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 (NCX1-3) proteins are essential for handling Ca2+ homeostasis in many cell-types. Eukaryotic NCX variants contain regulatory calcium-binding domains (CBD1 and CBD2), which are associated either with activation, inhibition or no response to regulatory Ca2+. 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+. Recent studies revealed that the Ca2+ binding to CBD1 (Ca3–Ca4) sites results in interdomain tethering of CBDs, which rigidifies CBDs movements with accompanied slow dissociation of “occluded” Ca2+. To resolve the structure-dynamic determinants of splicing-dependent regulation, we tested two-domain tandem (CBD12) constructs possessing either positive (CBD12-1.4), negative (CBD12-1.1) or no response (CBD12-1.2) to Ca2+ using hydrogen–deuterium exchange MS (HDX–MS). Combined 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. Remarkably, the extent and strength of Ca2+-dependent rigidification of CBD2 is splice-variant dependent; the main-chain rigidification spans from the Ca2+-binding sites of CBD1 and propagates up to the tip of CBD2 [>50 A (1 A=0.1 nm)] through α helix of CBD2 (positioned at the domains’ interface) in the splice variant exhibiting a positive response to regulatory Ca2+; on the other hands, the Ca2+-dependent rigidification stops at the α helix of CBD2 in the splice variant with an inhibitory response. 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.