title: Mapping of an ankyrin-sensitive, phosphatidylethanolamine/phosphatidylcholine mono- and bi-layer binding site in erythroid beta-spectrin.
abstract: It has been shown previously that binding of vesicles and monolayers containing PE (phosphatidylethanolamine) by either erythroid or non-erythroid spectrin proved sensitive to inhibition by purified erythrocyte ankyrin. We tested the lipid-binding affinities of the purified ankyrin-binding domain of beta-spectrin and of its truncated mutants in four ways, by analysing: (1) penetration of 'loose' PE/PC (phosphatidylcholine) monolayers; (2) binding to liposomes in suspension; (3) competition with spectrin for liposomes; and (4) binding of a PE/PC monolayer in a surface plasmon resonance system. The results obtained indicated that the full-length ankyrin-binding domain bound PE/PC mono- and bi-layers with moderate affinity, penetrated monolayers and competed with spectrin for liposomes. Moreover, its truncated mutants that retained the N-terminal part, in contrast with those lacking eight or 38 N-terminal residues (which bound lipid mono- and bi-layers with lower affinity), bound PE/PC mono- and bi-layers with an affinity and capacity comparable with those of the full-length ankyrin-binding domain, and this activity was inhibited by purified erythrocyte ankyrin. The full-length domain, in contrast with the mutant lacking 38 N-terminal residues, induced a small increase in the fluidity of PE/PC membranes when probed with 5'-doxyl stearate, similar to the effect of purified spectrin. Therefore we conclude that the binding site for PE-rich lipids, which is sensitive to ankyrin inhibition, is located in a 38-residue N-terminal fragment of the beta-spectrin ankyrin-binding domain, and that the first eight residues play a key role in this activity.
authors.affiliation: Institute of Biochemistry and Molecular Biology, University of Wrocław, ul. Przybyszewskiego 63/77, 51-148 Wrocław, Poland.
authors.forename: Anita
authors.initials: A
authors.lastname: Hryniewicz-Jankowska
authors.forename: Ewa
authors.initials: E
authors.lastname: Bok
authors.forename: Patrycja
authors.initials: P
authors.lastname: Dubielecka
authors.forename: Anna
authors.initials: A
authors.lastname: Chorzalska
authors.forename: Witold
authors.initials: W
authors.lastname: Diakowski
authors.forename: Adam
authors.initials: A
authors.lastname: Jezierski
authors.forename: Marek
authors.initials: M
authors.lastname: Lisowski
authors.forename: Aleksander F
authors.initials: AF
authors.lastname: Sikorski
mesh_terms: D017398:Alternative Splicing / Q000235:genetics; D017487:Ankyrins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002942:Circular Dichroism / Q000379:methods; D003001:Cloning, Molecular; D004912:Erythrocytes / Q000737:chemistry*; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D050356:Lipid Metabolism; D008560:Membrane Fluidity; D008567:Membranes, Artificial; D009154:Mutation / Q000235:genetics; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D010713:Phosphatidylcholines / Q000737:chemistry / Q000378:metabolism*; D010714:Phosphatidylethanolamines / Q000737:chemistry / Q000378:metabolism*; D017434:Protein Structure, Tertiary / Q000235:genetics; D013049:Spectrin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D017487:Ankyrins; D008051:Lipid Bilayers; D008567:Membranes, Artificial; D010455:Peptides; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D013049:Spectrin