Structure-dynamic determinants governing a mode of regulatory response and propagation of allosteric signal in splice variants of Na+/Ca2+ exchange (NCX) proteins

The Ca2+-dependent allosteric regulation of Na+/Ca2+ exchanger (NCX) proteins represents Ca2+ interaction with the cytosolic domains, CBD1 (calcium-binding domain 1) and CBD2, which is associated either with activation, inhibition or no response to regulatory Ca2+ in a given splice variant. CBD1 contains a high affinity Ca2+-sensor (which is highly conserved among splice variants), whereas primary information upon Ca2+ binding to CBD1 is modified by alternative splicing of CBD2, yielding the diverse regulatory responses to Ca2+. To resolve the structure-dynamic determinants of splicing-dependent regulation, we tested two-domain tandem (CBD12) constructs possessing either positive, negative or no response to Ca2+ using hydrogen–deuterium exchange MS (HDX–MS), SAXS, equilibrium 45Ca2+ binding and stopped-flow kinetics. Taken together with previously resolved crystallographic structures of CBD12, the data revealed that Ca2+ binding to CBD1 rigidifies the main-chain flexibility of CBD2 (but not of CBD1), whereas CBD2 stabilizes the apo-CBD1. Strikingly, the extent and strength of Ca2+-dependent rigidification of CBD2 is splice-variant dependent, where the main-chain rigidification spans from the Ca2+-binding sites of CBD1, through a helix of CBD2 (positioned at the domains’ interface) up to the tip of CBD2 [>50 Å (1 Å=0.1 nm)] or alternatively, it stops at the CBD2 helix in the splice variant exhibiting an inhibitory response to regulatory Ca2+. These results provide a structure-dynamic basis by which alternative splicing diversifies the regulatory responses to Ca2+ as well as controls the extent and strength of allosteric signal propagation over long distance.