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In addition to reducing hemoglobin-O2 affinity, 2,3-diphosphoglycerate (DPG) is known to modulate the mechanical properties of the erythrocyte membrane. By fluorescence spectroscopy and differential scanning calorimetry, we demonstrate that DPG binds the cytoplasmic domain of erythrocyte membrane band 3 in two stages characterized by apparent KD values of approximately approximately 2 and 12 mM. DPG was also shown to perturb the stability of ankyrin, protein 4.1, and protein 4.2 in situ and to directly bind to protein 4.1. In studies of membrane-skeleton interactions, DPG was observed to inhibit the fast and slow phases of ankyrin binding to band 3 and to reduce both the number of ankyrin sites and affinity of ankyrin for each class of site. The inhibition was biphasic, similar to the band 3-DPG binding isotherm; however, at physiological DPG concentrations a reduction in only 15% of the ankyrin sites was observed. In contrast, inhibition of protein 4.1 binding to the membrane reached 65% at physiological DPG concentrations (approximately approximately 5.9 mM); at more elevated concentrations, blockade was nearly quantitative, affecting glycophorin and band 3 sites alike. Taken together with previous observations, these data suggest that DPG's effect on O2 delivery may extend beyond its well recognized impact on hemoglobin-O2 affinity.

Of the several proteins that bind along the cytoplasmic domain of erythrocyte membrane band 3, only the sites of interaction of proteins 4.1 and 4.2 remain to be at least partially localized. Using five independent techniques, we have undertaken to map and characterize the binding site of band 4.1 on band 3. First, transfer of a radioactive cross-linker (125I-2-(p-azido-salicylamido)ethyl-1-3-dithiopropionate) from purified band 4.1 to its binding sites on stripped inside-out erythrocyte membrane vesicles (stripped IOVs) revealed major labeling of band 3, glycophorin C, and glycophorin A. Proteolytic mapping of the stripped IOVs then demonstrated that the label on band 3 was confined largely to a fragment comprising residues 1-201. Second, competitive binding experiments with Fab fragments of monoclonal and peptide-specific polyclonal antibodies to numerous epitopes along the cytoplasmic domain of band 3 displayed stoichiometric competition only with Fabs to epitopes between residues 1 and 91 of band 3. Weak competition was also observed with Fabs to a sequence of the cytoplasmic domain directly adjacent to the membrane-spanning domain, but only at 50-100-fold excess of Fab. Third, band 4.1 protected band 3 from chymotryptic hydrolysis at tyrosine 46 and to a much lesser extent at a site within the junctional peptide connecting the membrane-spanning and cytoplasmic domains of band 3. Fourth, ankyrin, which has been previously shown to interact with band 3 both near a putative central hinge and at the N terminus competed with band 4.1 for band 3 in stripped IOVs. Since band 4.1 does not associate with band 3 near the flexible central hinge, the competition with ankyrin can be assumed to derive from a mutual association with the N terminus. Finally, a synthetic peptide corresponding to residues 1-15 of band 3 was found to mildly inhibit band 4.1 binding to stripped IOVs. Taken together, these data suggest that band 4.1 binds band 3 predominantly near the N terminus, with a possible secondary site near the junction of the cytoplasmic domain and the membrane.