title: Specific phospholipid binding to Na,K-ATPase at two distinct sites.
abstract: Membrane protein function can be affected by the physical state of the lipid bilayer and specific lipid-protein interactions. For Na,K-ATPase, bilayer properties can modulate pump activity, and, as observed in crystal structures, several lipids are bound within the transmembrane domain. Furthermore, Na,K-ATPase activity depends on phosphatidylserine (PS) and cholesterol, which stabilize the protein, and polyunsaturated phosphatidylcholine (PC) or phosphatidylethanolamine (PE), known to stimulate Na,K-ATPase activity. Based on lipid structural specificity and kinetic mechanisms, specific interactions of both PS and PC/PE have been inferred. Nevertheless, specific binding sites have not been identified definitively. We address this question with native mass spectrometry (MS) and site-directed mutagenesis. Native MS shows directly that one molecule each of 18:0/18:1 PS and 18:0/20:4 PC can bind specifically to purified human Na,K-ATPase (α1β1). By replacing lysine residues at proposed phospholipid-binding sites with glutamines, the two sites have been identified. Mutations in the cytoplasmic αL8-9 loop destabilize the protein but do not affect Na,K-ATPase activity, whereas mutations in transmembrane helices (TM), αTM2 and αTM4, abolish the stimulation of activity by 18:0/20:4 PC but do not affect stability. When these data are linked to crystal structures, the underlying mechanism of PS and PC/PE effects emerges. PS (and cholesterol) bind between αTM 8, 9, 10, near the FXYD subunit, and maintain topological integrity of the labile C terminus of the α subunit (site A). PC/PE binds between αTM2, 4, 6, and 9 and accelerates the rate-limiting E1P-E2P conformational transition (site B). We discuss the potential physiological implications.
authors.affiliation: Department of Biomolecular Sciences, Weizmann Institute of Science, Rehovot 7610001, Israel.
authors.forename: Michael
authors.initials: M
authors.lastname: Habeck
authors.affiliation: Department of Biomolecular Sciences, Weizmann Institute of Science, Rehovot 7610001, Israel.
authors.forename: Einat
authors.initials: E
authors.lastname: Kapri-Pardes
authors.affiliation: Department of Biomolecular Sciences, Weizmann Institute of Science, Rehovot 7610001, Israel.
authors.forename: Michal
authors.initials: M
authors.lastname: Sharon
authors.affiliation: Department of Biomolecular Sciences, Weizmann Institute of Science, Rehovot 7610001, Israel Steven.Karlish@weizmann.ac.il.
authors.forename: Steven J D
authors.initials: SJ
authors.lastname: Karlish
mesh_terms: D001665:Binding Sites; D004789:Enzyme Activation; D006801:Humans; D013058:Mass Spectrometry; D008958:Models, Molecular; D008968:Molecular Conformation; D010743:Phospholipids / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D055550:Protein Stability; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D010743:Phospholipids; D000254:Sodium-Potassium-Exchanging ATPase
keywords: Na,K-ATPase; Na,K-ATPase activity; native mass spectrometry; protein stability; specific protein–lipid interaction