Crystal Structure of CBD2 from the Drosophila Na+/Ca2+ Exchanger: Diversity of Ca2+ Regulation and Its Alternative Splicing Modification

Na+/Ca2+ exchangers (NCXs) promote the extrusion of intracellular Ca2+ to terminate numerous Ca2+-mediated signaling processes. Ca2+ interaction at two Ca2+ binding domains (CBDs; CBD1 and CBD2) is important for tight regulation of the exchange activity. Diverse Ca2+ regulatory properties have been reported with several NCX isoforms; whether the regulatory diversity of NCXs is related to structural differences of the pair of CBDs is presently unknown. Here, we reported the crystal structure of CBD2 from the Drosophila melanogaster exchanger CALX1.1. We show that the CALX1.1-CBD2 is an immunoglobulin-like structure, similar to mammalian NCX1-CBD2, but the predicted Ca2+ interaction region of CALX1.1-CBD2 is arranged in a manner that precludes Ca2+ binding. The carboxylate residues that coordinate two Ca2+ in the NCX1-CBD1 structure are neutralized by two Lys residues in CALX1.1-CBD2. This structural observation was further confirmed by isothermal titration calorimetry. The CALX1.1-CBD2 structure also clearly shows the alternative splicing region forming two adjacent helices perpendicular to CBD2. Our results provide structural evidence that the diversity of Ca2+ regulatory properties of NCX proteins can be achieved by (1) local structure rearrangement of Ca2+ binding site to change Ca2+ binding properties of CBD2 and (2) alternative splicing variation altering the protein domain–domain conformation to modulate the Ca2+ regulatory behavior.