Asparagine 905 of the mammalian phospholipid flippase ATP8A2 is essential for lipid substrate–induced activation of ATP8A2 dephosphorylation

Abstract

The P-type ATPase protein family includes, in addition to ion pumps such as Ca 2؉-ATPase and Na ؉ ,K ؉-ATPase, also phospholipid flippases that transfer phospholipids between membrane leaflets. P-type ATPase ion pumps translocate their substrates occluded between helices in the center of the transmembrane part of the protein. The large size of the lipid substrate has stimulated speculation that flippases use a different transport mechanism. Information on the functional importance of the most centrally located helices M5 and M6 in the transmembrane domain of flippases has, however, been sparse. Using mutagenesis, we examined the entire M5-M6 region of the mammalian flippase ATP8A2 to elucidate its possible function in the lipid transport mechanism. This mutational screen yielded an informative map assigning important roles in the interaction with the lipid substrate to only a few M5-M6 residues. The M6 asparagine Asn-905 stood out as being essential for the lipid substrate-induced dephosphorylation. The mutants N905A/D/E/H/L/Q/R all displayed very low activities and a dramatic insensitivity to the lipid substrate. Strikingly, Asn-905 aligns with key ion-binding residues of P-type ATPase ion pumps, and N905D was recently identified as one of the mutations causing the neurological disorder cerebellar ataxia, mental retardation, and disequilibrium (CAMRQ) syndrome. Moreover, the effects of substitutions to the adjacent residue Val-906 (i.e. V906A/E/F/L/Q/S) suggest that the lipid substrate approaches Val-906 during the translocation. These results favor a flippase mechanism with strong resemblance to the ion pumps, despite a location of the translocation pathway in the periphery of the transmembrane part of the flippase protein. ATP8A2 is a membrane-bound enzyme, a "flippase," that utilizes the energy liberated by ATP hydrolysis to translocate (flip) the amino phospholipids phosphatidylserine (PS) 2 and phosphatidylethanolamine (PE), but not phosphatidylcholine (PC), from the exoplasmic leaflet to the cytoplasmic leaflet of cellular membrane lipid bilayers, thereby creating an essential asymmetric lipid distribution between the membrane leaflets (1). Flippases, also known as P4-ATPases, belong to the P-type ATPase family. Compared with the ion pump members of this enzyme family, flippases are poorly understood, although as many as 14 of the 36 P-type ATPases encoded by the human genome are flippases (1, 2), and certain mutations in flippases, including ATP8A2, are associated with severe disorders (1, 3-9). P4-ATPases are made up of three cytoplasmic domains, N (nucleotide-binding), P (phosphorylation), and A ("actuator"), linked to 10 transmembrane helices (M1-M10), like the ionpumping P-type ATPases Ca 2ϩ-ATPase, Na ϩ ,K ϩ-ATPase, H ϩ ,K ϩ-ATPase, and H ϩ-ATPase (P2-and P3-ATPases). In addition, most P4-ATPases are composed of an accessory subunit comparable in many respects to the ␤-subunit of Na ϩ ,K ϩand H ϩ ,K ϩ-ATPases. During the ATP hydrolysis cycle, ATP8A2, like other P-type ATPases, is phosphorylated at a ...

Crystal structure of a human plasma membrane phospholipid flippase

“…Evidently, correct size is important for gating residue Val357. Mutagenesis studies also reveal the important contribution of the conserved residues around the occlusion site (Phe72, Asn352) and TM5-6 (Lys880, Asn881 and Asn912), all alanine mutants showed significant reductions in either V max or apparent affinity for PtdSer and/or PtdEtn determined through ATPase activity profiles, and transport activities as well, in good agreement with previous studies of ATP8A2 27,43 . Interestingly, Phe343 is not conserved among P4-ATPases, despite its close position to the phospholipid head group.…”

“…The peak values of samples were compared to that of a fully purified sample used for the crystallization whose protein concentration was accurately determined by UV absorption, and protein concentrations for each measured sample were estimated. The phospholipid concentration-dependent ATPase activities were plotted, and data fitted as described previously 43 to estimate apparent affinities ( K 0.5 ) and V max using PRISM4 software. For all measurements, data were duplicated at twelve different phospholipid concentrations for a single measurement, and at least three independent measurements were conducted for each mutant.…”

Expression and functional characterization of missense mutations in ATP8A2 linked to severe neurological disorders

“…The p.I376M has been shown previously to be devoid of ATP‐dependent phospholipid flippase activity as well as PS and PE stimulated ATPase activity (Lee et al, ; Vestergaard et al, ). The p.N917D disease‐causing mutation (p.N905D in the bovine orthologue) can be added to this category since it expresses at a level comparable to non‐mutated ATP8A2 but lacks ATPase activity (Mikkelsen et al, ). Although these two categories of mutations display significant differences in levels of protein expression, they both can be considered as loss‐in‐function mutations since neither category displayed phospholipid activated ATPase activity.…”

“…Mutational studies together with molecular modeling and molecular simulation indicate that isoleucine 376 may be part of a hydrophobic gate that controls the translocation of PS and PE through a groove formed by transmembrane segments M1, M2, M4 and M6 of ATP8A2 (Andersen et al, ; Vestergaard et al, ). Asparagine 917, on the other hand, plays a crucial role in phospholipid induced dephosphorylation of ATP8A2 during phospholipid translocation (Mikkelsen et al, ). The p.K429M, p.K429N, p.A544P, p.R625W, and p.W702R mutations are located within domains important for the catalytic cycle.…”

“…The functional activity of two disease‐associated variants in ATP8A2 (c1128C>G, p.Ile376Met (p.I376M) missense mutation in transmembrane segment M4 and c2749A>G, p.Asn917Asp (p.N917D) mutation in transmembrane segment M6) have been examined at a biochemical level in the bovine orthologue (Mikkelsen et al, ; Vestergaard et al, ; Figure ). Both variants were found to be devoid of PS and PE activated ATPase activity (Mikkelsen et al, ; Vestergaard et al, ). The p.I376M variant was also shown to lack ATP‐dependent phospholipid flippase activity.…”