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.     

Biophysical studies of engineered mutant proteins based on calbindin D9k modified in the pseudo EF-hand

The genes for four mutant proteins from calbindin D9k, all with mutations in the N-terminal Ca2+-binding domain (pseudo EF-hand) have been synthesized and expressed in Escherichia coli. The purification scheme has been modified to minimize the formation of deamidated proteins. The set of modifications in the pseudo EF-hand is an attempt to turn this site into a structure resembling an archetypal EF-hand, with its characteristic 113Cd-NMR shift (-80 to -110 ppm) and high calcium-binding constants, whereas the C-terminal Ca2(+)-binding site (EF-hand) is kept intact in all mutant proteins. The mutant proteins studied here all have pseudo EF-hands with a lower calcium-binding constant and a higher calcium off-rate to the pseudo EF-hand than the wild-type protein. From the results obtained it is obvious that proline 20 in the pseudo EF-hand, which has been deleted or replaced by glycine in three of the mutants, has a stabilizing effect on calcium binding to that site. Furthermore, the modifications in the pseudo EF-hand seem to have only a local effect, leaving the tertiary structure of the protein and the calcium-binding properties of the unmodified site virtually unchanged.     

Brain S100A5 is a novel calcium-, zinc-, and copper ion-binding protein of the EF-hand superfamily

S100A5 is a novel member of the EF-hand superfamily of calcium-binding proteins that is poorly characterized at the protein level. Immunohistochemical analysis demonstrates that it is expressed in very restricted regions of the adult brain. Here we characterized the human recombinant S100A5, especially its interaction with Ca(2+), Zn(2+), and Cu(2+). Flow dialysis revealed that the homodimeric S100A5 binds four Ca(2+) ions with strong positive cooperativity and an affinity 20-100-fold higher than the other S100 proteins studied under identical conditions. S100A5 also binds two Zn(2+) ions and four Cu(2+) ions per dimer. Cu(2+) binding strongly impairs the binding of Ca(2+); however, none of these ions change the alpha-helical-rich secondary structure. After covalent labeling of an exposed thiol with 2-(4'-(iodoacetamide)anilino)-naphthalene-6-sulfonic acid, binding of Cu(2+), but not of Ca(2+) or Zn(2+), strongly decreased its fluorescence. In light of the three-dimensional structure of S100 proteins, our data suggest that in each subunit the single Zn(2+) site is located at the opposite side of the EF-hands. The two Cu(2+)-binding sites likely share ligands of the EF-hands. The potential role of S100A5 in copper homeostasis is discussed.     

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.     

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.     

X-ray structures of the microglia/macrophage-specific protein Iba1 from human and mouse demonstrate novel molecular conformation change induced by calcium binding

The ionized calcium-binding adaptor molecule 1 (Iba1) with 147 amino acid residues has been identified as a calcium-binding protein, expressed specifically in microglia/macrophages, and is expected to be a key factor in membrane ruffling, which is a typical feature of activated microglia. We have determined the crystal structure of human Iba1 in a Ca(2+)-free form and mouse Iba1 in a Ca(2+)-bound form, to a resolution of 1.9 A and 2.1 A, respectively. X-ray structures of Iba1 revealed a compact, single-domain protein with two EF-hand motifs, showing similarity in overall topology to partial structures of the classical EF-hand proteins troponin C and calmodulin. In mouse Iba1, the second EF-hand contains a bound Ca(2+), but the first EF-hand does not, which is often the case in S100 proteins, suggesting that Iba1 has S100 protein-like EF-hands. The molecular conformational change induced by Ca(2+)-binding of Iba1 is different from that found in the classical EF-hand proteins and/or S100 proteins, which demonstrates that Iba1 has an unique molecular switching mechanism dependent on Ca(2+)-binding, to interact with target molecules.     

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.     

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.     

Target selectivity in EF-hand calcium binding proteins

EF-hand calcium binding proteins have remarkable sequence homology and structural similarity, yet their response to binding of calcium is diverse and they function in a wide range of biological processes. Knowledge of the fine-tuning of EF-hand protein sequences to optimize specific biochemical properties has been significantly advanced over the past 10 years by determination of atomic resolution structures. These data lay the foundation for addressing how functional selectivity is generated from a generic ionic signal. This review presents current ideas about the structural mechanisms that provide the selectivity of different EF-hand proteins for specific cellular targets, using S100 and calmodulin family proteins to demonstrate the critical concepts. Three factors contribute significantly to target selectivity: molecular architecture, response to binding of Ca(2+) ions, and the characteristics of target binding surfaces. Comparisons of calmodulin and S100 proteins provide insights into the role these factors play in facilitating the variety of binding configurations necessary for recognizing a diverse set of targets.     

The crystal structure of metal-free human EF-hand protein S100A3 at 1.7-A resolution

S100A3 is a unique member of the EF-hand superfamily of Ca(2+)-binding proteins. It binds Ca(2+) with poor affinity (K(d) = 4-35 mm) but Zn(2+) with exceptionally high affinity (K(d) = 4 nm). This high affinity for Zn(2+) is attributed to the unusual high Cys content of S100A3. The protein is highly expressed in fast proliferating hair root cells and astrocytoma pointing toward a function in cell cycle control. We determined the crystal structure of the protein at 1.7 A. The high resolution structure revealed a large distortion of the C-terminal canonical EF-hand, which most likely abolishes Ca(2+) binding. The crystal structure of S100A3 allows the prediction of one putative Zn(2+) binding site in the C terminus of each subunit of S100A3 involving Cys and His residues in the coordination of the metal ion. Zn(2+) binding induces a large conformational change in S100A3 perturbing the hydrophobic interface between two S100A3 subunits, as shown by size exclusion chromatography and CD spectroscopy.     

Mapping the zinc ligands of S100A2 by site-directed mutagenesis

S100 family proteins are characterized by short individual N and C termini and a conserved central part, harboring two Ca(2+)-binding EF-hands, one of them highly conserved among EF-hand family proteins and the other characteristic for S100 proteins. In addition to Ca(2+), several members of the S100 protein family, including S100A2, bind Zn(2+). Two regions in the amino acid sequences of S100 proteins, namely the helices of the N-terminal EF-hand motif and the very C-terminal loop are believed to be involved in Zn(2+)-binding due to the presence of histidine and/or cysteine residues. Human S100A2 contains four cysteine residues, each of them located at positions that may be important for Zn(2+) binding. We have now constructed and purified 10 cysteine-deficient mutants of human S100A2 by site-directed mutagenesis and investigated the contribution of the individual cysteine residues to Zn(2+) binding. Here we show that Cys(1(3)) (the number in parentheses indicating the position in the sequence of S100A2) is the crucial determinant for Zn(2+) binding in association with conformational changes as determined by internal tyrosine fluorescence. Solid phase Zn(2+) binding assays also revealed that the C-terminal residues Cys(3(87)) and Cys(4(94)) mediated a second type of Zn(2+) binding, not associated with detectable conformational changes in the molecule. Cys(2(22)), by contrast, which is located within the first EF hand motif affected neither Ca(2+) nor Zn(2+) binding, and a Cys "null" mutant was entirely incapable of ligating Zn(2+). These results provide new information about the mechanism and the site(s) of zinc binding in S100A2.     

The regulatory role of EF-hand motifs of pig 80K diacylglycerol kinase as assessed using truncation and deletion mutants.

To elucidate the regulatory function of EF-hand motifs of pig 80K diacylglycerol (DG) kinase, we constructed and expressed several truncation and deletion mutants of the enzyme in E. coli or COS-7 cells. The bacterially expressed EF-hand region could bind Ca2+ and was suggested to undergo conformational change like calmodulin. A mutant enzyme lacking EF-hands lost Ca(2+)-binding activity, but could be fully activated by phosphatidylserine (PS) or deoxycholate in the absence of Ca2+. The full activation of the wild-type enzyme by PS, on the other hand, was totally dependent on Ca2+. Further, the wild-type enzyme expressed in COS-7 cells was exclusively soluble, whereas the EF-hand-deleted mutant was considerably associated with the membranes. The results suggest that under Ca(2+)-free condition, the EF-hand masks the PS-binding site of the DG kinase, and that the Ca(2+)-binding results in the exposure of the PS-binding site through the conformational change of the EF-hand region.     

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.     

Critical determinants of Ca(2+)-dependent inactivation within an EF-hand motif of L-type Ca(2+) channels

L-type (alpha(1C)) calcium channels inactivate rapidly in response to localized elevation of intracellular Ca(2+), providing negative Ca(2+) feedback in a diverse array of biological contexts. The dominant Ca(2+) sensor for such Ca(2+)-dependent inactivation has recently been identified as calmodulin, which appears to be constitutively tethered to the channel complex. This Ca(2+) sensor induces channel inactivation by Ca(2+)-dependent CaM binding to an IQ-like motif situated on the carboxyl tail of alpha(1C). Apart from the IQ region, another crucial site for Ca(2+) inactivation appears to be a consensus Ca(2+)-binding, EF-hand motif, located approximately 100 amino acids upstream on the carboxyl terminus. However, the importance of this EF-hand motif for channel inactivation has become controversial since the original report from our lab implicating a critical role for this domain. Here, we demonstrate not only that the consensus EF hand is essential for Ca(2+) inactivation, but that a four-amino acid cluster (VVTL) within the F helix of the EF-hand motif is itself essential for Ca(2+) inactivation. Mutating these amino acids to their counterparts in non-inactivating alpha(1E) calcium channels (MYEM) almost completely ablates Ca(2+) inactivation. In fact, only a single amino acid change of the second valine within this cluster to tyrosine (V1548Y) supports much of the functional knockout. However, mutations of presumed Ca(2+)-coordinating residues in the consensus EF hand reduce Ca(2+) inactivation by only approximately 2-fold, fitting poorly with the EF hand serving as a contributory inactivation Ca(2+) sensor, in which Ca(2+) binds according to a classic mechanism. We therefore suggest that while CaM serves as Ca(2+) sensor for inactivation, the EF-hand motif of alpha(1C) may support the transduction of Ca(2+)-CaM binding into channel inactivation. The proposed transduction role for the consensus EF hand is compatible with the detailed Ca(2+)-inactivation properties of wild-type and mutant V1548Y channels, as gauged by a novel inactivation model incorporating multivalent Ca(2+) binding of CaM.     

The DxDxDG motif for calcium binding: multiple structural contexts and implications for evolution

Calcium ions regulate many cellular processes and have important structural roles in living organisms. Despite the great variety of calcium-binding proteins (CaBPs), many of them contain the same Ca(2+)-binding helix-loop-helix structure, referred to as the EF-hand. In the canonical EF-hand, the loop contains three calcium-binding aspartic acid residues, which form the DxDxDG sequence motif, and is flanked by two alpha-helices. Recently, other CaBPs containing the same motif, but lacking one or both helices, have been described. Here, structural motif searches were used to analyse the full diversity of structural context in the known set of DxDxDG-containing CaBPs, including those where the structural resemblance of a given DxDxDG motif to that of EF-hands had not been noted. The results obtained indicate that the EF-hand represents but one, among many, structural context for the DxDxDG-like Ca(2+)-binding loops. While the structural similarity of the binuclear calcium-binding sites in anthrax protective antigen and human thrombospondin suggests that they are homologous, evolutionary relationships for mononuclear sites are harder to discern. The possible scenarios for the evolution of DxDxDG motif-containing calcium-binding loops in a variety of non-homologous proteins suggested loop transplant as a mechanism perhaps responsible for much of the diversity in structural contexts of present day DxDxDG-type CaBPs. Additionally, while it can be shown that existence of a DxDxDG sequence is not enough to confer a conformation suitable for calcium binding, local convergent evolution may still have a role. The analysis presented here has consequences for the prediction of calcium binding from sequence alone.     

S100A16, a novel calcium-binding protein of the EF-hand superfamily

S100A16 protein is a new and unique member of the EF-hand Ca(2+)-binding proteins. S100 proteins are cell- and tissue-specific and are involved in many intra- and extracellular processes through interacting with specific target proteins. In the central nervous system S100 proteins are implicated in cell proliferation, differentiation, migration, and apoptosis as well as in cognition. S100 proteins became of major interest because of their close association with brain pathologies, for example depression or Alzheimer's disease. Here we report for the first time the purification and biochemical characterization of human and mouse recombinant S100A16 proteins. Flow dialysis revealed that both homodimeric S100A16 proteins bind two Ca(2+) ions with the C-terminal EF-hand of each subunit, the human protein exhibiting a 2-fold higher affinity. Trp fluorescence variations indicate conformational changes in the orthologous proteins upon Ca(2+) binding, whereas formation of a hydrophobic patch, implicated in target protein recognition, only occurs in the human S100A16 protein. In situ hybridization analysis and immunohistochemistry revealed a widespread distribution in the mouse brain. Furthermore, S100A16 expression was found to be astrocyte-specific. Finally, we investigated S100A16 intracellular localization in human glioblastoma cells. The protein was found to accumulate within nucleoli and to translocate to the cytoplasm in response to Ca(2+) stimulation.     

Activation and inhibition of photoreceptor guanylyl cyclase by guanylyl cyclase activating protein 1 (GCAP-1): the functional role of Mg2+/Ca2+ exchange in EF-hand domains

Guanylyl cyclase activating protein 1 (GCAP-1), a Ca(2+)/Mg(2+) sensor protein that accelerates retinal guanylyl cyclase (RetGC) in the light and decelerates it in the dark, is inactive in cation-free form. Binding of Mg(2+) in EF-hands 2 and 3 was essential for RetGC activation in the conditions mimicking light adaptation. Mg(2+) binding in EF-hand 2 affected the conformation of a neighboring non-metal binding domain, EF-hand-1, and increased GCAP-1 affinity for RetGC nearly 40-fold compared with the metal-free EF-hand 2. Mg(2+) binding in EF-hand 3 increased GCAP-1 affinity for RetGC 5-fold and its maximal RetGC stimulation 2-fold. Mg(2+) binding in EF-hand 4 affected neither GCAP-1 affinity for RetGC, nor RetGC activation. Inactivation of Ca(2+) binding in EF-hand 4 was sufficient to render GCAP-1 a constitutive activator of RetGC, whereas the EF-hand 3 role in Ca(2+)-dependent deceleration of RetGC was likely to be through the neighboring EF-hand 4. Inactivation of Ca(2+) binding in EF-hand 2 affected cooperativity of RetGC inhibition by Ca(2+), but did not prevent the inhibition. We conclude that 1) Mg(2+) binding in EF-hands 2 and 3, but not EF-hand 4, is essential for the ability of GCAP-1 to activate RetGC in the light; 2) Mg(2+) or Ca(2+) binding in EF-hand 3 and especially in EF-hand 2 is required for high-affinity interaction with the cyclase and affects the conformation of the neighboring EF-hand 1, a domain required for targeting RetGC; and 3) RetGC inhibition is likely to be primarily caused by Ca(2+) binding in EF-hand 4.