Identification of the protein 4.1 binding site to phosphatidylserine vesicles

Abstract

Previous studies have shown that protein 4.1 is a multifunctional protein that binds to spectrin, actin, glycophorins, the anion channel protein, and phosphatidylserine (PS). In this report, we have characterized the binding of protein 4.1 and its major proteolytic fragments to phospholipid vesicles. Pure 125I-labeled protein 4.1 was incubated with PS liposomes, and the free protein 4.1 was separated by ultracentrifugation. Protein 4.1 bound to PS liposomes with a high affinity. At saturation, there was 9 .times. 10-3 pmol of protein 4.1 bound/pmol of PS with a Kd of 3.3 .times. 10-7 M. When the protein 4.1 containing lipoosomes were examined in an electron microscope, the protein 4.1 was found uniformly decorating the vesicles in a rosettelike fashion. Among peripheral membrane proteins tested (spectrin, actin, ankyrin, and protein 4.1), protein 4.1 showed the highest level of binding to PS. The binding of protein 4.1 to PS, one of the principal phospholipids of the inner half of the lipid bilayer, was considerably higher than the binding to phosphatidylcholine, that is principally located in the outer half of the lipid bilayer. To identify the structural domain of protein 4.1 involved in binding to the phospholipids, a mixture of proteolytic fragments of protein 4.1 was incubated with PS liposomes. The liposomes selectively retained 30-kilodalton (kDa) basic domain of the protein, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis/isoelectric focusing. The 30-kDa fragment was purified from chymotrypytic digests of protein 4.1 by ion-exchange chromatography on DEAE 52. The purified 30-kDa peptide of protein 4.1 competitively inhibited the binding of 125I-labeled protein 4.1 to PS. These data indicate that protein 4.1 is capable of forming high-affinity associations with PS. These associations may be important in the maintenance of normal red cell structure and function.