Crystal structure of Ca/H antiporter protein YfkE reveals the mechanisms of Ca efflux and its pH regulation

Ca efflux by Ca cation antiporter (CaCA) proteins is important for maintenance of Ca homeostasis across the cell membrane. Recently, the monomeric structure of the prokaryotic Na/Ca exchanger (NCX) antiporter NCX_Mj protein from Methanococcus jannaschii shows an outward-facing conformation suggesting a hypothesis of alternating substrate access for Ca efflux. To demonstrate conformational changes essential for the CaCAmechanism, we present the crystal structure of the Ca/H antiporter protein YfkE from Bacillus subtilis at 3.1-Å resolution. YfkE forms a homotrimer, confirmed by disulfide crosslinking. The protonated state of YfkE exhibits an inward-facing conformation with a large hydrophilic cavity opening to the cytoplasm in each protomer and ending in the middle of the membrane at the Ca-binding site. A hydrophobic “seal” closes its periplasmic exit. Four conserved α-repeat helices assemble in an X-like conformation to form a Ca/H exchange pathway. In the Ca-binding site, two essential glutamate residues exhibit different conformations compared with their counterparts in NCX_Mj, whereas several amino acid substitutions occlude the Na-binding sites. The structural differences between the inward-facing YfkE and the outward-facing NCX_Mj suggest that the conformational transition is triggered by the rotation of the kink angles of transmembrane helices 2 and 7 and is mediated by large conformational changes in their adjacent transmembrane helices 1 and 6. Our structural and mutational analyses not only establish structural bases for mechanisms of Ca/H exchange and its pH regulation but also shed light on the evolutionary adaptation to different energy modes in the CaCA protein family.