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.     

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.     

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+.     

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.     

Characterization of p26olf, a novel calcium-binding protein in the frog olfactory epithelium

We have previously shown that p26olf is a novel S100-like Ca(2+)-binding protein in the frog olfactory epithelium. In this paper, we characterized the Ca(2+) binding property of p26olf, examined the precise localization in the frog olfactory epithelium, and searched for the possible target proteins of p26olf. By flow dialysis experiments using (45)Ca, p26olf was suggested to bind approximately 4 Ca(2+). Circular dichroism measurement showed that binding of Ca(2+) to p26olf induces an increase in the apparent content of both alpha-helix and beta-sheet with an apparent K(d) value of 2.4 micrometer. Electron microscopic observation disclosed p26olf immunoreactivity in the cilia, dendritic knob, and dendrite of olfactory receptor cells. Blot overlay analysis and affinity purification of p26olf-binding proteins showed that p26olf binds to a frog beta-adrenergic receptor kinase-like protein in a Ca(2+)-dependent manner. These results suggested that p26olf has some roles in the olfactory transduction or adaptation.     

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).     

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.     

Cloning and characterization of Xenopus dicalcin, a novel S100-like calcium-binding protein in Xenopus eggs.

To contribute to the study of the calcium-signaling mechanism of egg, we cloned and characterized a 26 kDa Ca(2+)-binding protein from Xenopus laevis eggs, a homologue of Rana catesbeiana dicalcin (renamed from p26olf) that was isolated from the olfactory epithelium. The primary structure of Xenopus dicalcin shows approximately 61% identity to that of Rana dicalcin and consists of two S100-like regions aligned in tandem, as seen in Rana dicalcin. Genomic Southern blot analysis indicated that Xenopus dicalcin is a unique orthologue of Rana dicalcin. Northern blot analysis showed that Xenopus dicalcin mRNA is expressed in Xenopus eggs and also in other tissues. These results indicated that Xenopus dicalcin is a novel S100-like Ca(2+)-binding protein in Xenopus eggs.     

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.     

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.     

Localization and characterization of the inhibitory Ca2+-binding site of Physarum polycephalum myosin II

A myosin II is thought to be the driving force of the fast cytoplasmic streaming in the plasmodium of Physarum polycephalum. This regulated myosin, unique among conventional myosins, is inhibited by direct Ca2+ binding. Here we report that Ca2+ binds to the first EF-hand of the essential light chain (ELC) subunit of Physarum myosin. Flow dialysis experiments of wild-type and mutant light chains and the regulatory domain revealed a single binding site that shows moderate specificity for Ca2+. The regulatory light chain, in contrast to regulatory light chains of higher eukaryotes, is unable to bind divalent cations. Although the Ca2+-binding loop of ELC has a canonical sequence, replacement of glutamic acid to alanine in the -z coordinating position only slightly decreased the Ca2+ affinity of the site, suggesting that the Ca2+ coordination is different from classical EF-hands; namely, the specific "closed-to-open" conformational transition does not occur in the ELC in response to Ca2+. Ca2+- and Mg2+-dependent conformational changes in the microenvironment of the binding site were detected by fluorescence experiments. Transient kinetic experiments showed that the displacement of Mg2+ by Ca2+ is faster than the change in direction of cytoplasmic streaming; therefore, we conclude that Ca2+ inhibition could operate in physiological conditions. By comparing the Physarum Ca2+ site with the well studied Ca2+ switch of scallop myosin, we surmise that despite the opposite effect of Ca2+ binding on the motor activity, the two conventional myosins could have a common structural basis for Ca2+ regulation.     

Calcium affinity, cooperativity, and domain interactions of extracellular EF-hands present in BM-40

The structure and function of cytosolic Ca(2+)-binding proteins containing EF-hands are well understood. Recently, the presence of EF-hands in an extracellular protein was for the first time proven by the structure determination of the EC domain of BM-40 (SPARC (for secreted protein acidic and rich in cysteine)/osteonectin) (Hohenester, E., Maurer, P., Hohenadl, C., Timpl, R., Jansonius, J. N., and Engel, J. (1996) Nat. Struct. Biol. 3, 67-73). The structure revealed a pair of EF-hands with two bound Ca(2+) ions. Two unusual features were noted that distinguish the extracellular EF-hands of BM-40 from their cytosolic counterparts. An insertion of one amino acid into the loop of the first EF-hand causes a variant Ca(2+) coordination, and a disulfide bond connects the helices of the second EF-hand. Here we show that the extracellular EF-hands in the BM-40 EC domain bind Ca(2+) cooperatively and with high affinity. The EC domain is thus in the Ca(2+)-saturated form in the extracellular matrix, and the EF-hands play a structural rather than a regulatory role. Deletion mutants demonstrate a strong interaction between the EC domain and the neighboring FS domain, which contributes about 10 kJ/mol to the free energy of binding and influences cooperativity. This interaction is mainly between the FS domain and the variant EF-hand 1. Certain mutations of Ca(2+)-coordinating residues changed affinity and cooperativity, but others inhibited folding and secretion of the EC domain in a mammalian cell line. This points to a function of EF-hands in extracellular proteins during biosynthesis and processing in the endoplasmic reticulum or Golgi apparatus.     

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.     

Implications on zinc binding to S100A2

Human S100A2 is an EF-hand calcium-binding S100 protein that is localized mainly in the nucleus and functions as tumor suppressor. In addition to Ca2+ S100A2 binds Zn2+ with a high affinity. Studies have been carried out to investigate whether Zn2+ acts as a regulatory ion for S100A2, as in the case of Ca2+. Using the method of competition with the Zn2+ chelator 4-(2-pyridylazo)-resorcinol, an apparent Kd of 25 nM has been determined for Zn2+ binding to S100A2. The affinity lies close to the range of intracellular free Zn2+ concentrations, suggesting that S100A2 is able to bind Zn2+ in the nucleus. Two Zn2+-binding sites have been identified using site directed mutagenesis and several spectroscopic techniques with Cd2+ and Co2+ as probes. In site 1 Zn2+ is bound by Cys21 and most likely by His 17. The binding of Zn2+ in site 2 induces the formation of a tetramer, whereby the Zn(2+) is coordinated by Cys2 from each subunit. Remarkably, only binding of Zn2+ to site 2 substantially weakens the affinity of S100A2 for Ca2+. Analysis of the individual Ca2+-binding constants revealed that the Ca2+ affinity of one EF-hand is decreased about 3-fold, whereas the other EF-hand exhibits a 300-fold decrease in affinity. These findings imply that S100A2 is regulated by both Zn2+ and Ca2+, and suggest that Zn2+ might deactivate S100A2 by inhibiting response to intracellular Ca2+ signals.     

Interactions of calcineurin A, calcineurin B, and Ca2+.

Calcineurin B (CN-B) is the Ca(2+)-binding, regulatory subunit of the phosphatase calcineurin. Point mutations to Ca(2+)-binding sites in CN-B were generated to disable individual Ca(2+)-binding sites and evaluate contributions from each site to calcineurin heterodimer formation. Ca(2+)-binding properties of four CN-B mutants and wild-type CN-B were analyzed by flow dialysis confirming that each CN-B mutant binds three Ca2+ and that wild-type CN-B binds four Ca2+. Macroscopic dissociation constants indicate that N-terminal Ca(2+)-binding sites have lower affinity for Ca2+ than the C-terminal sites. Each CN-B mutant was coexpressed with the catalytic subunit of calcineurin, CN-A, to produce heterodimers with specific disruption of one Ca(2+)-binding site. Enzymes containing CN-B with a mutation in Ca(2+)-binding sites 1 or 2 have a lower ratio of CN-B to CN-A and a lower phosphatase activity than those containing wild-type CN-B or mutants in sites 3 or 4. Effects of heterodimer formation on Ca2+ binding were assessed by monitoring (45)Ca2+ exchange by flow dialysis. Enzymes containing wild-type CN-B and mutants in sites 1 and 2 exchange (45)Ca2+ slowly from two sites whereas mutants in sites 3 and 4 exchange (45)Ca2+ slowly from a single site. These data indicate that the Ca2+ bound to sites 1 and 2 is likely to vary with Ca2+ concentration and may act in dynamic modulation of enzyme function, whereas Ca(2+)-binding sites 3 and 4 are saturated at all times and that Ca2+ bound to these sites is structural.     

New perspectives on S100 proteins: a multi-functional Ca 2+ -, Zn 2+ - and Cu 2+ -binding protein family

S100 proteins (16 members) show a very divergent pattern of cell- and tissue-specific expression, of subcel-lular localizations and relocations, of post-translational modifications, and of affinities for Ca 2+ , Zn 2+ , and Cu 2+ , consistent with their pleiotropic intra- and extracellular functions. Up to 40 target proteins are reported to interact with S100 proteins and for S100A1 alone 15 target proteins are presently known. Therefore it is not surprising that many functional roles have been proposed and that several human disorders such as cancer, neurodegenerative diseases, cardiomyopathies, inflammations, diabetes, and allergies are associated with an altered expression of S100 proteins. It is not unlikely that their biological activity in some cases is regulated by Zn 2+ and Cu 2+ , rather than by Ca 2+ Despite the numerous putative functions of S100 proteins, their three-dimensional structures of, e.g., S100B, S100A6, and S100A7 are surprisingly similar. They contain a compact dimerization domain whose conformation is rather insensitive to Ca 2+ binding and two lateral a-helices III and III, which project outward of each subunit when Ca 2+ is bound. Target docking depends on the two hydrophobic patches in front of the paired EF-hand generated by the binding of Ca 2+. The selec-tivity in target binding is assured by the central linker between the two EF-hands and the C-terminal tail. It appears that the S100-binding domain in some target proteins contains a basic amphiphilic a-helix and that the mode of interaction and activation bears structural similarity to that of calmodulin.© Kluwer Academic Publishers     

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.     

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.     

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.     

Mapping sites in guanylyl cyclase activating protein-1 required for regulation of photoreceptor membrane guanylyl cyclases

Guanylyl cyclase activating protein (GCAP)-1 regulates photoreceptor membrane guanylyl cyclase, RetGC, in a Ca2+-sensitive manner. It contains four Ca2+-binding motifs, EF-hands, three of which are capable of binding Ca2+. GCAP-1 activates RetGC in low Ca2+ and inhibits it in high Ca2+. In this study we used deletion and substitution analysis to identify regions of GCAP-1 sequence that are specifically required for inhibition and activation. A COOH-terminal sequence within Met157 to Arg182 is required for activation but not for inhibition of RetGC. We localized one essential stretch to 5 residues from Arg178 to Arg182. Another sequence essential for activation is within the N-terminal residues Trp21 to Thr27. The region between EF-hands 1 and 3 of GCAP-1 also contains elements needed for activation of RetGC. Finally, we found that inhibition of RetGC requires the first 9 amino-terminal residues of GCAP-1, but none of the residues from Gln33 to the COOH-terminal Gly205 are specifically required for inhibition. The ability of GCAP-1 mutants to regulate RetGC was tested on total guanylyl cyclase activity present in rod outer segments. In addition, the key mutants were also shown to produce similar effects on recombinant bovine outer segment cyclases GC1 and GC2.