Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 114, Issue 11, Pages 2904-2909Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1620799114
Keywords
Na,K-ATPase; specific protein-lipid interaction; protein stability; Na,K-ATPase activity; native mass spectrometry
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Funding
- Minerva Foundation
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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-directedmutagenesis. 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 (alpha 1 beta 1). By replacing lysine residues at proposed phospholipid-binding sites with glutamines, the two sites have been identified. Mutations in the cytoplasmic alpha L8-9 loop destabilize the protein but do not affect Na,K-ATPase activity, whereas mutations in transmembrane helices (TM), alpha TM2 and alpha TM4, 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 alpha TM 8, 9, 10, near the FXYD subunit, and maintain topological integrity of the labile C terminus of the alpha subunit (site A). PC/PE binds between alpha TM2, 4, 6, and 9 and accelerates the rate-limiting E1P-E2P conformational transition (site B). We discuss the potential physiological implications.
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