Structure of Trimeric Calcium/Proton Antiporter Protein YfkE Reveals the Mechanisms of Calcium Efflux and its pH Regulation

Abstract

Ca2+ efflux by Ca2+ cation antiporter (CaCA) proteins is important to maintain Ca2+ homeostasis across the cell membrane in all kingdoms of life. CaCA proteins extrude Ca2+ out of the cells by utilizing a counter electrochemical gradient of other cations such as Na+ or H+ as energy. To understand Ca2+/cation exchange mechanism, we determined 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, and each protomer contains 11 transmembrane helices. The protonated state of YfkE is locked in an inward-facing conformation with a large hydrophilic cavity in each protomer opening to the cytoplasm and ending in the middle of the membrane at the Ca2+ binding site, whereas a hydrophobic "seal" closes its periplasmic exit. Two highly conserved helices, TMs 2 & 7, kink toward each other, forming a X-like Ca2+ translocation pathway, which is blocked by a histidine residue at the Ca2+ binding site. Our structural and functional analyses suggest that Ca2+/H+ alternating access on each side of the membrane induces the rotation of the kink angles of TMs 2 and 7 and the transition between inward- and outward-facing conformations is mediated by large conformational changes of TMs 1 and 6. These studies not only establish structural bases for the novel mechanisms for Ca2+ efflux and its pH regulation, but also shed light on the evolutionary adaptation of different energy modes, Na+ vs. H+, in the CaCA protein family.