Characterization of the ankyrin-band 3 interaction of the erythrocyte membrane
The interaction between ankyrin and band 3 of the human erythrocyte membrane constitutes the major linkage between the cytoskeletal infrastructure and the lipid bilayer. Despite the importance of this association in red cell mechanical properties, little is known concerning its dynamic and structural characteristics and especially its regulation. We have found that the ability of band 3 to associate with ankyrin is dependent on the redox state of its two cytoplasmic domain sulfhydryls, Cys 201 and 317, and that all affinity for ankyrin is lost upon their covalent derivatization or reversible oxidation. Secondly, we have shown that band 3 cytoplasmic cysteines are clustered in a pocket at the interface between the two subunits of the band 3 dimer and that this cysteine cluster is located just above the putative hinge (residues 176-191) around which the cytoplasmic domain undergoes major conformational changes. Then, we have confirmed that this sulfhydryl region is the most critical site for ankyrin interaction by mapping the binding regions of ankyrin on band 3 using competition assays with antibodies specific to various epitopes along its elongated structure. This study also revealed the unexpected importance of the N-terminal region of the domain for ankyrin binding, a result corroborated by the blockade of phosphorylation of band 3's N-terminal tyrosine residues upon association with ankyrin and by the ability of glycolytic enzymes to inhibit ankyrin binding. Finally, we have shown that band 3 exists in situ in an equilibrium among three binding forms having either no, low, or high affinity for ankyrin and corresponding, respectively, to the three native conformational states of the cytoplasmic domain of band 3. Since the dissociation of bound ankyrin is able to occur rapidly, these results suggest the importance of band 3's bending motion about its central hinge in modulating the cytoskeletal linkage to the membrane. ^
Major Professor: Philip S. Low, Purdue University.