The X-ray crystal structure of D.rerio secretagogin. (A) Secretagogin monomer consists of six EF-hand motifs arranged in pairs to form three globular domains (rendered consecutively in red, yellow and cyan). Linkers L1 and L2, highlighted in green, connect the individual domains. The apposed metal binding loops form antiparallel β-sheet on the outer surface of the V-shaped molecule. The helices of the individual EF-hand motifs are labeled for clarity. (B) A stereo view of Cα-trace of the superposed domains I, II and III (red, yellow and cyan, respectively) of secretagogin. The topologically equivalent motifs EF1, EF3 and EF5 overlap well, except for the Ca²⁺-binding loop which adopts the open, “Ca²⁺-ready” conformation in EF5-hand (cyan arrow) and closed, “Ca²⁺-free” conformation in EF1 and EF3 motifs. The EF2 hand differs from all the remaining EF-hands due to a break at Met63 in the helix E2 (red arrow). (C) A stereo image of the calcium-binding loop in EF5-hand motif of D. rerio secretagogin. A final 2mF_o-DF_c electron density map (blue mesh) is contoured at 1.2σ level. The refined protein model is shown in sticks. Residues of the calcium-binding motif at positions 1 (Asp206), 3 (Ser208), 5 (Thr210), 7 (Ala212) and 12 (Glu217) are labeled for clarity. A symmetry molecule of secretagogin from the crystalline lattice contributes Lys138 (cyan sticks), which mimics the positively charged calcium ion and stabilizes the loop in “Ca²⁺-ready”-like conformation.

Multiple sequence alignment of D. rerio secretagogin and selected hexa-EF-hand proteins. Amino acid sequences of proteins are labeled by their UniProt identifiers or pseudo identifiers (in italics): SEGN_DANRE, zebrafish secretagogin; SEGN_XENLA, African horned frog secretagogin; SEGN_CHICK, chicken secretagogin (Gene Bank gi:118086706); SEGN_OPOSS, opposum secretagogin (Gene Bank gi|126322175); SEGN_PIG, porcine secretagogin; SEGN_BOVIN, bovine secretagogin; SEGN_HUMAN, human secretagogin; SERGN_RAT, rat secretagogin; CALB1_HUMAN, human calbindin D_28K; CALB2_HUMAN, human calretinin. Individual domains and linker regions of hexa-EF-hand proteins are aligned in three blocks to reveal interdomain conservation patterns. Positions of helices in D. rerio secretagogin are depicted above the alignment and secondary structure elements are labeled and color-coded as in Fig. 1(A). The calcium binding loops are boxed and position of the residues within the calcium-binding consensus motif is given for easy orientation.

D. rerio secretagogin and rat calbindin D_28K have different quarternary domain arrangement. The domain II and III of D. rerio secretagogin (red) were superposed with the corresponding region of rat calbindin D_28K (blue; PDB ID 2f33). In the resulting overlay, the domain I of secretagogin is rotated by almost 180 degrees with respect to the core formed by domains II and III in both proteins. The green arrow points to the point from which structures diverge.