Intracellular Ca2+regulation of H+/Ca2+antiporter YfkE mediated by a Ca2+mini-sensor

Abstract

The H+/Ca2+(calcium ion) antiporter (CAX) plays an important role in maintaining cellular Ca2+homeostasis in bacteria, yeast, and plants by promoting Ca2+efflux across the cell membranes. However, how CAX facilitates Ca2+balance in response to dynamic cytosolic Ca2+perturbations is unknown. Here, we identified a type of Ca2+“mini-sensor” in YfkE, a bacterial CAX homolog fromBacillus subtilis.The mini-sensor is formed by six tandem carboxylate residues within the transmembrane (TM)5-6 loop on the intracellular membrane surface. Ca2+binding to the mini-sensor triggers the transition of the transport mode of YfkE from a high-affinity to a low-affinity state. Molecular dynamics simulation and fluorescence resonance energy transfer analysis suggest that Ca2+binding to the mini-sensor causes an adjacent segment, namely, the exchanger inhibitory peptide (XIP), to move toward the Ca2+translocation pathway to interact with TM2a in an inward-open cavity. The specific interaction was demonstrated with a synthetic peptide of the XIP, which inhibits YfkE transport and interrupts conformational changes mediated by the mini-sensor. By comparing the apo and Ca2+-bound CAX structures, we propose the following Ca2+transport regulatory mechanism of YfkE: Ca2+binding to the mini-sensor induces allosteric conformational changes in the Ca2+translocation pathway via the XIP, resulting in a rearrangement of the Ca2+-binding transport site in the midmembrane. Since the Ca2+mini-sensor and XIP sequences are also identified in other CAX homologs and/or Ca2+transporters, including the mammalian Na+/Ca2+exchanger (NCX), our study provides a regulatory mechanism for the Ca2+/cation transporter superfamily.