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 from <i>Bacillus subtilis.</i> 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.