Key residues controlling bidirectional ion movements in Na+/Ca2+ exchanger

Abstract

Prokaryotic and eukaryotic Na/Ca exchangers (NCX) control Ca homeostasis. NCX orthologs exhibit up to 10-fold differences in their turnover rates (k), whereas the ratios between the cytosolic (cyt) and extracellular (ext) K values (K = K/K) are highly asymmetric and alike (K ≤ 0.1) among NCXs. The structural determinants controlling a huge divergence in k at comparable K remain unclear, although 11 (out of 12) ion-coordinating residues are highly conserved among NCXs. The crystal structure of the archaeal NCX (NCX_Mj) was explored for testing the mutational effects of pore-allied and loop residues on k and K. Among 55 tested residues, 26 mutations affect either k or K, where two major groups can be distinguished. The first group of mutations (14 residues) affect k rather than K. The majority of these residues (10 out of 14) are located within the extracellular vestibule near the pore center. The second group of mutations (12 residues) affect K rather than k, whereas the majority of residues (9 out 12) are randomly dispersed within the extracellular vestibule. In conjunction with computational modeling-simulations and hydrogen-deuterium exchange mass-spectrometry (HDX-MS), the present mutational analysis highlights structural elements that differentially govern the intrinsic asymmetry and transport rates. The key residues, located at specific segments, can affect the characteristic features of local backbone dynamics and thus, the conformational flexibility of ion-transporting helices contributing to critical conformational transitions. The underlying mechanisms might have a physiological relevance for matching the response modes of NCX variants to cell-specific Ca and Na signaling.

Hydrogen-Deuterium Exchange Mass-Spectrometry of Secondary Active Transporters: From Structural Dynamics to Molecular Mechanisms

“…We detected a Na + -dependent decrease in deuterium uptake at S int , S Ca , and S ext , but not S mid , whereas in the presence of Ca 2+ the decrease in deuterium uptake was mainly observed at S Ca . This is consistent with the predictions made by the MD simulations and mutational analyses foreseeing the occupation of S mid by a water molecule but not by Na + or Ca 2+ in the ground state (Marinelli et al, 2014;Giladi et al, 2016a;Giladi et al, 2017;van Dijk et al, 2018). Notably, subsequent crystallographic studies have validated our binding sites' assignment (Liao et al, 2016).…”

“…Surprisingly, the crystal structure of NCX from Methanocaldococcus jannaschii (NCX_Mj) revealed four ion binding sites, simultaneously occupied by three Na + ions at sites termed S int , S mid , S ext , and one Ca 2+ ion at a site termed S Ca (Liao et al, 2012). Since this binding mode was inconsistent with previous studies, MD simulations and ion flux analyses of mutants were performed, suggesting that Na + ions occupy S int , S Ca , and S ext , whereas Ca 2+ occupies S Ca (Marinelli et al, 2014;Giladi et al, 2016b;Giladi et al, 2017;van Dijk et al, 2018). Thus, the Na + and Ca 2+ ions are bound in a mutually exclusive manner along the transport cycle.…”

Structure‐affinity insights into the Na+ and Ca2+ interactions with multiple sites of a sodium‐calcium exchanger

“…The ion-binding pocket, located in the middle of the ion passageway pore, contains four putative sites (S int , S mid , S ext , and S Ca ) with distinct ion selectivity: S int and S ext exhibit high selectivity to Na + , whereas S Ca shows a similar selectivity to Na + or Ca 2+ . Follow-up studies revealed that in the OF state, NCX_Mj can bind either 3Na + (at S int , S ext , and S Ca ) or 1Ca 2+ (at S Ca ), whereas the functional role of S mid remains unclear [30][31][32][33][34][35]. The lack of the NCX_Mj crystal structure in the inward-facing (IF) state prevents the structural delineation of respective sites in the IF state.…”

“…Several lines of evidence suggest that the prokaryotic and eukaryotic NCXs preferentially adopt the OF conformation [15][16][17][31][32][33][34], where these structure-dynamic mechanisms underlying the functional asymmetry can operate in the absence of any 'secondary' regulatory factors [36][37][38]. Previous mutational studies have identified the asymmetric contributions of distinct residues to rate-equilibrium relationships of bidirectional ion movements [31][32][33]. Consistent with these observations, the hydrogen-deuterium exchange mass spectrometry analysis of the apo-and ion-bound forms of NCX_Mj revealed that the local backbone dynamics are characteristically different at the cytosolic and extracellular vestibules [31,34].…”

“…Thus, eukaryotic NCXs contain two ioncoordinating carboxylates instead of three in NCX_Mj. Despite these structural differences, eukaryotic NCXs and NCX_Mj exhibit the same stoichiometry of ion exchange (3Na + :1Ca 2+ ), although the ionexchange rates of single transport cycle are 10 3 -10 4 faster in eukaryotic NCXs than in NCX_Mj [9][10][11][31][32][33]. How the relevant structural differences affect ion selectivity and binding affinity (at the extracellular and cytosolic vestibules), as well as the transport rates (catalysis), remains unclear.…”