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 a 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 a 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.