Structural Analysis Of Lipid Flippases,Type ? P-type ATPases

Lipid flippases are Type IV P-type ATPases(P4-ATPases),most of which form heterodimers with?-subunit from CDC50 protein family.They mediate phospholipid transportation from the exoplasmic leaflet to the cytoplasmic leaflet,which play an important role in lipid asymmetry,vesicle budding and trafficking,etc.Similar to other classic cation-transporting P-type ATPases,lipid flippases also works according to the Post-Albers cycle,self-phosphorylating/dephosphorylating the highly conserved aspartate residue during phospholipid transportation.It is still incompletely understood how P4-ATPases transport phospholipids.The structures of lipid flippases were not deciphered when the project was started.How P4-ATPases transport phospholipids and how the activity is regulated are critic questions to be answered in the field.Besides,as the lipid flippases are embedded in the phospholipid bilayers,it is of more physiological relevance to decide the structures in the membrane environment.In order to decipher the molecular mechanism of phospholipid transportation by P4-ATPases,this project employs cryo-EM single particle analysis to determine the structures of lipid flippases in lipid bilayers.To start with,the Chaetomium thermophilum(Ct)lipid flippase Ct Dnf1-Ct Cdc50 was over expressed in yeast cells,reconstituted into nanodiscs with yeast lipids and purified for structural investigation.Be F3^-or AMPPCP was added to the sample before grid preparation in order to capture the E2P and E1-ATP states respectively.Cryo-EM study indeed identified the two states with resolutions of 3.5?(E2P)and 3.4?(E1-ATP).The structures reveal that the actuator domain undergoes large displacement and TM1 becomes invisible in the E1-ATP state.Meanwhile,the local bilayers are distorted.TM2,TM4 and TM6 form a continuous hydrophilic transmembrane groove,in which two phospholipid binding sites are identified in the E2P state(one site not reported before).Thus,a new model is proposed:during phospholipid transportation,the transmembrane domain of Ct Dnf1-Ct Cdc50 undergoes large conformational changes with the movements of the cytoplasmic domains,and phospholipid molecules are relayed by the lipid binding sites and transported from the exoplasmic leaflet to the cytoplasmic leaflet after two working cycles.Next,the study focused on the yeast lipid flippase Dnf1p-Lem3p in Saccharomyces cerevisiae.The structures determined in detergent show that E2P(2.8?)and E1-ATP(3.1?)states are consistent with the reported P4-ATPase structures in the corresponding states.However,the structures determined in nanodiscs with yeast lipids show that Dnf1p adopts a conformation unreported before,either with the addition of Be F3^-(3.8?)or AMPPCP(3.2?).In this unique conformation,the actuator domain is away from the other two cytosolic domains.TM1 and TM2 swap their relative positions and TM4 is a continues helix rather than the commonly seen kinked helix.Thus,the hydrophilic environment within the lipid flipping groove is disrupted.Biochemical experiments show that this resting state does exist in yeast cells.When the lipid composition of nanodiscs changes to 90%PS-10%PC,Dnf1p can be captured in E2P(2.9?)and E1-ATP(3.7?)states again.We propose that the existence of the off-cycle resting state may be an activity regulation mechanism of Dnf1p.In summary,the project determined the structures of P4-ATPases in different states and proposed a possible phospholipid transportation model with a possible activity regulation mechanism.The results provide important structural clues for further research on the molecular mechanism of phospholipid flippases.The regulatory mechanism and substrate specificity of phospholipid flippases shall be investigated in the future.