Abstact

P-type ATPases represent an evolutionarily conserved superfamily of ion, lipid, and peptide pumps found across all domains of life. Among the substrates transported by P-type ATPases, Mg(2+) is of critical importance in bacterial, fungal, and plant cellular homeostasis. A bacterial P-type ATPase found in Gram-negative bacteria, Mg(2+) transporter A (MgtA), facilitates the transport of Mg(2+) from the periplasm to the cytoplasm under conditions of Mg(2+) starvation. MgtA is a cardiolipin-sensitive integral membrane ion-transporter that scavenges Mg(2+) during bacterial infection and pathogenesis. Here, we determined cryo-EM structures of MgtA capturing three distinct states along the Mg(2+) transport cycle, including a phosphorylated E2-P intermediate (2.6 A resolution), an E1-like conformation stabilized by the peptide regulator MgtR (2.7 A resolution), and an E1-like ATP-bound state (2.8 A resolution). These three conformations reveal the binding of Mg(2+) in the transmembrane domain coordinated in a novel site involving Ser(702) and Asn(706) on M5, Ser(773) and Asp(777) on M7, and Ser(821) and Thr(824) on M8. In the E2-P conformation, the phosphate analog BeF(3) is bound in close proximity to the catalytic aspartate, Asp(361), suggesting that it represents a covalent aspartylphosphate intermediate. In the presence of AMPPCP, Mg(2+) remains bound in the transmembrane domain and the ATP analog is bound in a catalytically competent conformation. Overall, the structures reveal distinct steps in the transport cycle of MgtA compared to other P-type ATPases, as well as lipid binding sites that fill gaps in our understanding of transport regulation.