Characterization of the reversible conformational equilibrium in the cytoplasmic domain of human erythrocyte membrane band 3

The cytoplasmic domain of erythrocyte membrane band 3 (cdb3) serves as a ce nter of membrane organization, interacting with such proteins as ankyrin, p rotein 4.1, protein 4.2, hemoglobin, several glycolytic enzymes, and a tyro sine kinase, p72(syk). cdb3 exists in a reversible, pH-dependent conformati onal equilibrium characterized by large changes in Stokes radius (I I A) an d intrinsic fluorescence (2-fold). Based on the crystallographic structure of the cdb3 dimer, we hypothesized that the above conformational equilibriu m might involve the movement of flanking peripheral protein binding domains away from a shared dimerization domain. To test this hypothesis, we have m utated both donor (W105L) and acceptor (D316A) residues of a prominent H bo nd that bridges the above two domains and have examined the effect on the r esulting conformational equilibrium. Analysis of the intrinsic fluorescence , Stokes radius, thermal stability, urea stability, and segmental mobility of these mutants reveals that the above H bond is indeed present in the low pH conformation of cdb3 and broken in a higher pH conformation. The data f urther reveal that cdb3 exists in three native pH-dependent conformations a nd that rupture of the aforementioned H bond occurs only during conversion of the low pH conformation to the mid-pH conformation. Conversion of the mi d-pH conformation to the high pH conformation would now appear to involve s tructural changes primarily in the peripheral protein binding domain. Becau se ankyrin associates avidly with the low pH conformation of cdb3, ankyrin occupancy should strongly influence this structural equilibrium and thereby affect band 3 and perhaps global membrane properties.