Crystal structure of Ca2+/H+ antiporter protein YfkE reveals the mechanisms of Ca2+ efflux and its pH regulation
Ca2+ efflux by Ca2+ cation antiporter (CaCA) proteins is important for maintenance of Ca2+ homeostasis across the cell membrane. Recently, the monomeric structure of the prokaryotic Na+/Ca2+ exchanger (NCX) antiporter NCX_Mj protein from Methanococcus jannaschii shows an outward-facing conformation suggesting a hypothesis of alternating substrate access for Ca2+ efflux. To demonstrate conformational changes essential for the CaCA mechanism, we present the crystal structure of the Ca2+/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 Ca2+-binding site. A hydrophobic "seal" closes its periplasmic exit. Four conserved α-repeat helices assemble in an X-like conformation to form a Ca2+/H+ exchange pathway. In the Ca2+-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 Ca2+/H+ exchange and its pH regulation but also shed light on the evolutionary adaptation to different energy modes in the CaCA protein family.