Phosphatidylserine Binding Sites in Erythroid Spectrin: Location and Implications for Membrane Stability

Abstract

The erythrocyte membrane is a composite structure consisting of a lipid bilayer tethered to the spectrin-based membrane skeleton. Two complexes of spectrin with other proteins are known to participate in the attachment. Spectrin has also been shown to interact with phosphatidylserine (PS), a component of the lipid bilayer, which is confined to its inner leaflet. That there may be multiple sites of interaction with PS in the spectrin sequence has been inferred, but they have not hitherto been identified. Here we have explored the interaction of PS-containing liposomes with native α- and β-spectrin chains and with recombinant spectrin fragments encompassing the entire sequences of both chains. We show that both α-spectrin and β-spectrin bind PS and that sites of high affinity are located within 8 of the 38 triple-helical structural repeats which make up the bulk of both chains; these are α8, α9−10, β2, β3, β4, β12, β13, and β14, and PS affinity was also found in the nonhomologous N-terminal domain of the β-chain. No other fragments of either chain showed appreciable binding. Binding of spectrin and its constituent chains to mixed liposomes of PS and phosphatidylcholine (PC) depended on the proportion of PS. Binding of spectrin dimers to PS liposomes was inhibited by single repeats containing PS binding sites. It is noteworthy that the PS binding sites in β-spectrin are grouped in close proximity to the sites of attachment both of ankyrin and of 4.1R, the proteins engaged in attachment of spectrin to the membrane. We conjecture that direct interaction of spectrin with PS in the membrane may modulate its interactions with the proteins and that (considering also the known affinity of 4.1R for PS) the formation of PS-rich lipid domains, which have been observed in the red cell membrane, may be a result.