acid pair hypothesis, the Ca(2+)-binding affinity would be maximized in an EF-hand motif that has four carboxylate ligands paired along the +/-x, and +/-z-axes [R.E. Reid, R.S. Hodges, J. Theor. Biol. 84 (1980) 401-444]. Molecular dynamics simulations and free energy calculations were employed to investigate the influence of Ser to Asp mutation at position 5 on calcium-binding affinity. We found that the Asp variant exhibited remarkable stability during the entire molecular dynamics simulation, with not only the retention of the Ca(2+)-binding site, but also increased compactness in the coordination sphere. The S55D fragment also accommodated Ca(2+) well. We conclude that the reason why Asp which is the most common residue at site 5 of calcium-binding loop in canonical EF-hands has never been identified at this position experimentally for PVs might be related to its physiological functions.     

Prokaryotic calcium-binding protein of the calmodulin superfamily. Calcium binding to a Saccharopolyspora erythraea 20 kDa protein.

The EF-hand calcium-binding protein from Saccharopolyspora erythraea has been shown, using 113Cd NMR, to possess three Cd(2+)-ion binding sites. This indicates that of the four EF-hand motifs in the molecule, one (probably site 2) is unable to bind Cd(2+)-ions. Data from the titration of the protein with Ca2+, in the presence of Quin2, were fitted to a curve calculated on the assumption that the protein contains three high affinity Ca2+ binding sites, two of which (pK1 = 8.0, pK2 = 9.0) are strongly cooperative, and one single site (pK3 = 7.5). Preliminary 1H NMR experiments indicate marked structural changes upon Ca(2+)-binding.     

Ca2+-dependent binding of calcium-binding protein 1 to presynaptic group III metabotropic glutamate receptors and blockage by phosphorylation of the receptors

Presynaptic group III metabotropic glutamate receptors (mGluRs) and Ca(2+) channels are the main neuronal activity-dependent regulators of synaptic vesicle release, and they use common molecules in their signaling cascades. Among these, calmodulin (CaM) and the related EF-hand Ca(2+)-binding proteins are of particular importance as sensors of presynaptic Ca(2+), and a multiple of them are indeed utilized in the signaling of Ca(2+) channels. However, despite its conserved structure, CaM is the only known EF-hand Ca(2+)-binding protein for signaling by presynaptic group III mGluRs. Because the mGluRs and Ca(2+) channels reciprocally regulate each other and functionally converge on the regulation of synaptic vesicle release, the mGluRs would be expected to utilize more EF-hand Ca(2+)-binding proteins in their signaling. Here I show that calcium-binding protein 1 (CaBP1) bound to presynaptic group III mGluRs competitively with CaM in a Ca(2+)-dependent manner and that this binding was blocked by protein kinase C (PKC)-mediated phosphorylation of these receptors. As previously shown for CaM, these results indicate the importance of CaBP1 in signal cross talk at presynaptic group III mGluRs, which includes many molecules such as cAMP, Ca(2+), PKC, G protein, and Munc18-1. However, because the functional diversity of EF-hand calcium-binding proteins is extraordinary, as exemplified by the regulation of Ca(2+) channels, CaBP1 would provide a distinct way by which presynaptic group III mGluRs fine-tune synaptic transmission.     

Secondary structure and calcium-induced folding of the Clostridium thermocellum dockerin domain determined by NMR spectroscopy

Assembly of the cellulosome, a large, extracellular cellulase complex, depends upon docking of a myriad of enzymatic subunits to homologous receptors, or cohesin domains, arranged in tandem along a noncatalytic scaffolding protein. Docking to the cohesin domains is mediated by a highly conserved domain, dockerin (DS), borne by each enzymatic subunit. DS consists of two 22-amino-acid duplicated sequences, each bearing homology to the EF-hand calcium-binding loop. To compare the DS structure with that of the EF-hand helix-loop-helix motif, we analyzed the solution secondary structure of the DS from the cellobiohydrolase CelS subunit of the Clostridium thermocellum cellulosome using multidimensional heteronuclear NMR spectroscopy. The effect of Ca(2+)-binding on the DS structure was first investigated by using 2D (15)N-(1)H HSQC NMR spectroscopy. Changes in the spectra during Ca(2+) titration revealed that Ca(2+) induces folding of DS into its tertiary structure. This Ca(2+)-induced protein folding distinguishes DS from typical EF-hand-containing proteins. Sequential backbone assignments were determined for 63 of 69 residues. Analysis of the NOE connectivities and H(alpha) chemical shifts revealed that each half of the dockerin contains just one alpha-helix, comparable to the F-helix of the EF-hand motif. Thus, the structure of the DS Ca(2+)-binding subdomain deviates from that of the canonical EF-hand motif.     

Backbone dynamics of the regulatory domain of calcium vector protein, studied by 15N relaxation at four fields, reveals unique mobility characteristics of the intermotif linker

CaVP is a calcium-binding protein from amphioxus. It has a modular composition with two domains, but only the two EF-hand motifs localized in the C-terminal domain are functional. We recently determined the solution structure of this regulatory half (C-CaVP) in the Ca(2+)-saturated form and characterized the stepwise ion binding. This paper reports the (15)N nuclear relaxation rates of the Ca(2+)-saturated C-CaVP, measured at four different NMR fields (9.39, 11.74, 14.1, and 18.7 T), which were used to map the spectral density function for the majority of the amide H(N)-N vectors. Fitting the spectral density values at eight frequencies by a model-free approach, we obtained the microdynamic parameters characterizing the global and internal movements of the polypeptide backbone. The two EF-hand motifs, including the ion binding loops, behave like compact structural units with restricted mobility as reflected in the quite uniform order parameter and short internal correlation time (< 20 nsec). Comparative analysis of the two Ca(2+) binding sites shows that site III, having a larger affinity for the metal ion, is generally more rigid, and the amide vector in the second residue of each loop is significantly less restricted. The linker fragment is animated simultaneously by a larger amplitude fast motion and a slow conformational exchange on a microsecond to millisecond time scale. The backbone dynamics of C-CaVP characterized here is discussed in relation with other well-characterized Ca(2+)-binding proteins. Supplemental material: See www.proteinscience.org     

Identification of a Ca2+-binding domain in the rubella virus nonstructural protease

The rubella virus (RUB) nonstructural protein (NS) open reading frame (ORF) encodes a polypeptide precursor that is proteolytically self cleaved into two replicase components involved in viral RNA replication. A putative EF-hand Ca(2+)-binding motif that was conserved across different genotypes of RUB was predicted within the nonstructural protease that cleaves the precursor by using bioinformatics tools. To probe the metal-binding properties of this motif, we used an established grafting approach and engineered the 12-residue Ca(2+)-coordinating loop into a non-Ca(2+)-binding scaffold protein, CD2. The grafted EF-loop bound to Ca(2+) and its trivalent analogs Tb(3+) and La(3+) with K(d)s of 214, 47, and 14 microM, respectively. Mutations (D1210A and D1217A) of two of the potential Ca(2+)-coordinating ligands in the EF-loop led to the elimination of Tb(3+) binding. Inductive coupled plasma mass spectrometry was used to confirm the presence of Ca(2+) ([Ca(2+)]/[protein] = 0.7 +/- 0.2) in an NS protease minimal metal-binding domain, RUBCa, that spans the EF-hand motif. Conformational studies on RUBCa revealed that Ca(2+) binding induced local conformational changes and increased thermal stability (Delta T(m) = 4.1 degrees C). The infectivity of an RUB infectious cDNA clone containing the mutations D1210A/D1217A was decreased by approximately 20-fold in comparison to the wild-type (wt) clone, and these mutations rapidly reverted to the wt sequence. The NS protease containing these mutations was less efficient at precursor cleavage than the wt NS protease at 35 degrees C, and the mutant NS protease was temperature sensitive at 39 degrees C, confirming that the Ca(2+)-binding loop played a structural role in the NS protease and was specifically required for optimal stability under physiological conditions.     

Sequential calcium binding to the regulatory domain of calcium vector protein reveals functional asymmetry and a novel mode of structural rearrangement

Calcium vector protein (CaVP) from amphioxus is a two-domain, calcium-binding protein (18.3 kDa) of the calmodulin superfamily. Only two of the four EF-hand motifs (sites III and IV) have a significant binding affinity for calcium ions. We determined the solution structure of the domain containing these active sites (C-CaVP: W81-S161), in the Ca(2+)-saturated state, using NMR spectroscopy and restrained molecular dynamics. The tertiary structure is similar to other Ca(2+)-binding domains containing a pair of EF-hand motifs. The apo state has spectroscopic and thermodynamic characteristics of a molten globule, with conserved secondary structure but highly fluctuating tertiary organization. Titration of C-CaVP with Ca(2+) revealed a stepwise ion binding, with a stable equilibrium intermediate in which only site III binds a calcium ion. Despite a highly fluctuating structure of the free site IV, the calcium-bound site III has a persistent structure, with similar secondary elements but different interhelix angle and hydrophobic packing relative to the fully calcium-saturated state.     

Solution structure of a type I dockerin domain, a novel prokaryotic, extracellular calcium-binding domain

The type I dockerin domain is responsible for incorporating its associated glycosyl hydrolase into the bacterial cellulosome, a multienzyme cellulolytic complex, via its interaction with a receptor domain (cohesin domain) of the cellulosomal scaffolding subunit. The highly conserved dockerin domain is characterized by two Ca(2+)-binding sites with sequence similarity to the EF-hand motif. Here, we present the three-dimensional solution structure of the 69 residue dockerin domain of Clostridium thermocellum cellobiohydrolase CelS. Torsion angle dynamics calculations utilizing a total of 728 NOE-derived distance constraints and 79 torsion angle restraints yielded an ensemble of 20 structures with an average backbone r.m.s.d. for residues 5 to 29 and 32 to 66 of 0.54 A from the mean structure. The structure consists of two Ca(2+)-binding loop-helix motifs connected by a linker; the E helices entering each loop of the classical EF-hand motif are absent from the dockerin domain. Each dockerin Ca(2+)-binding subdomain is stabilized by a cluster of buried hydrophobic side-chains. Structural comparisons reveal that, in its non-complexed state, the dockerin fold displays a dramatic departure from that of Ca(2+)-bound EF-hand domains. A putative cohesin-binding surface, comprised of conserved hydrophobic and basic residues, is proposed, providing new insight into cellulosome assembly.     

Structure of a sarcoplasmic calcium-binding protein from Nereis diversicolor refined at 2.0 A resolution.

The crystal structure of a sarcoplasmic Ca(2+)-binding protein (SCP) from the sandworm Nereis diversicolor has been determined and refined at 2.0 A resolution using restrained least-squares techniques. The two molecules in the crystallographic asymmetric unit, which are related by a non-crystallographic 2-fold axis, were refined independently. The refined model includes all 174 residues and three calcium ions for each molecule, as well as 213 water molecules. The root-mean-square difference in co-ordinates for backbone atoms and calcium ions of the two molecules is 0.51 A. The final crystallographic R-factor, based on 18,959 reflections in the range 2.0 A less than or equal to d less than or equal to 7.0 A, with intensities exceeding 2.0 sigma, is 0.182. Bond lengths and bond angles in the molecules have root-mean-square deviations from ideal values of 0.013 A and 2.2 degrees, respectively. SCP has four distinct domains with the typical helix-loop-helix (EF-hand) Ca(2+)-binding motif, although the second Ca(2+)-binding domain is not functional due to amino acid changes in the loop. The structure shows several unique features compared to other Ca(2+)-binding proteins with four EF-hand domains. The overall structure is highly compact and globular with a predominant hydrophobic core, unlike the extended dumbbell-shaped structure of calmodulin or troponin C. A hydrophobic tail at the COOH terminus adds to the structural stability by packing against a hydrophobic pocket created by the folding of the NH2 and COOH-terminal Ca(2+)-binding domain pairs. The first and second domains show different helix-packing arrangements from any previously described for Ca(2+)-binding proteins.     

Calcium binding to the photosystem II subunit CP29

We have identified a Ca(2+)-binding site of the 29-kDa chlorophyll a/b-binding protein CP29, a light harvesting protein of photosystem II most likely involved in photoregulation. (45)Ca(2+) binding studies and dot blot analyses of CP29 demonstrate that CP29 is a Ca(2+)-binding protein. The primary sequence of CP29 does not exhibit an obvious Ca(2+)-binding site therefore we have used Yb(3+) replacement to analyze this site. Near-infrared Yb(3+) vibronic side band fluorescence spectroscopy (Roselli, C., Boussac, A., and Mattioli, T. A. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 12897-12901) of Yb(3+)-reconstituted CP29 indicated a single population of Yb(3+)-binding sites rich in carboxylic acids, characteristic of Ca(2+)-binding sites. A structural model of CP29 presents two purported extra-membranar loops which are relatively rich in carboxylic acids, one on the stromae side and one on the lumenal side. The loop on the lumenal side is adjacent to glutamic acid 166 in helix C of CP29, which is known to be the binding site for dicyclohexylcarbodiimide (Pesaresi, P., Sandonà, D., Giuffra, E. , and Bassi, R. (1997) FEBS Lett. 402, 151-156). Dicyclohexylcarbodiimide binding prevented Ca(2+) binding, therefore we propose that the Ca(2+) in CP29 is bound in the domain including the lumenal loop between helices B and C.     

Five members of a novel Ca(2+)-binding protein (CABP) subfamily with similarity to calmodulin

Five members of a novel Ca(2+)-binding protein subfamily (CaBP), with 46-58% sequence similarity to calmodulin (CaM), were identified in the vertebrate retina. Important differences between these Ca(2+)-binding proteins and CaM include alterations within their second EF-hand loop that render these motifs inactive in Ca(2+) coordination and the fact that their central alpha-helixes are extended by one alpha-helical turn. CaBP1 and CaBP2 contain a consensus sequence for N-terminal myristoylation, similar to members of the recoverin subfamily and are fatty acid acylated in vitro. The patterns of expression differ for each of the various members. Expression of CaBP5, for example, is restricted to retinal rod and cone bipolar cells. In contrast, CaBP1 has a more widespread pattern of expression. In the brain, CaBP1 is found in the cerebral cortex and hippocampus, and in the retina this protein is found in cone bipolar and amacrine cells. CaBP1 and CaBP2 are expressed as multiple, alternatively spliced variants, and in heterologous expression systems these forms show different patterns of subcellular localization. In reconstitution assays, CaBPs are able to substitute functionally for CaM. These data suggest that these novel CaBPs are an important component of Ca(2+)-mediated cellular signal transduction in the central nervous system where they may augment or substitute for CaM.     

Instead of binding calcium, one of the EF-hand structures in guanylyl cyclase activating protein-2 is required for targeting photoreceptor guanylyl cyclase

Guanylyl cyclase activator proteins (GCAPs) are calcium-binding proteins closely related to recoverin, neurocalcin, and many other neuronal Ca(2+)-sensor proteins of the EF-hand superfamily. GCAP-1 and GCAP-2 interact with the intracellular portion of photoreceptor membrane guanylyl cyclase and stimulate its activity by promoting tight dimerization of the cyclase subunits. At low free Ca(2+) concentrations, the activator form of GCAP-2 associates into a dimer, which dissociates when GCAP-2 binds Ca(2+) and becomes inhibitor of the cyclase. GCAP-2 is known to have three active EF-hands and one additional EF-hand-like structure, EF-1, that deviates form the EF-hand consensus sequence. We have found that various point mutations within the EF-1 domain can specifically affect the ability of GCAP-2 to interact with the target cyclase but do not hamper the ability of GCAP-2 to undergo reversible Ca(2+)-sensitive dimerization. Point mutations within the EF-1 region can interfere with both the activation of the cyclase by the Ca(2+)-free form of GCAP-2 and the inhibition of retGC basal activity by the Ca(2+)-loaded GCAP-2. Our results strongly indicate that evolutionary conserved and GCAP-specific amino acid residues within the EF-1 can create a contact surface for binding GCAP-2 to the cyclase. Apparently, in the course of evolution GCAP-2 exchanged the ability of its first EF-hand motif to bind Ca(2+) for the ability to interact with the target enzyme.     

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.