Structure of a Ca2+ bound phosphoenzyme intermediate in the inward-to-outward transition of Ca2+-ATPase 1 from Listeria monocytogenes

Active transport by Ca2+-ATPases of the P-type ATPase family maintain a very low cytosolic calcium concentration and steep electrochemical gradients. Detailed mechanisms of this transport have been described from structures of mammalian sarco/endoplasmic reticulum Ca2+-ATPases (SERCA) stabilized by inhibitors at specific intermediate steps of the transport cycle. An essentially irreversible step is crucial to prevent reflux in active transport against steep gradients. Single-molecule FRET (smFRET) study of the bacterial Ca2+-ATPase LMCA1 revealed an intermediate of the transition between so-called [Ca]E1P and E2P states, suggesting that calcium release from this intermediate is the irreversible step. Here, we present a 3.5A cryo-EM structure for a four-glycine insertion mutant (G4-LMCA1) in a lipid nanodisc obtained under turnover conditions and adopting such a calcium-bound intermediate, denoted [Ca]E2P. The cytosolic domains are positioned in the E2P-like conformation, while the calcium-binding transmembrane (TM) domain is similar to calcium-bound E1P-ADP like conformation of SERCA. Missing density for the E292 residue at the calcium site (equivalent of SERCA1a E309) suggests flexibility and a site poised for calcium release and proton uptake. The structure suggests a mechanism for the inward-to-outward transition in Ca2+-ATPases, where ADP release and re-organisation of the cytoplasmic domains precede calcium release.