Ca2+-dependent and Ca2+-independent Calmodulin Binding Sites in Erythrocyte Protein 4.1

In vitro protein binding assays identified two distinct calmodulin (CaM) binding sites within the NH2-terminal 30-kDa domain of erythrocyte protein 4.1 (4.1R): a Ca2+-independent binding site (A264KKLWKVCVEHHTFFRL) and a Ca2+-dependent binding site (A181KKLSMYGVDLHKAKDL). Synthetic peptides corresponding to these sequences bound CaM in vitro; conversely, deletion of these peptides from a 30-kDa construct reduced binding to CaM. Thus, 4.1R is a unique CaM-binding protein in that it has distinct Ca2+-dependent and Ca2+-independent high affinity CaM binding sites. CaM bound to 4.1R at a stoichiometry of 1:1 both in the presence and absence of Ca2+, implying that one CaM molecule binds to two distinct sites in the same molecule of 4.1R. Interactions of 4.1R with membrane proteins such as band 3 is regulated by Ca2+ and CaM. While the intrinsic affinity of the 30-kDa domain for the cytoplasmic tail of erythrocyte membrane band 3 was not altered by elimination of one or both CaM binding sites, the ability of Ca2+/CaM to down-regulate 4.1R-band 3 interaction was abrogated by such deletions. Thus, regulation of protein 4.1 binding to membrane proteins by Ca2+ and CaM requires binding of CaM to both Ca2+-independent and Ca2+-dependent sites in protein 4.1.