Structural Basis of Substrate-Independent Phosphorylation in a P4-ATPase Lipid Flippase

P4-ATPases define a eukaryotic subfamily of the P-type ATPases, and are responsible for transverse flip specific lipids from extracellular or luminal leaflet to cytosolic cell membranes. The enzymatic cycle ATPases is divided into autophosphorylation dephosphorylation half-reactions. Unlike most other transport their substrate during only, i.e. phosphorylation half-reaction not associated with transport. To study structural basis distinct mechanisms P4-ATPases, we have determined cryo-EM structures Drs2p-Cdc50p Saccharomyces cerevisiae covering multiple intermediates cycle. We identify several motifs Drs2p in general that decrease movements flexibility domains as compared such Na+/K+-ATPase Ca2+-ATPase. These include linkers connect transmembrane region actuator (A) domain, which dephosphorylation. Additionally, mutation Tyr380, interacts conserved Asp340 DGET loop highlights functional role these P4-ATPase A-domain. Finally, (TM) transport, also undergoes less extensive conformational changes, ensured both by longer segment connecting TM helix 4 site, possible stabilization auxiliary subunit Cdc50p. Collectively adaptions becoming transport-independent.