Population Shift Underlies Ca2+-induced Regulatory Transitions in the Sodium-Calcium Exchanger (NCX)

In eukaryotic Na+/Ca2+ exchangers (NCX) the Ca2+ binding CBD1 and CBD2 domains form a two-domain regulatory tandem (CBD12). An allosteric Ca2+ sensor (Ca3-Ca4 sites) is located on CBD1, whereas CBD2 contains a splice-variant segment. Recently, a Ca2+-driven interdomain switch has been described, albeit how it couples Ca2+ binding with signal propagation remains unclear. To resolve the dynamic features of Ca2+-induced conformational transitions we analyze here distinct splice variants and mutants of isolated CBD12 at varying temperatures by using small angle x-ray scattering (SAXS) and equilibrium Ca-45(2+) binding assays. The ensemble optimization method SAXS analysis demonstrates that the apo and Mg2+-bound forms of CBD12 are highly flexible, whereas Ca2+ binding to the Ca3-Ca4 sites results in a population shift of conformational landscape to more rigidified states. Population shift occurs even under conditions in which no effect of Ca2+ is observed on the globally derived D-max (maximal interatomic distance), although under comparable conditions a normal [Ca2+]-dependent allosteric regulation occurs. Low affinity sites (Ca1-Ca2) of CBD1 do not contribute to Ca2+-induced population shift, but the occupancy of these sites by 1 mM Mg2+ shifts the Ca2+ affinity (K-d) at the neighboring Ca3-Ca4 sites from similar to 50 nM to similar to 200 nM and thus, keeps the primary Ca2+ sensor (Ca3-Ca4 sites) within a physiological range. Thus, Ca2+ binding to the Ca3-Ca4 sites results in a population shift, where more constraint conformational states become highly populated at dynamic equilibrium in the absence of global conformational transitions in CBD alignment.