Specific phospholipid binding to Na,K-ATPase at two distinct sites

Area of Science:

• Biochemistry
• Molecular Biology
• Membrane Biophysics

Background:

• Membrane protein function is influenced by lipid bilayer properties and specific lipid-protein interactions.
• Na,K-ATPase activity is modulated by bilayer properties and specific lipids like phosphatidylserine (PS) and polyunsaturated phospholipids (PC/PE).
• While specific lipid interactions with Na,K-ATPase have been inferred, definitive binding sites remained unidentified.

Purpose of the Study:

• To identify the specific binding sites of phosphatidylserine (PS) and phosphatidylcholine (PC) on human Na,K-ATPase.
• To elucidate the functional consequences of these lipid-protein interactions on Na,K-ATPase activity and stability.

Main Methods:

• Native mass spectrometry (MS) was employed to directly observe lipid binding to purified human Na,K-ATPase (α1β1).
• Site-directed mutagenesis, specifically replacing lysine residues with glutamines, was used to pinpoint phospholipid-binding sites.
• Analysis was integrated with existing crystal structure data.

Main Results:

• Native MS confirmed specific binding of one molecule each of 18:0/18:1 PS and 18:0/20:4 PC to Na,K-ATPase.
• Mutations in the cytoplasmic αL8-9 loop affected protein stability but not activity.
• Mutations in transmembrane helices (αTM2 and αTM4) abolished PC stimulation without impacting stability, identifying these as key sites for PC interaction.

Conclusions:

• Two distinct lipid binding sites on Na,K-ATPase were identified: Site A (between αTM 8, 9, 10) for PS, stabilizing the protein.
• Site B (between αTM2, 4, 6, and 9) for PC, accelerating the rate-limiting E1P-E2P conformational transition.
• These findings provide a mechanistic understanding of how specific lipids regulate Na,K-ATPase function and have potential physiological implications.