Mechanism of extracellular ion exchange and binding-site occlusion in a sodium/calcium exchanger

Abstract

Na+/Ca2+ exchangers utilize the Na+ electrochemical gradient across the plasma membrane to extrude intracellular Ca2+, and play a central role in Ca2+ homeostasis. Here, we elucidate their mechanisms of extracellular ion recognition and exchange through a structural analysis of the exchanger from Methanococcus jannaschii (NCX_Mj) bound to Na+, Ca2+ or Sr2+ in various occupancies and in an apo state. This analysis defines the binding mode and relative affinity of these ions, establishes the structural basis for the anticipated 3Na+:1Ca2+ exchange stoichiometry, and reveals the conformational changes at the onset of the alternating-access transport mechanism. An independent analysis of the dynamics and conformational free-energy landscape of NCX_Mj in different ion-occupancy states, based on enhanced-sampling molecular-dynamics simulations, demonstrates that the crystal structures reflect mechanistically relevant, interconverting conformations. These calculations also reveal the mechanism by which the outward-to-inward transition is controlled by the ion-occupancy state, thereby explaining the emergence of strictly-coupled Na+/Ca2+ antiport.

Structure-Functional Basis of Ion Transport in Sodium–Calcium Exchanger (NCX) Proteins

“…Recently, a thorough structural analysis of ion recognition in the outward-facing state was performed by soaking NCX_Mj crystals in solutions with different ionic compositions [27]. These studies unequivocally confirm the model derived from the molecular dynamics and the ion-flux assays mentioned above.…”

“…However, only recently has a major advancement in understanding the structural basis for this mechanism been achieved by solving the crystal structure of NCX_Mj from the arachaeabacterium Methanococcus jannaschii [24]. In combination with known crystal structures, specially designed MD simulations and extended kinetic analyses of ion-fluxes in mutants revealed that the simultaneous occupation of 3Na + and 1Ca 2+ is thermodynamically forbidden [25,26,27]. Similarly to mammalian NCX, the NCX_Mj protein also translocates Na + and Ca 2+ with a stoichiometry of 3:1, although in contrast with NCX_Mj, the three mammalian genes NCX1-3 contain a large cytosolic regulatory loop (~520 aa) between helices 5 and 6 [28,29,30].…”

“…Notably, NCX_Mj was crystallized in the outward-facing occluded state in the presence of both Na + and Ca 2+ [24,27]. This structure reveals 10 transmembrane helices (TM) arranged in two pseudosymmetrical halves (TM1-5 and TM6-10) (Figure 1A).…”

“…The structural information gained from the X-ray studies of NCX_Mj makes this protein an ideal system for studying ion transport by NCX proteins, since NCX_Mj lacks regulatory domains, while exhibiting striking similarities with mammalian NCXs in ion transport machinery [24,25,26,27,35]. The Khananshvili laboratory has extensively studied the functional properties of NCX_Mj by using 45 Ca 2+ -uptake assays in Escherichia coli -derived vesicles containing overexpressed NCX_Mj protein and its mutants (Figure 3A).…”

“…By definition, only the transition state (and not the occluded and/or semi-open ground states) is capable of initiating the movement of the TM1/TM6 gating bundle toward OF/IF swapping (Figure 4B,C). Even though no experimental approaches/techniques are currently available for direct inspection of the transition state [7,8], a rational combination of X-ray crystallography, HDX-MS, kinetic analysis of mutants and MD simulations may lead to meaningful conclusions that may help in defining concrete objectives for resolving future challenges [24,25,26,27]. We describe below successful multidisciplinary approaches applied to the NCX_Mj protein, which can be explored for other transporters as well.…”