Table 1.
List of eleven non-redundant, representative calcium-bound X-ray and NMR protein complexes, which represent eleven different families of EF-hand domains.
Figure 1.
The structural elements of the EF-hand fold.
Conserved elements of the EF-hand domains include (A) the flanking “incoming” and “exiting” α-helices (helix I to helix IV), the two interacting nodes, where the flanking α-helices interact and form interacting clusters I and II (shown in black and grey circles), position of the DxDxDG calcium binding loops, and the central β-sheet. (B) Structural alignment of the Odd and Even EF-hand motifs from the eleven fold representative structures. (C) Structural superposition of amino acids from Figure 1B. In 1K94_A, B, Figure 1B, “G3” designates two different three amino acid long insertions after Gly201 in 1K94_A and after Gly501 in 1K94_B. The structural frame of reference, “X”, “Y”, “Z”, “-X”, “-Y”, “-Z” and “X-4”, designate the seven key equivalent structural positions within all EF-hand domains, as numbered in Kretsinger and Nockolds [40]. The Odd EF-hand helix-loop-helix supersecondary structure is shown in grey. In panel A, B and C, the residues that belong to cluster I and cluster II are respectively highlighted in black and grey.
Figure 2.
Example interactions within cluster I.
(A) and (B) illustrate two types of interactions between the flanking α-helices I and IV (cluster I in Figure 1). The interactions occur via amino acids at positions -X+1, X-4 and -Z+1, which are also shown as black circles in Figures 1A and 1C and highlighted in black in the alignment in Figure 1B. Because in cluster I, positions X-4 and -Z+1 are purely aromatic (Φ) in all EF-hand representative structures, cluster I is called aromatic. (C) Contains the description of interactions for the eleven EF-hand fold representatives. The pattern “(-X+1)helixIV CH-π Φ(X-4)helixI CH-π Φ(-Z+1)helixIV weak HB (-X+1)helixIV” indicates a circular interaction, where a side-chain atom of the –X+1 residue from the flanking α-helix IV forms a CH-π interaction with the ring of the X-4 aromatic amino acid from helix I, which, in turn, forms a CH-π interaction with the ring of the –Z+1 aromatic amino acid from helix IV, which interacts with the initial –X+1 residue from the flanking α-helix IV by means of a weak CH-O hydrogen bond.
Table 2.
Summarized areas of interacting surfaces among the amino acids within clusters I and II (black and grey clusters in Figure 1, respectively), and the interacting area between the two clusters (I/II), within the EF-domains of the eleven representative structures.
Table 3.
Effects of calcium binding on the conformation of all EF-hand domains whose structures are known in the apo-form and with bound Ca2+ ions, and target protein ligands.
Table 4.
Effects of calcium binding on the conformation of all EF-hand domains, whose structures are known in the apo-form and with bound Ca2+ ions, and target protein ligands (where they exist).
Table 5.
Single calcium binding EF-hand domains with known apo- and holo-form structures.
Figure 3.
Interaction of cluster I with the central β sheet.
The side chain of the aromatic residue at position X-4 in cluster I is perpendicular and directly interacts with the β-sheet linker of the EF-hand domain, contributing to its hydrophobic core. (A) Interactions between residue X-4 of cluster I (F47) and the β-sheet linker in 2PVB. (B) The interactions in the fold-representative structures include two classic, weak CH-O hydrogen bonds, whose detailed parameters are given in Table S6 in File S1. The (X-4)odd – O/(-Y-1)odd interaction shows the weak CH-O hydrogen bond between residue X-4 of cluster I and residue -Y-1 (G56 in 2PVB) of the “odd” EF-hand motif, while the (X-4)odd – O/(-Y+1)even interaction shows the weak CH-O hydrogen bond between the same residue X-4 of cluster I and residue -Y-1 (I97 in 2PVB) of the “even” EF-hand motif.
Figure 4.
Five distinguishable groups of EF-hand domains based on the degree of structural rearrangements within clusters I and II, and the inter-cluster interactions that take place upon calcium binding: (1) Open Static (open domain conformation and no conformational changes); (2) Closed Static (closed domain conformation and no conformational changes); (3) Local Dynamic (simultaneous conformational changes in clusters I and II and the entire domain); (4) Dynamic (only global conformational changes, but not in clusters I and II); and (5) Local Static (stable open domain conformation, conformational changes only in clusters I and II, but not the entire domain).
Domain level conformational changes, are shown by the small (“closed” domains) and large (“open” domains) distance between clusters I and II (triangles, ellipses and inverted triangles). Domain types (3) and (4) do undergo domain opening, while domain types (1), (2) and (5) do not. The conformation of the domains of type (2) remains “closed”, while the conformation the domains of types (1) and (5) remains open. Local conformational changes in clusters I and II are shown by normal triangles (compact conformation of cluster I), inverted triangles (compact conformation of cluster II), and ellipses (less compact, more open conformation of cluster II). The conformation of cluster I does not change in all of the known structures and is the same in buffers and sensors, such as in calbindin 9K, calmodulin and troponin C. The conformation of cluster II does change from being less compact to more compact in the domains of types (3) and (5). In group (4), EF-hand domains undergo domain opening, but the conformations of the conserved clusters I and II remain intact.