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 (αβ). 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 EP-EP conformational transition (site B). We discuss the potential physiological implications.

Direct Binding of Phosphatidylglycerol at Specific Sites Modulates Desensitization of a Pentameric Ligand-Gated Ion Channel

“…This is supported by enrichment of POPG compared to POPE in phospholipids that are co-purified with ELIC, and coarse-grained simulations which show enrichment of POPG among the boundary phospholipids of ELIC. Second, native MS also allows determination of the stoichiometry and sites of lipid binding (32, 45). By relating binding stoichiometry and thermal stability, the data estimate that 32 POPG lipids, which is the average number of annular lipids in ELIC from MD simulations, results in greater than 80% of the stabilizing effect against thermal denaturation (Supplementary Fig.…”

Renal Mg handling, FXYD2 and the central role of the Na,K-ATPase

“…3 Figure 2A provides an overview of the whole Na,K-pump molecule including a1b1 and FXYD2 subunits with the bound lipids, while Figure 2B depicts the bound phospholipid plus cholesterol in greater detail. As shown in Kapri-Pardes et al 2011;Mishra et al 2011;Habeck et al 2015Habeck et al , 2017, the bound phosphatidyl serine and cholesterol interact with each other and residues in the transmembrane segments of FXYD2 and aM8, aM9 and aM10 to protect against thermal inactivation or excess detergent-mediated inactivation. FXYD2 protects against thermal inactivation by amplifying the mutual a/FXYD2, SOPS-cholesterol interactions (Kapri-Pardes et al 2011;Mishra et al 2011).…”

“…; Habeck et al. , ), the bound phosphatidyl serine and cholesterol interact with each other and residues in the transmembrane segments of FXYD2 and α M8, α M9 and α M10 to protect against thermal inactivation or excess detergent‐mediated inactivation. FXYD2 protects against thermal inactivation by amplifying the mutual α /FXYD2, SOPS–cholesterol interactions (Kapri‐Pardes et al.…”

“…The molecular mechanism of stabilization has now been established. It involves a specific binding interaction between the Na,K-ATPase and the acid phospholipid stearoyl oleoyl phosphatidyl serine (SOPS, 18:0-18:1PS) and cholesterol within the membrane domain (Cohen et al 2005;Haviv et al 2007Haviv et al , 2013Lifshitz et al 2007;Kapri-Pardes et al 2011;Cornelius et al 2015;Habeck et al 2015Habeck et al , 2017. This is depicted in Figure 2 (in the so-called site A -see (Cornelius et al 2015)).…”

Expression of multidrug transporter P‐glycoprotein in Pichia pastoris affects the host's methanol metabolism

“…Pichia pastoris is an efficient producer of recombinant membrane proteins for structural and functional studies (Bill et al ., ; Lee et al ., ; Habeck et al ., ). Unlike prokaryotes, this eukaryotic organism can produce correctly folded proteins with all the required disulfide bond and post‐translational modifications (Wang et al ., ), and the transgene is stably integrated in the host genome ensuring production stability (Athmaram et al ., ).…”