Identification of a critical ankyrin-binding loop on the cytoplasmic domain of erythrocyte membrane band 3 by crystal structure analysis and site-directed mutagenesis

The cytoplasmic domain of erythrocyte membrane band 3 (cdb3) serves as a center of membrane organization, interacting with such proteins as ankyrin, protein 4.1, protein 4.2, hemoglobin, several glycolytic enzymes, a tyrosine phosphatase, and a tyrosine kinase, p72^syk. The crystallographic structure of the cdb3 dimer has revealed that residues 175-185 assume β-hairpin loop similar to a putative ankyrin-binding motif at the cytoplasmic surface of the Na⁺/K⁺-ATPase. To test whether this hairpin loop constitutes an ankyrin-binding site on cdb3, we have deleted amino acids 175-185 and substituted the 11-residue loop with a Gly-Gly dipeptide that bridges the deletion without introducing strain into the structure. Although the deletion mutant undergoes the same native conformational changes exhibited by wild type cdb3 and binds other peripheral proteins normally, the mutant exhibits no affinity for ankyrin. This suggests that the exposed β-hairpin turn indeed constitutes a major ankyrin-binding site on cdb3. Other biochemical studies suggest that ankyrin also docks at the NH₂ terminus of band 3. Thus, antibodies to the NH₂ terminus of cdb3 block ankyrin binding to the cdb3, and ankyrin binding to cdb3 prevents p72^syk phosphorylation of cdb3 at its NH₂ terminus (predominantly at Tyr-8). However, a truncation mutant of cdb3 lacking the NH₂-terminal 50 residues displays the same binding affinity as wild type cdb3. These data thus suggest that the NH₂ terminus of cdb3 is proximal to but not required for the cdb3. ankyrin interaction.