Mapping of an ankyrin-sensitive, phosphatidylethanolamine/phosphatidylcholine mono- and bi-layer binding site in erythroid beta-spectrin.
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.
Hryniewicz-Jankowska
Anita
A
Institute of Biochemistry and Molecular Biology, University of Wrocław, ul. Przybyszewskiego 63/77, 51-148 Wrocław, Poland.
Bok
Ewa
E
Dubielecka
Patrycja
P
Chorzalska
Anna
A
Diakowski
Witold
W
Jezierski
Adam
A
Lisowski
Marek
M
Sikorski
Aleksander F
AF
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
D017487:Ankyrins; D008051:Lipid Bilayers; D008567:Membranes, Artificial; D010455:Peptides; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D013049:Spectrin