Hierarchical integration of mitochondrial and nuclear positioning pathways by the Num1 EF hand.

Positioning organelles at the right place and time is critical for their function and inheritance. In budding yeast, mitochondrial and nuclear positioning require the anchoring of mitochondria and dynein to the cell cortex by clusters of Num1. We have previously shown that mitochondria drive the assembly of cortical Num1 clusters, which then serve as anchoring sites for mitochondria and dynein. When mitochondrial inheritance is inhibited, mitochondrial-driven assembly of Num1 in buds is disrupted and defects in dynein-mediated spindle positioning are observed. Using a structure-function approach to dissect the mechanism of mitochondria-dependent dynein anchoring, we found that the EF hand-like motif (EFLM) of Num1 and its ability to bind calcium are required to bias dynein anchoring on mitochondria-associated Num1 clusters. Consistently, when the EFLM is disrupted, we no longer observe defects in dynein activity following inhibition of mitochondrial inheritance. Thus, the Num1 EFLM functions to bias dynein anchoring and activity in nuclear inheritance subsequent to mitochondrial inheritance. We hypothesize that this hierarchical integration of organelle positioning pathways by the Num1 EFLM contributes to the regulated order of organelle inheritance during the cell cycle.

Role of the EF-hand and coiled-coil domains of human Rab44 in localisation and organelle formation.

Rab44 is a large Rab GTPase that contains an amino-terminal EF-hand domain, a coiled-coil domain, and a carboxyl-terminal Rab GTPase domain. However, the roles of the EF-hand and coiled-coil domains remain unclear. Here, we constructed various deletion and point mutants of human Rab44. When overexpressed in HeLa cells, the wild-type Rab44 (hWT) formed ring-like structures, and partially localised to lysosomes. The dominant negative mutant, hT847N, localised to lysosomes and the cytosol, while the constitutively active mutant, hQ892L, formed ring-like structures, and partially localised to the plasma membrane and nuclei. The hΔEF, hΔcoil, and h826-1021 mutants also formed ring-like structures; however, their localisation patterns differed from hWT. Analysis of live imaging with LysoTracker revealed that the size of LysoTracker-positive vesicles was altered by all other mutations than the hC1019A and hΔEF. Treatment with ionomycin, a Ca2+ ionophore, induced the translocation of hWT and hΔcoil into the plasma membrane and cytosol, but had no effect on the localisation of the hΔEF and h826-1021 mutants. Thus, the EF- hand domain is likely required for the partial translocation of Rab44 to the plasma membrane and cytosol following transient Ca2+ influx, and the coiled-coil domain appears to be important for localisation and organelle formation.

High-affinity cooperative Ca2+ binding by MICU1-MICU2 serves as an on-off switch for the uniporter.

The mitochondrial calcium uniporter is a Ca2+-activated Ca2+ channel that is essential for dynamic modulation of mitochondrial function in response to cellular Ca2+ signals. It is regulated by two paralogous EF-hand proteins-MICU1 and MICU2, but the mechanism is unknown. Here, we demonstrate that both MICU1 and MICU2 are stabilized by Ca2+ We reconstitute the MICU1-MICU2 heterodimer and demonstrate that it binds Ca2+ cooperatively with high affinity. We discover that both MICU1 and MICU2 exhibit affinity for the mitochondria-specific lipid cardiolipin. We determine the minimum Ca2+ concentration required for disinhibition of the uniporter in permeabilized cells and report a close match with the Ca2+-binding affinity of MICU1-MICU2. We conclude that cooperative, high-affinity interaction of the MICU1-MICU2 complex with Ca2+ serves as an on-off switch, leading to a tightly controlled channel, capable of responding directly to cytosolic Ca2+ signals.

Calcium Binding and Disulfide Bonds Regulate the Stability of Secretagogin towards Thermal and Urea Denaturation.

Secretagogin is a calcium-sensor protein with six EF-hands. It is widely expressed in neurons and neuro-endocrine cells of a broad range of vertebrates including mammals, fishes and amphibia. The protein plays a role in secretion and interacts with several vesicle-associated proteins. In this work, we have studied the contribution of calcium binding and disulfide-bond formation to the stability of the secretagogin structure towards thermal and urea denaturation. SDS-PAGE analysis of secretagogin in reducing and non-reducing conditions identified a tendency of the protein to form dimers in a redox-dependent manner. The denaturation of apo and Calcium-loaded secretagogin was studied by circular dichroism and fluorescence spectroscopy under conditions favoring monomer or dimer or a 1:1 monomer: dimer ratio. This analysis reveals significantly higher stability towards urea denaturation of Calcium-loaded secretagogin compared to the apo protein. The secondary and tertiary structure of the Calcium-loaded form is not completely denatured in the presence of 10 M urea. Reduced and Calcium-loaded secretagogin is found to refold reversibly after heating to 95°C, while both oxidized and reduced apo secretagogin is irreversibly denatured at this temperature. Thus, calcium binding greatly stabilizes the structure of secretagogin towards chemical and heat denaturation.

Oncomodulin, an EF-Hand Ca2+ Buffer, Is Critical for Maintaining Cochlear Function in Mice.

Oncomodulin (Ocm), a member of the parvalbumin family of calcium binding proteins, is expressed predominantly by cochlear outer hair cells in subcellular regions associated with either mechanoelectric transduction or electromotility. Targeted deletion of Ocm caused progressive cochlear dysfunction. Although sound-evoked responses are normal at 1 month, by 4 months, mutants show only minimal distortion product otoacoustic emissions and 70-80 dB threshold shifts in auditory brainstem responses. Thus, Ocm is not critical for cochlear development but does play an essential role for cochlear function in the adult mouse. SIGNIFICANCE STATEMENT: Numerous proteins act as buffers, sensors, or pumps to control calcium levels in cochlear hair cells. In the inner ear, EF-hand calcium buffers may play a significant role in hair cell function but have been very difficult to study. Unlike other reports of genetic disruption of EF-hand calcium buffers, deletion of oncomodulin (Ocm), which is predominately found in outer hair cells, leads to a progressive hearing loss after 1 month, suggesting that Ocm critically protects hearing in the mature ear.

Regulation of the transient receptor potential channel TRPA1 by its N-terminal ankyrin repeat domain.

The transient receptor potential channel A1 (TRPA1) is unique among ion channels of higher vertebrates in that it harbors a large ankyrin repeat domain. The TRPA1 channel is expressed in the inner ear and in nociceptive neurons. It is involved in hearing as well as in the perception of pungent and irritant chemicals. The ankyrin repeat domain has special mechanical properties, which allows it to function as a soft spring that can be extended over a large range while maintaining structural integrity. A calcium-binding site has been experimentally identified within the ankyrin repeats. We built a model of the N-terminal 17 ankyrin repeat structure, including the calcium-binding EF-hand. In our simulations we find the calcium-bound state to be rigid as compared to the calcium-free state. While the end-to-end distance can change by almost 50% in the apo form, these fluctuations are strongly reduced by calcium binding. This increase in stiffness that constraints the end-to-end distance in the holo form is predicted to affect the force acting on the gate of the TRPA1 channel, thereby changing its open probability. Simulations of the transmembrane domain of TRPA1 show that residue N855, which has been associated with familial episodic pain syndrome, forms a strong link between the S4-S5 connecting helix and S1, thereby creating a direct force link between the N-terminus and the gate. The N855S mutation weakens this interaction, thereby reducing the communication between the N-terminus and the transmembrane part of TRPA1.

In silico prediction and analysis of Caenorhabditis EF-hand containing proteins.

Calcium (Ca⁺²) is a ubiquitous messenger in eukaryotes including Caenorhabditis. Ca⁺²-mediated signalling processes are usually carried out through well characterized proteins like calmodulin (CaM) and other Ca⁺² binding proteins (CaBP). These proteins interact with different targets and activate it by bringing conformational changes. Majority of the EF-hand proteins in Caenorhabditis contain Ca⁺² binding motifs. Here, we have performed homology modelling of CaM-like proteins using the crystal structure of Drosophila melanogaster CaM as a template. Molecular docking was applied to explore the binding mechanism of CaM-like proteins and IQ1 motif which is a ∼25 residues and conform to the consensus sequence (I, L, V)QXXXRXXXX(R,K) to serve as a binding site for different EF hand proteins. We made an attempt to identify all the EF-hand (a helix-loop-helix structure characterized by a 12 residues loop sequence involved in metal coordination) containing proteins and their Ca⁺² binding affinity in Caenorhabditis by analysing the complete genome sequence. Docking studies revealed that F165, F169, L29, E33, F44, L57, M61, M96, M97, M108, G65, V115, F93, N104, E144 of CaM-like protein is involved in the interaction with IQ1 motif. A maximum of 170 EF-hand proteins and 39 non-EF-hand proteins with Ca⁺²/metal binding motif were identified. Diverse proteins including enzyme, transcription, translation and large number of unknown proteins have one or more putative EF-hands. Phylogenetic analysis revealed seven major classes/groups that contain some families of proteins. Various domains that we identified in the EF-hand proteins (uncharacterized) would help in elucidating their functions. It is the first report of its kind where calcium binding loop sequences of EF-hand proteins were analyzed to decipher their calcium affinities. Variation in Ca⁺²-binding affinity of EF-hand CaBP could be further used to study the behaviour of these proteins. Our analyses postulated that Ca⁺² is likely to be key player in Caenorhabditis cell signalling.

Caltubin, a novel molluscan tubulin-interacting protein, promotes axonal growth and attenuates axonal degeneration of rodent neurons.

Axotomized central neurons of most invertebrate species demonstrate a strong regenerative capacity, and as such may provide valuable molecular insights and new tools to promote axonal regeneration in injured mammalian neurons. In this study, we identified a novel molluscan protein, caltubin, ubiquitously expressed in central neurons of Lymnaea stagnalis and locally synthesized in regenerating neurites. Reduction of caltubin levels by gene silencing inhibits the outgrowth and regenerative ability of adult Lymnaea neurons and decreases local α- and ß-tubulin levels in neurites. Caltubin binds to α- and/or ß-tubulin in both Lymnaea and rodent neurons. Expression of caltubin in PC12 cells and mouse cortical neurons promotes NGF-induced axonal outgrowth and attenuates axonal retraction after injury. This is the first study illustrating that a xenoprotein can enhance outgrowth and prevent degeneration of injured mammalian neurons. These results may open up new avenues in molecular repair strategies through the insertion of molecular components of invertebrate regenerative pathways into mammalian neurons.

Cellular roles of neuronal calcium sensor-1 and calcium/calmodulin-dependent kinases in fungi.

The neuronal calcium sensor-1 (NCS-1) possesses a consensus signal for N-terminal myristoylation and four EF-hand Ca(2+)-binding sites, and mediates the effects of cytosolic Ca(2+). Minute changes in free intracellular Ca(2+) are quickly transformed into changes in the activity of several kinases including calcium/calmodulin-dependent protein kinases (Ca(2+)/CaMKs) that are involved in regulating many eukaryotic cell functions. However, our current knowledge of NCS-1 and Ca(2+)/CaMKs comes mostly from studies of the mammalian enzymes. Thus far very few fungal homologues of NCS-1 and Ca(2+)/CaMKs have been characterized and little is known about their cellular roles. In this minireview, we describe the known sequences, interactions with target proteins and cellular roles of NCS-1 and Ca(2+)/CaMKs in fungi.

Protein phosphatase with EF-hand domains 2 (PPEF2) is a potent negative regulator of apoptosis signal regulating kinase-1 (ASK1).

The function of protein phosphatases with EF-hand domains (PPEF) in mammals is not known. Large-scale expression profiling experiments suggest that PPEF expression may correlate with stress protective responses, cell survival, growth, proliferation, or neoplastic transformation. Apoptosis signal regulating kinase-1 (ASK1) is a MAP kinase kinase kinase implicated in cancer, cardiovascular and neurodegenerative diseases. ASK1 is activated by oxidative stress and induces pro-apoptotic or inflammatory signalling, largely via sustained activation of MAP kinases p38 and/or JNK. We identify human PPEF2 as a novel interacting partner and a negative regulator of ASK1. In COS-7 or HEK 293A cells treated with H(2)O(2), expression of PPEF2 abrogated sustained activation of p38 and one of the JNK p46 isoforms, and prevented ASK1-dependent caspase-3 cleavage and activation. PPEF2 efficiently suppressed H(2)O(2)-induced activation of ASK1. Overexpessed as well as endogenous ASK1 co-immunoprecipitated with PPEF2. PPEF2 was considerably more potent both as a suppressor of ASK1 activation and as its interacting partner as compared to protein phosphatase 5 (PP5), a well-known negative regulator of ASK1. PPEF2 was found to form complexes with endogenous Hsp70 and to a lesser extent Hsp90, which are also known interacting partners of PP5. These data identify, for the first time, a possible downstream signalling partner of a mammalian PPEF phosphatase, and suggest that, despite structural divergence, PPEF and PP5 phosphatases may share common interacting partners and functions.

Protein 4.2 binds to the carboxyl-terminal EF-hands of erythroid alpha-spectrin in a calcium- and calmodulin-dependent manner.

Spectrin and protein 4.1 cross-link F-actin protofilaments into a network called the membrane skeleton. Actin and 4.1 bind to one end of beta-spectrin. The adjacent end of alpha-spectrin, called the EF-domain, is calmodulin-like, with calcium-dependent and calcium-independent EF-hands. It has no known function. However, the sph(1J)/sph(1J) mouse has very fragile red cells and lacks the last 13 amino acids in the EF-domain, suggesting the domain is critical for skeletal integrity. Using pulldown binding assays, we find the alpha-spectrin EF-domain either alone or incorporated into a mini-spectrin binds native and recombinant protein 4.2 at a previously identified region of 4.2 (G(3) peptide). Native 4.2 binds with an affinity comparable with other membrane skeletal interactions (K(d) = 0.30 microM). EF-domains bearing the sph(1J) mutation are inactive. Binding of protein 4.2 to band 3 (K(d) = 0.45 microM) does not interfere with the spectrin-4.2 interaction. Spectrin-4.2 binding is amplified by micromolar concentrations of Ca(2+) (but not Mg(2+)) by three to five times. Calmodulin also binds to the EF-domain (K(d) = 17 microM), and Ca(2+)-calmodulin blocks Ca(2+)-dependent binding of protein 4.2 but not Ca(2+)-independent binding. The data suggest that protein 4.2 is located near protein 4.1 at the spectrin-actin junctions. Because proteins 4.1 and 4.2 also bind to band 3, the erythrocyte anion channel, we suggest that one or both of these proteins cause a portion of band 3 to localize near the spectrin-actin junctions and provide another point of attachment between the membrane skeleton and the lipid bilayer.

The IQ domains in neuromodulin and PEP19 represent two major functional classes.

The affinities of Ca(2+)-saturated and Ca(2+)-free calmodulin for a fluorescent reporter construct containing the PEP19 IQ domain differ by a factor of approximately 100, with K(d) values of 11.0 +/- 1.2 and 1128.4 +/- 176.5 muM, respectively, while the affinities of a reporter containing the neuromodulin IQ domain are essentially identical, with K(d) values of 2.9 +/- 0.3 and 2.4 +/- 0.3 muM, respectively. When Ca(2+) is bound only to the C-terminal pair of Ca(2+)-binding sites in calmodulin, the K(d) value for the PEP19 reporter complex is decreased approximately 5-fold, while the value for the neuromodulin reporter complex is increased by the same factor. When Ca(2+) is bound only to the N-terminal pair of Ca(2+)-binding sites, the K(d) value for the PEP19 reporter complex is unaffected, but the value for the complex with the neuromodulin reporter is increased approximately 12-fold. These functional differences are largely ascribed to three differences in the CaM-binding sequences of the two reporters. Replacement of a central Gly in the neuromodulin IQ domain with a Lys at this position in PEP19 almost entirely accounts for the distinctive patterns of Ca(2+)-dependent stability changes exhibited by the two complexes. Replacement of a Lys immediately before the "IQ" amino acid pair in the neuromodulin sequence with the Ala in PEP19 accounts for the remaining Ca(2+)-dependent differences. Replacement of an Ala in the N-terminal half of the neuromodulin sequence with the Gln in PEP19 accounts for approximately half of the Ca(2+)-independent difference in the stabilities of the two reporter complexes, with the Ca(2+)-independent effect of the Lys replacement accounting for most of the remainder. Since the central Gly in the neuromodulin sequence is conserved in half of all known IQ domains, these results suggest that the presence or absence of this residue defines two major functional classes.

Membrane localization of RasGRP1 is controlled by an EF-hand, and by the GEF domain.

RasGRP1 is an exchange factor for membrane-localized Ras GTPases. Activation of RasGRP1 requires its translocation to membranes, which can be directly mediated by either its PT or C1 domains. RasGRP1 also has a pair of EF-hands which have been proposed to regulate RasGRP1 by sensing receptor-induced calcium fluxes. We determined that one of these EF-hands, EF1, is required for receptor-induced translocation of RasGRP1 to the plasma membrane in B cell lines. EF1 enables plasma membrane targeting of RasGRP1 by counteracting the SuPT domain, a negative regulator of the PT domain. Contrary to expectations, EF1-mediated translocation of RasGRP1 does not involve antigen receptor-induced intracellular calcium flux. Instead, alternative splicing affecting EF1 serves to modulate RasGRP1 localization. Excision of an exon encoding part of EF1 selectively disables PT domain-mediated plasma membrane targeting of RasGRP1, without affecting C1 domain-mediated localization to endomembranes. While EF1 specifically controls PT-mediated plasma membrane targeting, the Ras binding site in the catalytic GEF domain of RasGRP1 is required for both PT-mediated plasma membrane targeting and C1-mediated localization to endomembranes. Positive feedback between its GEF domain and membrane-binding domains could be important for full activation of RasGRP1, with occupation of the Ras binding sites in the GEF domain resulting in functional liberation of the PT and C1 domains, and membrane binding by these domains serving to maintain the Ras-GEF interaction.

What determines the degree of compactness of a calcium-binding protein?

The EF-hand calcium-binding proteins may exist either in an extended or a compact conformation. This conformation is sometimes correlated with the function of the calcium-binding protein. For those proteins whose structure and function are known, calcium sensors are usually extended and calcium buffers compact; hence, there is interest in predicting the form of the protein starting from its sequence. In the present study, we used two different procedures: one that already exists in the literature, the sosuidumbbell algorithm, mainly based on the charges of the two EF-hand domains, and the other comprising a novel procedure that is based on linker average hydrophilicity. The linker consists of the residues that connect the domains. The two procedures were tested on 17 known-structure calcium-binding proteins and then applied to 59 unknown-structure centrins. The sosuidumbbell algorithm yielded the correct conformations for only 15 of the known-structure proteins and predicted that all centrins should be in a closed form. The linker average hydrophilicity procedure discriminated well between all the extended and non-extended forms of the known-structure calcium-binding proteins, and its prediction concerning centrins reflected well their phylogenetic classification. The linker average hydrophilicity criterion is a simple and powerful means to discriminate between extended and non-extended forms of calcium-binding proteins. What is remarkable is that only a few residues that constitute the linker (between 2 and 20 in our tested sample of proteins) are responsible for the form of the calcium-binding protein, showing that this form is mainly governed by short-range interactions.

Regulation of bestrophin Cl channels by calcium: role of the C terminus.

Human bestrophin-1 (hBest1), which is genetically linked to several kinds of retinopathy and macular degeneration in both humans and dogs, is the founding member of a family of Cl(-) ion channels that are activated by intracellular Ca(2+). At present, the structures and mechanisms responsible for Ca(2+) sensing remain unknown. Here, we have used a combination of molecular modeling, density functional-binding energy calculations, mutagenesis, and patch clamp to identify the regions of hBest1 involved in Ca(2+) sensing. We identified a cluster of a five contiguous acidic amino acids in the C terminus immediately after the last transmembrane domain, followed by an EF hand and another regulatory domain that are essential for Ca(2+) sensing by hBest1. The cluster of five amino acids (293-308) is crucial for normal channel gating by Ca(2+) because all but two of the 35 mutations we made in this region rendered the channel incapable of being activated by Ca(2+). Using homology models built on the crystal structure of calmodulin (CaM), an EF hand (EF1) was identified in hBest1. EF1 was predicted to bind Ca(2+) with a slightly higher affinity than the third EF hand of CaM and lower affinity than the second EF hand of troponin C. As predicted by the model, the D312G mutation in the putative Ca(2+)-binding loop (312-323) reduced the apparent Ca(2+) affinity by 20-fold. In addition, the D312G and D323N mutations abolished Ca(2+)-dependent rundown of the current. Furthermore, analysis of truncation mutants of hBest1 identified a domain adjacent to EF1 that is rich in acidic amino acids (350-390) that is required for Ca(2+) activation and plays a role in current rundown. These experiments identify a region of hBest1 (312-323) that is involved in the gating of hBest1 by Ca(2+) and suggest a model in which Ca(2+) binding to EF1 activates the channel in a process that requires the acidic domain (293-308) and another regulatory domain (350-390). Many of the approximately 100 disease-causing mutations in hBest1 are located in this region that we have implicated in Ca(2+) sensing, suggesting that these mutations disrupt hBest1 channel gating by Ca(2+).

Structural basis for Ca2+ -dependent formation of ALG-2/Alix peptide complex: Ca2+/EF3-driven arginine switch mechanism.

ALG-2 belongs to the penta-EF-hand (PEF) protein family and interacts with various intracellular proteins, such as Alix and TSG101, that are involved in endosomal sorting and HIV budding. Through X-ray crystallography, we solved the structures of Ca(2+)-free and -bound forms of N-terminally truncated human ALG-2 (des3-20ALG-2), Zn(2+)-bound form of full-length ALG-2, and the structure of the complex between des3-23ALG-2 and the peptide corresponding to Alix799-814 in Zn(2+)-bound form. Binding of Ca(2+) to EF3 enables the side chain of Arg125, present in the loop connecting EF3 and EF4, to move enough to make a primary hydrophobic pocket accessible to the critical PPYP motif, which partially overlaps with the GPP motif for the binding of Cep55 (centrosome protein 55 kDa). Based on these results, together with the results of in vitro binding assay with mutant ALG-2 and Alix proteins, we propose a Ca(2+)/EF3-driven arginine switch mechanism for ALG-2 binding to Alix.

Birth of normal offspring from mouse eggs activated by a phospholipase Czeta protein lacking three EF-hand domains.

Sperm-specific phospholipase C, PLCzeta, is a candidate for the Ca(2+) oscillation-inducing factor that is introduced into the ooplasm upon sperm-egg fusion. In addition to the 647-residue full-length PLCzeta, s-PLCzeta lacking the N-terminal 110 amino acids is known to be present in the mouse testis. In this study, we attempted to obtain full-term offspring from s-PLCzeta-activated eggs by round spermatid injection. Metaphase II-arrested eggs injected with a high RNA concentration of s-PLCzeta RNA normally developed to blastocysts. When the round spermatid nucleus was injected into telophase II-stage eggs previously activated by s-PLCzeta RNA, three live offspring were successfully obtained by transfer of the developed 4-cell embryos to pseudopregnant mice. These three offspring all grew to be normal adults and reproduced healthy second-generation mice.

A novel calcium-binding protein is associated with tau proteins in tauopathy.

Tauopathies are a group of neurological disorders characterized by the presence of intraneuronal hyperphosphorylated and filamentous tau. Mutations in the tau gene have been found in kindred with tauopathy. The expression of the human tau mutant in transgenic mice induced neurodegeneration, indicating that tau plays a central pathological role. However, the molecular mechanism leading to tau-mediated neurodegeneration is poorly understood. To gain insights into the role that tau plays in neurodegeneration, human tau proteins were immunoprecipitated from brain lysates of the tauopathy mouse model JNPL3, which develops neurodegeneration in age-dependent manner. In the present work, a novel EF-hand domain-containing protein was found associated with tau proteins in brain lysate of 12-month-old JNPL3 mice. The association between tau proteins and the novel identified protein appears to be induced by the neurodegeneration process as these two proteins were not found associated in young JNPL3 mice. Consistently, the novel protein co-purified with the pathological sarkosyl insoluble tau in terminally ill JNPL3 mice. Calcium-binding assays demonstrated that this protein binds calcium effectively. Finally, the association between tau and the novel calcium-binding protein is conserved in human and enriched in Alzheimer's disease brain. Taken together, the identification of a novel calcium-binding protein associated with tau protein in terminally ill tauopathy mouse model and its confirmation in human brain lysate suggests that this association may play an important physiological and/or pathological role.

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.