Structures and metal-ion-binding properties of the Ca²⁺-binding helix–loop–helix EF-hand motifs

The canonical EF-loop

In the canonical EF-loop, five of Ca²⁺'s seven co-ordinating groups are provided by the nine-residue loop. The remaining two come from a bidentate carboxylate ligand supplied by the side chain of an acidic amino acid located in the exiting helix, three residues removed from the loop's C-terminus. Though this residue is not structurally part of the loop, it is commonly referred to as the EF-loop's twelfth residue. Of the ligands provided by the loop itself, three or four come from the side-chain carboxy groups of negatively charged amino acids and one from a backbone carbonyl group. Frequently, the co-ordination sphere of the Ca²⁺ ion is completed by a water molecule hydrogen-bonded to one of the side chains of the loop (Figure 3B). The chelating residues of the loop are notated in two ways, the first based on linear position and the second on the tertiary geometry imposed by their alignment on the axes of a pentagonal bipyramid: 1(+X), 3(+Y), 5(+Z), 7(−Y), 9(−X), 12(−Z). The Y- and Z-axis pairs align along the vertices of an approximately planar pentagon, whereas the X-axis pair takes up an axial position perpendicular to the Y/Z plane [10]. In addition to the Ca–O bonds formed through the chelating interactions, the canonical EF-loop also contains an extensive network of hydrogen bonds between both chelating and non-chelating residues. These bonds help to stabilize the otherwise unfavourably close proximity of the negatively charged oxygen atoms in the co-ordination sphere of the bound Ca²⁺.

Several of the loop's residues contribute individual and identifiable roles to both the stabilization and fold of this structure. Through numerous intraloop hydrogen bonds, the first residue plays an important part in defining a precise sterochemical arrangement for the loop (Figure 3C). The conserved glycine residue found at the sixth position facilitates the unusual main-chain conformation (ϕ and ψ∼60° and 20° respectively) which results in a 90° turn that enables the remaining Ca²⁺ ligands to take up co-ordinating positions. The eighth residue of the loop is a highly conserved hydrophobic residue [11], the main chain NH and CO groups of which face away from the Ca²⁺-binding site towards the loop of the paired EF-hand. This forms the short antiparallel β-sheet with the corresponding groups of the paired loop's eighth position. As well as chelating the bound Ca²⁺ ion either directly or indirectly via a bridging water molecule, the hydrogen-bonding pattern formed by the side chain of loop position 9 initiates the exiting helix, a conformation stabilized through additional hydrogen bonds with the side-chain carboxylate group of position 12. Finally, the bidentate side-chain ligands supplied by the twelfth loop position are critical to both the structure and function of the EF-loop. The role played by the ligands of the twelfth loop position is discussed in depth in this review.

As the above discussion reflects EF-loop structural information predominantly obtained through X-ray crystal structures, it represents a static view. MD (molecular dynamics) simulations corroborate many of these points, but they have shed light on the transitory nature of the Ca²⁺ ligands. Although EF1 (EF-hand 1) remained essentially as observed in the crystal structure, in the course of simulations the bound Ca²⁺ ion in EF2 of CaM had a tendency to gain an extra water molecule as a ligand, the appearance of which coincided with a decreased contribution from the main-chain carbonyl group of the −Y ligand [12]. This ligand exchange results in two water molecules in the co-ordination sphere, a liganding scheme that increases the flexibility of the loop and perhaps the Ca²⁺ off-rate [13]. A second short equilibrium simulation on Ca²⁺-bound EF3 of the C-terminal domain of CaM found that the co-ordination number of the Ca²⁺ ion fluctuated between seven and eight as the Asp⁹⁵ (+Y ligand) flipped between monodentate and bidentate [14]. As this same phenomenon was observed in EF2 of calbindin D_9K [15], it appears that the current van der Waals parameters of Ca²⁺ that are used in the simulations, co-ordination numbers of 7 and 8, are in a quasi-equilibrium.

Non-canonical EF-loops

Although the Ca²⁺ chelation scheme outlined above is employed by the majority of EF-hands, the composition and length of functional binding loops vary significantly in the ‘EF-handome’, a term first coined by Haiech et al. [16] (Figure 4). The different non-canonical EF-loops can be classified into four groups, three of which demonstrate different ways whereby the pentagonal bipyramidal co-ordination is achieved. The first group contains EF-hands that, although they are the same length as the canonical sequence (12 residues), do not use canonical ligands to bind the Ca²⁺ ion (Figure 3C). This group contains two types of deviation. In approx. 10% of known EF-hands, an aspartic acid residue instead of a glutamic acid residue occupies loop position 12. These ‘Asp12’ EF-loops tend to be smaller and more compact than the canonical loop [17,18] a characteristic that has effects on Ca²⁺ co-ordination and Mg²⁺ selectivity (discussed later). As seen in the crystal structure of EF3 from CIB (calcium- and integrin-binding protein), the side chain of Asp12 is too short for both oxygen atoms to co-ordinate Ca²⁺, and although one does, the second is replaced by a bridging water molecule [19]. The second deviation in this group are EF-loops that bind Ca²⁺ through an increased use of main-chain carbonyl groups such as EF4 of AtCBL2 [Arabidopsis thaliana (thale cress) calcineurin B-like protein] [20]. This deviation is most likely a response to the substitution of otherwise disabling amino acids, since, through a shift in the entering helix, the main-chain carbonyl oxygen atom substitutes for the missing side-chain carboxy group.

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Figure 4 Non-canonical EF-loops

(A) Sequences of representative non-canonical EF-loops. The Ca²⁺ ligands at the co-ordinating positions are indicated in bold with those ligands provided by the carbonyl group of the backbone underlined. Indicated in parentheses is the length of the loop. Again a water molecule directly co-ordinates the bound Ca²⁺ ion at the −X position. (B) The loop of CaM EF1 (PDB code 1EXR) indicating the canonical arrangement. (C) EF-loops that have the canonical length, but do not chelate the Ca²⁺ ion with the canonical ligands: i, CIB EF3 which, due to the short length of Asp¹², has a water ligand as the second co-ordinating group at the −Z position (PDB code 1XO5); ii, AtCBL2 EF4 has a lysine residue at the +Y position and as such chelates the Ca²⁺ ion through the backbone carbonyl group (PDB code 1UHN). (D) EF-loops with insertions: i, the Ψ-hand of calbindin D_9K overcomes a two-residue insertion by turning inside out and using the backbone carbonyl groups for co-ordination (PDB code 3ICB); ii, scallop myosin ELC EF1 uses backbone carbonyl groups and co-ordination from ligands provided by the entering helix to overcome an unusual separation of aspartic acid residues and a substitution of a lysine residue for the C-terminal glutamic acid ligand (PDB code 1WDC). (E) EF1 of calpain domain VI is shorter than the canonical sequence and has an additional ligand provided by a backbone carbonyl group as well as an additional water molecule (PDB code 1DVI). (F) EF5 of ALG-2 binds the Ca²⁺ ion through octahedral co-ordination since, because of an insertion, the too-distant C-terminal ligand potentially provided by a glutamine residue is replaced by a single water molecule (1HQV). In (B)–(F) the Ca²⁺ ion is represented by a yellow sphere, water molecules by blue spheres, the chelating groups as red sticks and the side chain and backbone carbonyl groups as black sticks. For clarity, position 9 of the loop, which is hydrogen-bonded to the co-ordinating water molecule, is not shown [175].

The second group of non-canonical EF-loops are those with insertions (Figure 4D). An entire subfamily, the S100s and related proteins including calbindin D_9K, contain a non-canonical EF-loop in EF1 termed the ‘pseudo-EF-loop’ forming an EF-hand variant known as the Ψ-hand (‘pseudo-EF-hand’). To accommodate a two-residue insertion in the first part of the loop, this segment is turned inside-out with the dipoles of the main-chain carbonyl groups providing the chelating +X, +Y and +Z ligands. Interestingly, the presence of these unusual liganding groups does not prevent the formation of several favourable hydrogen bonds and, at least in the case of calbindin D_9K, high-affinity Ca²⁺ binding still occurs. Main-chain co-ordinating groups are also used to overcome a three-residue insertion between the +X and +Y positions and enable pentagonal bipyramidal geometry in EF1 of AtCBL2 [20] and a one-residue insertion between these same positions in EF1 of the extracellular glycoprotein BM40 (also known as SPARC and osteonectin) [21]. Interestingly, the loops of EF1 and the canonical EF2 of BM40 can be superimposed with an rmsd (root mean square deviation) of only 0.25 Å (1 Å=0.1 nm), demonstrating that the one-residue insertion of EF1 results only in a very localized effect. The final non-canonical EF-hand that falls into this subgroup is the single functional EF-hand found in the ELC (essential light chain) of scallop (Argopecten irradians) myosin. The EF-loop of its EF1 is unusual in a number of ways: (i) although there is a significant number of aspartic acid residues present, owing to their unusual separation, main-chain carbonyl groups provide a number of ligands; (ii) a substitution of lysine for the C-terminal glutamic acid ligand is compensated for by an insertion of two residues after the first ligand, enabling Ca²⁺ co-ordination entirely by the N-terminal part of the loop; and (iii) instead of coming from a glutamic acid residue found in the exiting helix, the final two ligands are provided by two side chains of the entering helix [22].

Members of the third group of non-canonical EF-hands are rarer. At present there is only one example of a Ca²⁺ chelation loop shorter than the canonical sequence: the eleven-residue loop of EF1 in most members of the penta-EF-hand subfamily (Figure 4E). In these EF-loops, as is the case for several of the loops mentioned above, the lack of prototypic side chains in the X and Y positions is compensated for by both an alternative loop conformation that allows for the oxygen of a main-chain carbonyl group to act as a ligand and by co-ordination through an additional water molecule [23].

Of course, one way in which a non-canonical EF-loop can try to bind Ca²⁺ is to abandon the pentagonal bipyramidal co-ordination scheme. An octahedral coordination scheme is seen in EF5 of the apoptosis-linked protein ALG-2 (apoptosis-linked gene-2) (Figure 4F). The loop of this EF-hand in all known members of the penta-EF-hand subfamily contains a two-residue insertion in the C-terminal part of the loop that inactivates the site in all members except ALG-2 [24]. In this protein the Ca²⁺ is co-ordinated only by the N-terminal part of the loop and has an octahedral geometry, as the potential glutamine ligand is too distant and it is replaced by a single water molecule [25]. Octahedral co-ordination is also seen in EF1 of the conventional myosin ELC from the acellular true slime mould Physarum polycephalum [26]. The unusual conformation of this EF-loop prevents the loop's C-terminal ligands from participating in the co-ordination scheme.

The EF-hand pair

The EF-hand motif almost always occurs in pairs, creating an 11 Å distance between the two bound Ca²⁺ ions [27] (Figure 2B). Stacked against one another in a face-to-face manner, this pair forms a four-helix bundle with the amphipathic helices packed together to make a hydrophobic core. The structural integrity of the two EF-hand motifs is further stabilized through the short antiparallel β-sheet formed between the pairs' EF-loops [3]. Even though the two EF-hands in the domain are related by an approximate 2-fold axis of symmetry that passes through the eighth position of the loop [10], they are not identical. In fact, an EF-hand's position in a pair is strictly defined and designated as either ‘odd’ or ‘even’. This asymmetry translates into asymmetric dynamic and Ca²⁺-binding properties well documented in CaM [28,29] and calbindin D_9K [30,31].

Two pieces of evidence point to the pervasiveness of this pairing. Firstly, it is maintained even if one of the EF-hands is non-functional, a good example of which is seen in the Ca²⁺-bound forms of the SCPs (sarcoplasmic Ca²⁺-binding proteins) of the invertebrates Nereis diversicolor (a ragworm) and Branchiostoma (formerly Amphioxus; the lancelet). For Nereis SCP, although EF2 is non-functional, owing to an extensive insertion, it is still paired with a functional EF1 and the two-fold symmetry is approximately preserved [18]. In the case of Branchiostoma SCP, the non-functional EF4 is again paired with a Ca²⁺-bound EF3, but in this case the antiparallel β-sheet between the two EF-hands is lost [17].

The second piece of evidence is provided by peptide studies on individual EF-hands. The hydrophobic packing of the EF-hand motif provides a strong driving force for peptide dimerization, the extent of which has been demonstrated in experiments using isolated EF-hands from several EF-hand containing proteins, including CaM [32], TnC (troponin C) [33,34], calbindin D_9K [35] and Nereis SCP [36]. These peptides are observed to form homodimers in the presence of Ca²⁺, which includes a short β-sheet formed between the loops. Tellingly, when a homodimer is mixed with its natural partner, a heterodimer is preferentially formed at the expense of the homodimer. This suggests a highly specific packing of the core [11], a suggestion corroborated by the 200-fold increased affinity of the heterodimer compared with the homodimer for Ca²⁺ [37,38]. Interestingly, in addition to dimer formation, there is a cation-induced transition from a random-coil structure to a helix–loop–helix conformation, a transition which may have important implications for protein folding [39]. Finally, the interdependent folding of a pair of Ca²⁺-binding sites is supported by calorimetric studies showing that the Ca²⁺-saturated forms of TnC and CaM unfold as a ‘co-operative block’ [40].

Even though the structure of the EF-hand pair is highly conserved throughout this protein family, diversity is introduced through both structural additions to the N-terminus of the Ca²⁺-binding domain and by modifications to both the length and composition of the linker connecting the two EF-hands. An additional and significant level of diversity is also introduced through the topic of the next subsection – the organization of the EF-hand domains in a protein.

EF-hand domain organization

The smallest member of this family is calbindin D_9K, a 9 kDa protein which contains a single EF-hand pair (Figure 5). Although the other members of the S100 subfamily to which this protein belongs also contain a single pair of EF-hands, these members occur as part of either homo- or hetero-dimers, giving each of the S100 proteins four EF-hands. Most of the proteins of the EF-hand family contain two pairs, each similar to calbindin D_9K, totalling four EF-hand motifs. Three different arrangements of the two domains have been observed, depending on the conformation and length of the linker between the domains of paired EF-hands. In the first, two independent globular domains are connected by a flexible linker. As seen for sTnC (skeletal-muscle TnC) [41], and extensively for CaM [42], the plasticity of the linker in this arrangement allows the two EF-hand domains to take up a variety of orientations with respect to each other, enabling an extensive array of target interaction arrangements. In the second conformation, which is seen in the structure of recoverin and other members of the NCS (neuronal Ca²⁺-sensor) subfamily, a U-shaped linker between the domains places the four EF-hands in a compact tandem array on one face of the protein [43,44]. Here, the relative disposition of the domains is fixed by intra- and inter-domain contacts. The final observed arrangement for the four EF-hands is the compact globular fold seen in the structure of the invertebrate SCPs [17,18]. In this conformation, the two pairs of EF-hands reside on opposite faces of the compact molecule. In fact, the highly apolar surfaces of the two domains exhibit such an affinity and specificity for each other that they preclude recognition and binding to the hydrophobic surface of a target protein [45]. Finally, there are those members of the EF-hand superfamily which contain six EF-hands, including the neuroprotective protein calbindin D_28K and its homologue calretinin, as well as a subfamily of ER regulatory proteins, including reticulocalbindin (see Supplementary Table 1 and references cited therein). For calbindin D_28K the three pairs of EF-hands form a compact and ellipsoid-type conformation similar to that of the NCS subfamily [46]. In these larger EF-hand-containing proteins, rarely are all the EF-hands active in binding Ca²⁺.

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Figure 5 EF-hand domain organization

(A) Calbindin D_9K is the smallest member of this family and contains a single pair of EF-hands (PDB code 3ICB). (B) Parvalbumin is a three-EF-hand protein with the non-functional EF-hand serving to stabilize the Ca²⁺-binding pair (PDB code 4CPV). (C) EF-hand proteins with four EF-hands: i, the two independent domains of CaM are connected by a flexible linker (PDB code 1EXR); ii, in recoverin the two domains are placed in tandem on the same face of the protein (PDB code 1JSA); iii, in Nereis SCP the EF-hands form a compact globular fold with the pairs of EF-hands on opposite sides of the molecule (PDB code 2SCP). (D) Domain VI of calpain is a member of the PEF (penta-EF-hand) subfamily and has five Ca²⁺-binding motifs, although the fifth EF-hand does not remain unpaired, since it promotes dimerization to create a ten-EF-hand structural unit (PDB code 1DVI). (E) Calbindin D_28K contains three paired EF-hand domains totaling six EF-hands (PDB code 2G9B). As this is an NMR structure, no direct information on the Ca²⁺ ions is obtained. In (A)–(E) the first EF-hand pair is coloured purple/pink, the second is blue/turquoise and the third is brown/orange. Helices that are not part of the EF-hands are coloured grey [175].

Because the EF-hand motif has a strong tendency to be part of a pair, examples of EF-hand proteins that contain an odd number of EF-hands are rare. A single, functional EF-hand is found in the C-terminal domain of the CaV1.2 subunit of the voltage-gated Ca²⁺ channel. Despite the non-canonical sequence found in its EF-loop, this motif is believed to bind bivalent cations and may function in Ca²⁺ homoeostasis [47]. For the parvalbumins, a three-EF-hand protein subfamily, the unpaired EF1 lacks several characteristic features of the EF-hand motif. Functionally, this site does not bind Ca²⁺, but serves to stabilize the Ca²⁺-binding EF2/3 pair by behaving like an endogenous peptide bound to the C-terminal domain in a manner reminiscent of Ca-CaM–peptide interactions [48]. Interestingly, substitution of a canonical loop into this inactivated EF-hand does not restore Ca²⁺ binding and causes an overall destabilization of the structure [49]. Finally, an entire subfamily contains five EF-hands. However, the function of this fifth hand is to promote self-association to create a ten-EF-hand structural unit.

This dimerization property of the EF-hand is particularly interesting, as it suggests that, in addition to being a Ca²⁺-binding unit, it can also serve as a dimerization motif. In structures of penta-EF-hand subfamily members, EF5 of the first monomer is packed against the equivalent section of the second, burying one-fifth of the total surface of each monomer and forming a tightly associated four-helix bundle [23,25,50,51] (Figure 6A). Pairing between fifth EF-hands is also used to mediate heterodimer formation between domains IV and VI of calpain as well as ALG-2 and peflin [52]. Interestingly, the structure of the EF5/5′ pair mimics that of the traditional EF-hand pair, including the antiparallel β-sheet.

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Figure 6 Intermolecular organization of EF-hand proteins

(A) The homodimer of calpain domain VI formed through interactions between EF5/5′ (PDB code 1DVI). One monomer is in purple; the other is in blue. (B) The structure of apo-calpain, a multidomain protein with the different domains highlighted by different colours (PDB code 1DF0). Domains IV and VI (orange and magenta respectively) are members of the PEF subfamily and interact via their unpaired EF5 [175].

In the previously discussed proteins, the EF-hand is the major structural unit. This is not always the case, as this motif also occurs as one domain of a multidomain protein (see Supplementary Table 1). In many instances, such as that seen for domains IV and VI of calpain (Figure 6B), the EF-hand pair mediates domain–domain interactions. Similarly, the EF-hand domain in both the P. polycephalum protein CBP40 [53] and Cb1 [54] is near the centre of the protein, where it creates a large interface (∼1500 and ∼800 Å respectively) that serves to position the other domains.

The topics discussed in this subsection, the Ca²⁺ ion co-ordination scheme, the structural unit of the EF-hand pair, as well as the organization of this pair in EF-hand containing proteins, highlights the adaptability of the EF-hand Ca²⁺-binding motif. Owing to the plasticity of the loop, site-specific mutations, insertions and deletions introduced over time are accommodated in many cases through non-canonical Ca²⁺ co-ordination mechanisms. Variation in the amino acid composition of the EF-hand helices leads to different helix packing interactions between the EF-hands as well as between a given EF-hand domain and the rest of the protein. Finally, an additional level of complexity to this motif is provided by the diversity of the response to Ca²⁺ binding, a topic that is discussed in the next section.

Ca²⁺ binding and induced structure formation

Traditionally, the role of Ca²⁺ binding has been examined through the lens of signal transduction, which focuses on the Ca²⁺-induced conformational changes and their effects on target interactions. Although this consequence of Ca²⁺ binding is important and will be discussed in the next paragraph, Ca²⁺ can also play a de novo structural role and, in many cases, the binding of this cation is key to the structural integrity of the protein. EF-hands that serve a structural function have a Ca²⁺ affinity high enough for Ca²⁺ to be bound even in the resting cell. Proteins such as the sarcoplasmic CaBPs (the invertebrate SCPs [55,56]), the C-terminal domain of CaVP (calcium vector protein) [57] and the prokaryotic protein calerythrin [58] all exhibit an unfolded, molten-globule-like state in the absence of Ca²⁺ in vitro. Another example is the bilobal TnC, in which the two EF-hands of the C-terminal domain have an approx. 100-fold higher affinity for Ca²⁺ than have the N-terminal EF-hands – an affinity high enough to ensure Ca²⁺ saturation in the resting cell. Like the aforementioned proteins, the C-terminal domain of TnC is unfolded in the absence of Ca²⁺ [59]. When compared with the N-terminal domain, this lack of structure is thought to be due to the absence of stabilizing hydrophobic interactions that overcome the destabilizing electrostatic interactions, the main source of which are the carboxy ligands of the EF-hand motif [60]. Interestingly, a structural role for the EF-hand has also been suggested for several multidomain signalling proteins, since for both phospholipase Cδ [61] and EH (Eps15-homology) domains [62], Ca²⁺ binding has no effect on the function of the protein. In contrast with these proteins, in which Ca²⁺-binding induces structure formation, most EF-hand-containing proteins are structured in the apo state, and the binding of this cation leads to a conformational change through which this protein transduces the message of an increased Ca²⁺ concentration.

Ca²⁺ binding and conformational change

Initially this Ca²⁺-induced conformational transformation was considered in terms of a closed-to-open domain transition quantified through the change in interhelical angle of vectors defined by the helices that bind Ca²⁺. For the members of the CaM subfamily and several other EF-hand-containing proteins, these helices, antiparallel in the apo structure with interhelical angles of 130–140°, open upon Ca²⁺ binding to become roughly perpendicular with an angle that approximates to 90° [63,64] (Figure 7A). Through this change in angle there is a loss of interhelical contacts between the entering and exiting helices of the EF-hand as the top where Ca²⁺ binds is ‘pinched’ together and the bottom moved apart – a conformational change that exposes once-buried hydrophobic side chains and creates an extensive target-protein interaction site. In CaM these surfaces are approx. 10 Å×12.5 Å in each domain [65].

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Figure 7 Ca²⁺-induced conformational changes

(A) The closed-to-open transition as seen in the N-terminal domain of CaM. The helices move from a more antiparallel arrangement in the apo form (i) to being nearly perpendicular in the Ca²⁺-bound state (ii). Hydrophobic residues that were buried in the apo form are now exposed to the solvent. (B) Changes in the loop structure upon Ca²⁺ binding. The apo loop from EF1 of CaM is indicated in magenta, with the Ca²⁺ ligands in red. The Ca²⁺-bound loop is in turquoise, with the ligands in green. The bound Ca²⁺ ion is represented by the yellow sphere, and the water ligand is omitted for clarity. In the apo structure, most of the N-terminal ligands of the loop are in place to bind the Ca²⁺ ion. The major difference between the structures is seen in the placement of Glu12, which must move several angstroms to chelate the Ca²⁺ ion [PDB code for (A)–(B) 1EXR] [175].

At a molecular level this conformational change appears to be a consequence of Ca²⁺ chelation by the EF-loop. Initially the Ca²⁺ ion is bound by ligands in the N-terminal part of the loop (1, 3, 5 and 7), as this region is quite flexible and ‘waves around’ to catch the ion [14]. At this point the C-terminal ligands, predominantly those provided by the twelfth position, are too far away to chelate the bound Ca²⁺ ion directly (Figure 7B). In order for the loop to complete the ion's co-ordination sphere (an energetically favourable process), the exiting helix must be repositioned by ∼2Å. It is the movement of this helix that causes the conformation change in the EF-hand.

The repositioning of Glu12 is stabilized not only through the Ca–O ‘bonds’ that form, but also through two new hydrogen bonds between this side chain and the backbone at positions 2 and 9. The importance of these hydrogen bonds to the Ca²⁺-induced conformational change has been seen in structural studies in which this glutamic acid residue is replaced with a glutamine residue [28,66,67]. The inability of the glutamine residue's side chain to simultaneously form both of these hydrogen bonds leads to a conformational exchange between two conformations similar to those of the closed and open states. This conformational exchange suggests that, unlike in the wild-type protein, the equilibrium here is not being pushed towards the open structure and that one additional hydrogen bond (∼12 kJ/mol) is required to shift the balance [66].

This two-step binding pathway suggests an explanation for the observed asymmetry in the EF-hand pair mentioned above. As the linker between EF1 and EF2 provides a strong covalent coupling between helices II and III of the pair, rotation of helix II will cause pulling on helix III, forcing it to follow and re-orient. By contrast, as there is no covalent link between helices I and IV, a rotation of helix IV by itself can occur more readily. The consequence of this smaller conformational cost of Ca²⁺ binding to EF2 compared with EF1 is seen in several EF-hand proteins, including CaM, TnC and CIB, where the second EF-hand of the pairs is filled first, since the binding affinity is higher.

The closed-state-to-open-state conformational change seen in the CaM subfamily is not observed in all EF-hand subfamilies. For the S100s, Ca²⁺ binding induces a ‘change in hand’-type motion, as the interhelical angle between the entering and exiting helices of EF2 is modified from a negative angle to a positive one similar to that seen in CaM [68]. For recoverin and other members of the NCS subfamily, Ca²⁺ binding results in domain rotation. Through this rotation a myristoyl group that had been buried within protein core was exposed, leading these proteins to translocate to the plasma membrane [64]. In sorcin and potentially all other members of the penta-EF-hand subfamily, though Ca²⁺ binding seems to have little structural effect on the EF-hand domain itself, due to altered interhelical interactions, a large conformational change is thought to occur as the contacts between the C-terminal EF-hand domain and the N-terminal domain are weakened. This results in the exposure of a large hydrophobic protein target binding site [69,70]. Interestingly, for the aforementioned proteins, and as in the CaM subfamily, mutational studies indicate that it is still chelation by the ligands provided by the 12-loop position that trigger the Ca²⁺-induced conformational change. Finally, the Ca²⁺-buffer proteins calbindin D_9K and the parvalbumins experience minimal structural changes upon Ca²⁺ binding.

The diversity of EF-hand conformations has been attributed to the torsional flexibility of a region of the loop termed the ‘EFβ-scaffold’ [3]. Encompassing positions 6–8 the EFβ-scaffold forms a well-defined hinge around which the rotation of a few bonds enables the Ca²⁺-induced conformational change. Amazingly, orientations of the exiting helix differing by as much as 30° may be compatible with Glu12 binding in the Ca²⁺-co-ordinating position. Significantly for the latter proteins mentioned in the previous paragraph, through rotation of these bonds the EFβ-scaffold is able to close the loop on its own without requiring a large change in the interhelical angle.

The above paragraphs describe fixed conformations for the Ca²⁺-bound and -unbound EF-hand pair which, owing to protein dynamics, probably does not reflect the reality of the situation. From the anisotropic and anharmonic disorder seen in the 1.0 Å crystal structure of Ca-CaM [71] and NMR relaxation data from the structures of both apo and Ca-CaM [29,65,72–74], a model was proposed whereby CaM samples quasi-continuous substates rather than cycling between two well-defined conformations that depend on the Ca²⁺-bound status. Both apo- and Ca-CaM sample very large and partially intersecting volumes of conformational space, explaining the open-type conformation sometimes seen for apo-CaM and the closed-type conformation sometimes seen for Ca-CaM. Similar to the classical Monod–Wyman–Changeux model, the binding of Ca²⁺ to the unbound form stabilizes certain substates at the expense of others, as it results in structural changes in the EF-hands which decrease the frequency with which the closed substates are sampled. This suggests that, at least for CaM, the Ca²⁺-bound and apo forms seem to be less distinct structurally, which helps to explain the plasticity of this protein in the presence of its target proteins. The dynamics that enable the sampling of many substates is attributed to the flexibility in the backbone angles of the loop residues, predominantly those of position 8.

The Ca²⁺-induced conformational changes discussed in this section have energetic consequences on the ability of each EF-hand to bind this cation. These energetic consequences will be discussed in the next section.

Determinants of intrinsic Ca²⁺ affinity

The EF-hand presents an intriguing mystery, as the Ca²⁺ dissociation constants found in this protein family range from 10⁻⁹ to 10⁻⁴ M, with no major correlation between affinity and the nature or position of the ligands (Supplementary Table 2). However, if one considers thermodynamics and the following well-known equations, a few conclusions can be made: (2) Where R is the gas constant and T is the temperature. (3) Where where ΔH is the change in the enthalpy of the system, T is the temperature in degrees kelvin and ΔS is the change in entropy. The intrinsic Ca²⁺ affinity is determined by the difference in the Gibbs' free energy (ΔG) between the unbound and bound states. Clearly, the larger and more negative the ΔG, the higher the affinity. At a constant temperature, phenomena that increase ΔS or decrease ΔH between the unbound and bound states should lead to higher affinity (Figure 9).

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Figure 9 Determinants of the intrinsic Ca²⁺-binding ability of the EF-hands

(A) The effect on affinity (ΔG_tot) of water molecules remaining in the co-ordination sphere. As the increase in solvent entropy is thought to be the major contributor to the free energy of EF-hand Ca²⁺-binding, those EF-hands in which two water molecules remain will bind Ca²⁺ with a lower affinity than those in which all of the Ca²⁺ ligands are provided by the protein. (B) The intrinsic Ca²⁺ affinity of an EF-hand (ΔG_tot) is dependent upon a balance between favourable and unfavourable enthalpic and entropic factors. For each EF-hand the characteristics of these factors and the balance between them are unique. (C) The relative stability of both the apo and the Ca²⁺-bound states also affect an EF-hand's intrinsic Ca²⁺ affinity. The more stable the apo state or unstable the Ca²⁺-bound state compared with a reference denatured state (ii versus i), the lower the Ca²⁺ affinity of a given EF-hand.

EF-hands and Mg²⁺ selectivity

In the cell, the EF-hand selectively binds Ca²⁺ in the presence of 10³–10⁵-fold higher concentrations of K⁺ and Na⁺. Explained through the electrostatic repulsion model [111], the negative charge of the EF-hand co-ordinating array is adjusted so that these univalent cations lack sufficient positive charge to stabilize the repulsion between the co-ordinating oxygen atoms. Owing to its greater charge, Ca²⁺ and other similar-sized bivalent and tervalent cations are able to overcome this O–O repulsion. In the test tube, this motif has been shown to bind spherical metal cations from groups Ia, IIa and IIIa, including the lanthanides [112], a property that has been exploited in several in vitro experiments. As most of the cations found in these groups have extremely low concentrations in vivo, competition between Ca²⁺ and these ions is not a significant factor for EF-hands. The high concentration of free Mg²⁺ in the cell (0.5–2.0 mM) does, however, pose a problem, as competition from this cation decreases the observed affinity of many EF-hands [113].

Ca²⁺-specific and Ca²⁺/Mg²⁺-binding EF-hands

The similar properties of Ca²⁺ and Mg²⁺ lead to a complicated case of cation selectivity, as CaBPs must be able to discriminate against a 10²–10⁴-fold excess of Mg²⁺. The selectivity of Ca²⁺-specific sites is thought to be provided by two distinct properties of these metal ions, namely (i) the stringent requirement of Mg²⁺ for six ligands arranged in an octahedral geometry and (ii) Mg²⁺'s smaller ionic radius, resulting in a higher energetic cost of dehydration [114]. By controlling ligand flexibility, EF-hands can exclude Mg²⁺ by preventing the required sixfold co-ordination or the preferred formation of a smaller binding site. Despite these selectivity mechanisms, owing to the excess Mg²⁺ concentration, many Ca²⁺-specific EF-hands bind Mg²⁺ in the resting cell. NMR studies indicate that the EF-hands of CaM bind Mg²⁺ with dissociation constants in the millimolar range [115], implying that the protein is almost 50% saturated with this cation at resting Ca²⁺ levels [116]. Although Mg²⁺ binds the same sites as Ca²⁺, in the Ca²⁺-specific sites Mg²⁺ appears to bind only the ligands of the N-terminal part of the loop (1, 3, 5 and 7) (Figure 10A). By not engaging the C-terminal residues of the loop, including the critical twelfth residue, Mg²⁺ binding has no effect on the overall conformation of the EF-hand, and these sites remain ‘off’. This inconsequential binding is supported by the fact that Mg²⁺ causes only localized conformational rigidity in the EF-loop, and the structure of Mg-CaM is nearly the same as apo-CaM [115]. A similar effect is seen with Mg²⁺ binding to the Ca²⁺-specific EF-hands of calbindin D_9K [117].

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Figure 10 Mg²⁺ binding to EF-loops

(A) The Ca²⁺-specific EF-loop of calbindin D_9K EF2. In this EF-loop, Mg²⁺ is only bound to the N-terminal ligands of the loop. The potential ligand provided by Glu12 is too far from the bound cation to be used; instead a water molecule is found here (PDB code 3ICB). (B) The Ca²⁺/Mg²⁺ loop of parvalbumin EF3. With Mg²⁺ bound, the side chain of Glu12 is rotated 120° to provide only one ligand (i), contrasting with the bidentate ligand used for Ca²⁺ co-ordination (ii) (PDB codes 4PAL and 4CPV respectively). (C) The Ca²⁺/Mg²⁺ loop of scallop myosin RLC. The side chain at position 12 of this loop is provided by an aspartic acid residue, creating a smaller binding site that more greatly favours binding of Mg²⁺ (PDB code 1WDC). In (A)–(C) the Mg²⁺ ion is represented by a magenta sphere, the water ligand as a blue sphere, the protein ligands as red sticks and the backbone and side chain carbonyl groups are black. For clarity, position 9 of the loop, which is hydrogen-bonded to the co-ordinating water molecule, is not shown [175].

In contrast with the Ca²⁺-specific EF-hands, Ca²⁺/Mg²⁺ sites have an Mg²⁺-affinity severalfold higher and, at least in the resting cell, functionally bind the cation. This second class of site binds Mg²⁺ in almost the same manner as it binds Ca²⁺, including the use of a ligand provided by the twelfth loop residue. In fact, this side chain is thought to play a key role in adapting the number of co-ordination ligands of the Ca²⁺/Mg²⁺ EF-loop for the different cations. Seen in the Mg²⁺-bound structures of the Ca²⁺/Mg²⁺ sites of parvalbumin [118] and a molecular-dynamics simulation of the C-terminal domain of sTnC [119], through a 120° rotation of the C_α–C_β bond of Glu12, this amino acid provides a bidentate ligand for Ca²⁺ and a monodentate ligand for Mg²⁺ (Figure 10B). This bond rotation has also been observed in the Asp¹² EF-loop of molluscan myosin RLC (regulatory light chain) [22,120] (Figure 10C). In Ca²⁺/Mg²⁺ EF-hands, the flexibility of this side chain is critical, since, if it is sterically blocked into a bidentate configuration, these EF-hands become Ca²⁺-specific [121]. As rotation of a side chain is limited by the local environment, it is difficult, if not impossible, to predict from the primary structure whether a given EF-hand is a Ca²⁺- or a Ca²⁺/Mg²⁺-specific site. More commonly for Asp12 loops, rotation of the C_α–C_β bond at this position does not occur, and this residue provides a single ligand for both Ca²⁺ and Mg²⁺ [19,122]. As this ligand arrangement favours Mg²⁺ binding at the expense of Ca²⁺, it leads to a reduction in cation selectivity and explains the tendency of Asp12 loops to be Ca²⁺/Mg²⁺ sites. Finally, unlike Ca²⁺-specific EF-hands, Mg²⁺ binding to Ca²⁺/Mg²⁺ sites induces conformational changes that typically lead to structure formation from a molten globule. It is thought that, as for Ca²⁺ binding, the ligand provided by the twelfth position is probably key to this structural change.

EF-hands and co-operativity

The third factor that influences an EF-hand's ability to bind Ca²⁺ is co-operativity of ligand binding. Through interactions with their partner, many EF-hands bind Ca²⁺ with positive co-operativity, since binding of Ca²⁺ to the first site enhances the affinity of the second site for this ion. The effect of this phenomenon on the EF-hand pair is a greater sensitivity to an increased Ca²⁺ concentration when compared with that of an isolated, independent EF-hand, since it enables a small change in the Ca²⁺ concentration to control the physiological response (Figure 11A). Electrostatically, Ca²⁺ binding to an EF-hand pair would be expected to be an example of negative co-operativity, since the presence of the first cation not only neutralizes charged groups, but, owing to the close proximity of the binding sites, would repel the incoming Ca²⁺. As positive co-operativity is observed, the favourable structural effects of the first Ca²⁺ binding must outweigh the unfavourable electrostatic factors.

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Figure 11 Positive co-operativity of Ca²⁺ binding

(A) Comparison curves for sequential (circles) and co-operative (squares) Ca²⁺ binding. The curves were created from the Ca²⁺ binding constants for CIB (sequential) [77] and the C-terminal domain of CaM (co-operative) [104]. Also indicated is the free Ca²⁺-concentration in the resting and activated cell. (B) The different pathways of Ca²⁺-binding to an EF-hand pair, with the corresponding ΔG and affinity constants specified. (C) The short β-sheet that links residues 7 and 8 of both EF-loops. Indicated are the hydrogen-bond and dipole interactions that are thought to enable the Ca²⁺-binding sites to communicate and enable positive co-operativity. (C) is taken from [27] and is reproduced with the approval of the authors.

Positive co-operativity is an affinity enhancement and can be defined in terms of free energy where ΔΔG_co-op reflects the difference in affinity of one site in the presence and absence of a Ca²⁺ ion in the other [104]: (4) Of course, as most proteins are asymmetrical ΔG_(II,I)≠ΔG_{(I, II)}, ΔG_(I)≠ΔG_(II), and ΔG_{I, II}−ΔG_II≠ΔG_II,I−ΔG_I [81] (Figure 11B). The ΔΔG_co-op is related to the observed cation affinity through the microscopic binding constants in the following equation (K_I and K_II are the intrinsic affinity constants): (5) ΔΔG_co-op is the free energy of inter-site interactions, a coupling term that in many publications is simply defined as ΔΔG. If the affinity for the second ion is greater than the first, this term is negative, indicating positive co-operativity; however, some degree of co-operativity could still take place if this value is greater than zero [6]. As ΔΔG_co-op is an energetic term, it is more useful than the traditional Hill coefficient for examining the effects of positive co-operativity on Ca²⁺ binding. For calbindin D_9K the free energy of co-operativity is −5 kJ/mol at moderate salt concentrations (0.15 M) [134], a value that results from many contributions and tends to be small because of the cancellation among large terms. For example, compensation for the electrostatic repulsion between the two Ca²⁺ ions implies that the sum of the favourable structural effects associated with ion binding is larger (more negative) than the net free energy of co-operativity [135].

The mechanisms of co-operativity are varied and hard to determine, since this requires characterization of the various metal-bound states along the different binding pathways – a requirement difficult to achieve owing to the co-operative nature of Ca²⁺ binding. However, the intermediate states of a number of EF-hand proteins have been characterized and conclusions have been drawn regarding the conformational effects and molecular mechanisms of positive co-operativity.

Effects of protein–protein interactions

The fourth and final factor that affects the observed Ca²⁺ affinity of several EF-hands is the presence or absence of intermolecular interactions. For several EF-hand domains that are part of multidomain structures, the other domains are required for high-affinity Ca²⁺ binding. In isolation, the Ca²⁺-specific EF-hand in the ELC does not bind Ca²⁺, since this binding site requires a co-operative stabilizing interaction with both the RLC and the heavy chain of the intact myosin hexamer [156]. For BM40, high-affinity Ca²⁺-binding requires the paired EF-hand domain to be embedded in the larger EC domain [157]. In this manner BM40 contrasts with the small cytosolic EF-hand proteins in which the EF-hand pair itself is a stable unit capable of high-affinity Ca²⁺ binding.

An increase in Ca²⁺-affinity is seen to result from the interaction between Ca²⁺ sensors and their targets. This effect has been well-documented through the use of peptide models to represent target- binding domains of the CaM subfamily members CaM [158–162], sTnC [125], calmodulin-like skin protein [163], the Ca²⁺ regulatory subunit of the phosphatase calcineurin, calcineurin B [164] and CaVP [57]. It is thought that there is a transfer of the free energy of target binding to that of Ca²⁺ binding, since the presence of different target peptides has been seen to increase the affinity of these proteins for Ca²⁺ by up to two orders of magnitude [165]. This free energy coupling (ΔΔG_interact) provides a quantitative measure of the enhancement in Ca²⁺ affinity in the presence of the peptide, or, equivalently, the affinity enhancement for the peptide when Ca²⁺ is bound. In fact, ΔΔG_interact is high enough to restore the Ca²⁺-binding abilities of E12Q loop mutants [166]. However, in these cases the affinity of target binding is weakened, which likely reflects the energetic cost of generating the appropriate Ca²⁺-binding structure in the mutant loop. Interestingly, different target peptides affect the sensors differently, indicating another method whereby EF-hand proteins can tune their Ca²⁺-affinity.

A phenomenon that seems to be the result of intermolecular interactions, the increase in Ca²⁺ affinity is probably due to three consequences of target binding. First, the amphipathic nature of the peptides allows them to shield the exposed hydrophobic patches that result from Ca²⁺-binding sensor proteins such as CaM [167]. As the exposure of these patches is entropically unfavourable and takes away from the energy of Ca²⁺binding (Figure 9B), the fact that the presence of the peptide covers them causes the Ca²⁺ affinity to increase. Secondly, owing to intermolecular interactions, there is a decrease in the Ca²⁺ dissociation rate, which leads to an increase in affinity [158]. This decrease in the dissociation rate may reflect a steric hindrance to Ca²⁺ dissociation and functionally serve to extend the lifetime of the activated protein complex long after the Ca²⁺ transient has subsided. The third mechanism whereby target binding increases Ca²⁺ affinity is through stabilization of the Ca²⁺-bound state. As mentioned above, in the absence of the target, even with Ca²⁺ bound, there is conformational exchange between open and closed structures. However, the presence of the target shifts this equilibrium towards the open conformation, i.e. the closed conformation is now more rarely sampled, if ever [165]. This stabilization of the Ca²⁺bound state itself would lead to a marked decrease in the Ca²⁺ dissociation rate.

The presence of the protein target also has effects on the positive co-operativity. For the interaction between CaVP and CaVPT [168], myristoylated recoverin and rhodopsin kinase [169], and CaM with mastoparan or caldesmon [170], the presence of the target increases the observed co-operativity. That this occurs is not surprising, since, for CaM especially, the presence of the peptide decreases the entropic costs of subsequent Ca²⁺ binding as, once bound to the C-terminal domain, it will cover any exposed hydrophobic surfaces in the N-terminal domain that result from Ca²⁺ binding here, thus increasing the observed affinity. In addition, the more compact structure that forms as CaM collapses around the target enables communication between the helices of the two lobes (perhaps as in yCaM) [171].

This effect of target binding on the observed Ca²⁺ affinity has significant functional consequences. The first is that, for CaBP–target interactions that are constitutively bound together in the cell such as CaVP and its target [57], the unbound Ca²⁺ affinities of these proteins are not physiologically relevant. The second is that, because of this increase in Ca²⁺ affinity, there is a decrease in the effect of Mg²⁺ competition.

There is evidence, however, that the extent of the in vitro effect of the target peptide on Ca²⁺ affinity may not be quite as large in vivo. For the interaction between skMLCK (skeletal-muscle myosin light-chain kinase) and CaM, the peptide enhances affinity 220-fold, whereas the intact protein only enhances affinity 13-fold. These affinities result in only partial Ca²⁺ occupancy of the CaM–skMLCK complex at resting cellular Ca²⁺ levels, a state required for effective Ca²⁺-CaM-mediated activation in vivo. By contrast, the much higher affinities observed for the skMLCK–peptide complex would predict significant Ca²⁺ occupancy and activation, even in the absence of a Ca²⁺ signal. This difference in effect likely stems, at least in part, from the energetic cost of removing an autoinhibitory domain from the intact protein, an effect also seen for the full-length Ca²⁺-ATPase molecule [165].

Finally, target proteins can have a negative effect on the observed Ca²⁺ affinity. In the complex of the Ca²⁺-activated K⁺-channel bound to Ca-CaM, Ca²⁺ binds only to the N-terminal domain EF-hands, since the higher-affinity sites in the C-terminal domain have their affinities reduced so that Ca²⁺ can no longer bind. In the structure formed upon target interactions, the EF-hands in the C-terminal domain are found in the apo state, since a severe disruption in the Ca²⁺-co-ordination spheres of this domain causes Glu12 of both EF-loops to be removed from their Ca²⁺-binding positions [172]. This prevents Ca²⁺ from binding to these EF-hands. The evolutionary pressures of target-protein interaction may also be responsible for the different combinations of functional EF-hands in subfamilies such as the NCS subfamily, as adaptation to different target proteins leads to the inactivation of some EF-hands [173].

In this final part of this review we have tried to link thermodynamic studies on Ca²⁺ affinity to the molecular structures talked about in the above two sections. This final part has also stressed the importance of the many contributing factors to general EF-hand affinity. The Ca²⁺-binding abilities of each specific EF-hand is determined by an individual combination of these factors. The consequence of this is that it is nearly impossible from the primary sequence alone to predict the Ca²⁺ affinity of a given EF-hand containing protein.