Phosphatidylinositol-4,5 bisphosphate (PIP2) inhibits apo-calmodulin binding to protein 4.1

Membrane skeletal protein 4.1R80 plays a key role in regulation of erythrocyte plasticity. Protein 4.1R80 interactions with transmembrane proteins, such as glycophorin C (GPC), are regulated by Ca2+-saturated calmodulin (Ca2+/CaM) through simultaneous binding to a short peptide (pep11; A264KKLWKVCVEHHTFFRL) and a serine residue (Ser185), both located in the N-terminal 30 kDa FERM domain of 4.1R80 (H·R30). We have previously demonstrated that CaM binding to H·R30 is Ca2+-independent and that CaM binding to H·R30 is responsible for the maintenance of H·R30 β-sheet structure. However, the mechanisms responsible for the regulation of CaM binding to H·R30 are still unknown. To investigate this, we took advantage of similarities and differences in the structure of Coracle, the Drosophila sp. homologue of human 4.1R80, i.e. conservation of the pep11 sequence but substitution of the Ser185 residue with an alanine residue. We show that the H·R30 homologue domain of Coracle, Cor30, also binds to CaM in a Ca2+-independent manner and that the Ca2+/CaM complex does not affect Cor30 binding to the transmembrane protein GPC. We also document that both H·R30 and Cor30 bind to phosphatidylinositol-4,5 bisphosphate (PIP2) and other phospholipid species and that that PIP2 inhibits Ca2+-free CaM but not Ca2+-saturated CaM binding to Cor30. We conclude that PIP2 may play an important role as a modulator of apo-CaM binding to 4.1R80 throughout evolution.