Structure of the [Ca]E2P intermediate of Ca2+-ATPase 1 from Listeria monocytogenes.

Abstract

Active transport by P-type Ca²⁺-ATPases maintain internal calcium stores and a low cytosolic calcium concentration. Structural studies of mammalian sarco/endoplasmic reticulum Ca²⁺-ATPases (SERCA) have revealed several steps of the transport cycle, but a calcium-releasing intermediate has remained elusive. Single-molecule FRET studies of the bacterial Ca²⁺-ATPase LMCA1 revealed an intermediate of the transition between so-called [Ca]E1P and E2P states and suggested that calcium release from this intermediate was the essentially irreversible step of transport. Here, we present a 3.5 Å resolution cryo-EM structure for a four-glycine insertion mutant of LMCA1 in a lipid nanodisc obtained under conditions with calcium and ATP and adopting such an intermediate state, denoted [Ca]E2P. The cytosolic domains are positioned in the E2P-like conformation, while the calcium-binding transmembrane (TM) domain adopts a calcium-bound E1P-ADP-like conformation. Missing density for the E292 residue at the calcium site (the equivalent of SERCA1a E309) suggests flexibility and a site poised for calcium release and proton uptake. The structure suggests a mechanism where ADP release and re-organization of the cytoplasmic domains precede calcium release.