(A) A single EF-hand from the N-terminal domain of CaM. Extending for 29 consecutive amino acid residues, the EF-hand consists of a nine-residue entering helix, a nine-residue loop and an 11-residue exiting helix. (B) A pair of EF-hands from the N-terminal domain of CaM. An additional secondary-structural element is observed in the pair as a small β-sheet formed between residues in the latter part of the loops, through which passes the pseudo-2-fold axis of symmetry that relates the EF-hand pair, in most cases the functional unit of Ca2+ binding (PDB code 1EXR) .
Figure 3The co-ordination sphere of the canonical EF-loop
(A) The sequence preference of the EF-hand loop. The Ca2+ ligands are indicated by both their position in the EF-loop and in the co-ordinating array with whether or not co-ordination occurs via the side chain (sc) or through the backbone (bb) indicated below. The asterisk (*) highlights the ligand typically provided by a water molecule that is hydrogen-bonded to the side chain of the amino acid found at position 9. Also noted in the Figure are the most common amino acids at each position, with their corresponding percentages of occurrence, and those that occur with a frequency greater than 5% in known EF-loops . (B) A schematic diagram of the Ca2+ co-ordination sphere with the entering and exiting helices in red, the co-ordinating protein ligands in blue and co-ordinating water molecule (W) in teal (dark blue). Light green corresponds to the conserved glycine residue that provides the bend in the loop. Purple highlights the conserved hydrophobic residue that forms the short β-sheet in the paired EF-hand. Also indicated are the most common amino acids found at the critical positions. (C) Ca2+ co-ordination by the canonical EF-hand (EF1 of CaM) illustrating both the pentagonal bipyramidal co-ordination of the Ca2+ ion (continuous lines) and the extensive hydrogen bonding pattern found in the loop (broken lines) . The backbone NH groups are indicated in black, the side-chain oxygen atoms in red, the Ca2+ ion in yellow and the co-ordinating water in blue (PDB code 1EXR) . An interactive three-dimensional version of (C) is available at http://www.BiochemJ.org/bj/405/0199/bj4050199add.htm.
(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 D9K 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 .
(A) Calbindin D9K 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 D28K 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 .
Figure 6Intermolecular 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 .
(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] .
Figure 9Determinants of the intrinsic Ca2+-binding ability of the EF-hands
(A) The effect on affinity (ΔGtot) 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 (ΔGtot) 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.
(A) The Ca2+-specific EF-loop of calbindin D9K 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 .
(A) Comparison curves for sequential (circles) and co-operative (squares) Ca2+ binding. The curves were created from the Ca2+ binding constants for CIB (sequential)  and the C-terminal domain of CaM (co-operative) . 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  and is reproduced with the approval of the authors.