Abstract
Na+/Ca2+ exchanger (NCX) proteins extrude Ca2+ from the cell to maintain cellular homeostasis. Since NCX proteins contribute to numerous physiological and pathophysiological events, their pharmacological targeting has been desired for a long time. This intervention remains challenging owing to our poor understanding of the underlying structure-dynamic mechanisms. Recent structural studies have shed light on the structure-function relationships underlying the ion-transport and allosteric regulation of NCX. The crystal structure of an archaeal NCX (NCX_Mj) along with molecular dynamics simulations and ion flux analyses, have assigned the ion binding sites for 3Na+ and 1Ca2+, which are being transported in separate steps. In contrast with NCX_Mj, eukaryotic NCXs contain the regulatory Ca2+-binding domains, CBD1 and CBD2, which affect the membrane embedded ion-transport domains over a distance of ~80 Å. The Ca2+-dependent regulation is ortholog, isoform, and splice-variant dependent to meet physiological requirements, exhibiting either a positive, negative, or no response to regulatory Ca2+. The crystal structures of the two-domain (CBD12) tandem have revealed a common mechanism involving a Ca2+-driven tethering of CBDs in diverse NCX variants. However, dissociation kinetics of occluded Ca2+ (entrapped at the two-domain interface) depends on the alternative-splicing segment (at CBD2), thereby representing splicing-dependent dynamic coupling of CBDs. The HDX-MS, SAXS, NMR, FRET, equilibrium 45Ca2+ binding and stopped-flow techniques provided insights into the dynamic mechanisms of CBDs. Ca2+ binding to CBD1 results in a population shift, where more constraint conformational states become highly populated without global conformational changes in the alignment of CBDs. This mechanism is common among NCXs. Recent HDX-MS studies have demonstrated that the apo CBD1 and CBD2 are stabilized by interacting with each other, while Ca2+ binding to CBD1 rigidifies local backbone segments of CBD2, but not of CBD1. The extent and strength of Ca2+-dependent rigidification at CBD2 is splice-variant dependent, showing clear correlations with phenotypes of matching NCX variants. Therefore, diverse NCX variants share a common mechanism for the initial decoding of the regulatory signal upon Ca2+ binding at the interface of CBDs, whereas the allosteric message is shaped by CBD2, the dynamic features of which are dictated by the splicing segment.
Highlights
Calcium (Ca2+) is the most important and versatile secondary messenger in the cell; it carries vital information to virtually all processes important to cell life and function
Two possibilities were raised: (i) additional structural elements in the regulatory f-loop and/or membrane domain are involved in decoding and specifying the regulatory effects or alternatively, (ii) the conformational dynamics differ between the Na+/Ca2+ exchanger (NCX) splice variants and CALX, despite the similar orientations observed in the crystal structures and the small angle X-ray scattering (SAXS)-ensemble optimization method (EOM) data obtained for diverse splice variants, because there are either positive, negative, or no responses to regulatory Ca2+
The structure of NCX from the archaebacterium Methanococcus jannaschii (NCX_Mj) (Liao et al, 2012), along with molecular dynamics (MD) simulations and ion-flux analyses (Marinelli et al, 2014), verified the exchange mechanism and stoichiometry and provided important clues regarding the molecular basis of NCX ion selectivity (Figure 1)
Summary
Calcium (Ca2+) is the most important and versatile secondary messenger in the cell; it carries vital information to virtually all processes important to cell life and function (e.g., it couples excitation to contraction, hormone secretion, gene transcription, and controls enzyme activity through protein phosphorylationdephosphorylation involving numerous biochemical reactions). Biochemical studies utilizing transport assays in proteoliposomes (Khananshvili, 1990), followed by electrophysiological studies (Hilgemann et al, 1991; Niggli and Lederer, 1991), have concluded that NCX operates through a ping-pong mechanism in which one Ca2+ and three Na+ ions are translocated sequentially in separate steps rather than simultaneously across the membrane This mechanism implies the alternating access mechanism of the NCX ion binding sites in the inward (cytosolic) and outward (extracellular) conformations (Figure 1E). Three crystal structures of Ca2+/H+ exchangers were determined and revealed striking similarities with NCX_Mj, suggesting that the sliding mechanisms could be a general feature of the gene families belonging to the Ca/CA superfamily (Nishizawa et al, 2013; Waight et al, 2013) It remains unclear how ion binding drives the sliding of the gating bundle (the TM1/TM6 cluster) to initiate alternating access. The resolution of this mechanism is essential for understanding how Ca2+ binding to the regulatory CBDs in eukaryotic NCX orthologs accelerates the ion transport cycle
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