Structure‐affinity insights into the Na+ and Ca2+ interactions with multiple sites of a sodium‐calcium exchanger. (2nd March 2020) - Iwaki, Masayo
Abstract : Selective recognition and transport of Na + and Ca 2+ ions by sodium–calcium exchanger (NCX) proteins is a primary prerequisite for Ca 2+ signaling and homeostasis. Twelve ion‐coordinating residues are highly conserved among NCXs, and distinct NCX orthologs contain two or three carboxylates, while sharing a common ion‐exchange stoichiometry (3Na + :1Ca 2+ ). How these structural differences affect the ion‐binding affinity, selectivity, and transport rates remains unclear. Here, the mutational effects of three carboxylates (E54, E213, and D240) were analyzed on the ion‐exchange rates in the archaeal NCX from Methanococcus jannaschii and ion‐induced structure–affinity changes were monitored by attenuated total reflection–Fourier‐transform infrared spectroscopy (ATR‐FTIR). The D240N mutation elevated the ion‐transport rates by twofold to threefold, meaning that the deprotonation of D240 is not essential for transport catalysis. In contrast, mutating E54 or E213 to A, D, N, or Q dramatically decreased the ion‐transport rates. ATR‐FTIR revealed high‐ and low‐affinity binding of Na + or Ca 2+ with E54 and E213, but not with D240. These findings reveal distinct structure–affinity states at specific ion‐binding sites in the inward‐facing (IF) and outward‐facing orientation. Collectively, two multidentate carboxylate counterparts (E54 and E213) play a critical role in determining the ion coordination/transport in prokaryotic and eukaryotic NCXs, whereas the ortholog substitutions in prokaryotes (aspartate) and eukaryotes (asparagine) at the 240 position affect the ion‐transport rates differently ( k cat ), probably due to the structural differences in the transition state. Abstract : The multidentate (E54 and E213) carboxylates of the Na + /Ca 2+ exchanger (NCX) are essential for sequential binding/transport of 3Na + or 1Ca 2+ . The monodentate carboxylate (D240) is not involved in the ground state binding of Na + or Ca 2+, although it limits the transport rates. The mammalian NCXs (containing N240, instead D240) exhibit much higher transport rates, while sharing a common stoichiometry of ion exchange (3Na + :1Ca 2+ ) among evolutionary distant NCX orthologs.