Identification of adducin-binding residues on the cytoplasmic domain of erythrocyte membrane protein, band 3

Abstract

Two major complexes form structural bridges that connect the erythrocyte membrane to its underlying spectrin-based cytoskeleton. Although the band 3-ankyrin bridge may account for most of the membrane-to-cytoskeleton interactions, the linkage between the cytoplasmic domain of band 3 (cdb3) and adducin has also been shown to be critical to membrane integrity. In the present paper, we demonstrate that adducin, a major component of the spectrin-actin junctional complex, binds primarily to residues 246-264 of cdb3, and mutation of two exposed glutamic acid residues within this sequence completely abrogates both α- and β-adducin binding. Because these residues are located next to the ankyrin-binding site on cdb3, it seems unlikely that band 3 can bind ankyrin and adducin concurrently, reducing the chances of an association between the ankyrin and junctional complexes that would significantly compromise erythrocyte membrane integrity. We also demonstrate that adducin binds the kidney isoform of cdb3, a spliceoform that lacks the first 65 amino acids of erythrocyte cdb3, including the central strand of a large β-pleated sheet. Because kidney cdb3 is not known to bind any of the common peripheral protein partners of erythrocyte cdb3, including ankyrin, protein 4.1, glyceraldehyde-3-phosphate dehydrogenase, aldolase, and phosphofructokinase, retention of this affinity for adducin was unexpected.

Keywords: adducin; anion exchanger 1; cytoplasmic domain of band 3; erythrocytes; junctional complexes; membrane structure.

Figure 1

The association of GST-β-adducin tail with band 3 is not affected by the absence of the first 65 amino acids of cdb3. His-tagged kidney-cdb3 (triangles) or BSA (open squares) was immobilized on nickel beads and incubated with increasing concentrations of GST-β-adducin tail. Beads were pelleted, washed 5 times, and eluted with 250 mM imidazole in PBS. GST activity was then quantified as a measure of the amount of bound adducin. (a.u. represents arbitrary units).

Figure 2

Constellation of surface protruding residues located in the binding region comprising residues 246–264 of the lower pH conformation of cdb3. Accessible surface amino acids residing within 12Å of the geometric center of the above sequence were determined from the crystal structure of cdb3, and are shown with an (A) en face view and (B) right-rotated view. The two most exposed residues, Glu 252 and Glu 254, are highlighted in red. Less prominently exposed residues are highlighted in blue.

Figure 3

Binding of β-adducin tail and ankyrin to wild-type and mutant cdb3. Increasing concentrations of GST-β-adducin tail or GST-D3D4-ankyrin were incubated with immobilized wild-type or mutated cdb3 in which glutamates 252 and 254 were mutated to lysines. Beads were washed with PBS and eluted with 250 mM imidazole, and GST activity was quantified as a measure of bound adducin or ankyrin. (A) Binding of β-adducin tail domain to wild type cdb3 (blue, K_d = 260 nM), mutated cdb3 (red), or BSA (purple) is shown as a function of GST-β-adducin tail domain concentration. Binding of D3D4-ankyrin (green) to mutated cdb3 is shown as a function of GST-D3D4-ankyrin concentration. Individual data points (n=2) are plotted with estimated binding curves. (B) Dot blot analysis of β-adducin tail binding to purified wild-type and mutant cdb3. Wild-type or mutated band 3 was immobilized on nickel beads and incubated with increasing concentrations of GST-β-adducin tail domain. Beads were pelleted, washed 4×, and eluted with 250 mM imidazole in PBS. Eluted proteins were transferred to a nitrocellulose membranes and probed for GST-β-adducin tail with an anti-GST polyclonal antibody.

Figure 4

α-adducin tail binds kidney and erythrocyte cdb3, but not mutant cdb3, in a dose-dependent manner. α-adducin tail domain was reacted with Affi-Gel 15 beads. Different concentrations of His-tagged kidney (K-cdb3), erythrocyte (wt-cdb3), and mutant cdb3 (mt-cdb3) were incubated with the derivatized beads, and beads were pelleted, washed 4×, eluted and separated by SDS-PAGE. Cdb3 was then crudely quantitated by western blotting using anti-cdb3 antibody.

Figure 5

Mutation of cdb3 does not affect other protein functions. (A) Dot blot analysis of GST-D3D4-ankyrin interaction with wild-type and mutant cdb3. 1) Empty bead control, 2) wild-type cdb3, or 3) mutant cdb3 were immobilized on nickel beads and incubated with increasing concentrations of GST-D3D4-ankyrin. After washing and elution of proteins with 250 mM imidazole, eluted proteins were dot blotted on to nitrocellulose and probed with anti-GST antibody. (B) Effect of wild type and mutant cdb3 on GAPDH activity in solution. Wild-type erythrocyte band 3 has already been established to bind and inhibit GAPDH [39], whereas BSA has no effect on GAPDH activity. Data points represent mean ± S.D., n = 4.

Figure 6

Cdb3 mutants that cannot bind adducin retain normal structural properties. (A) Comparison of the pH-dependent conformational changes of wild-type and mutant cdb3. The intrinsic fluorescence (emission at 335 nm) of purified recombinant wild-type and mutant cdb3 was measured following excitation 290 nm and plotted as a function of pH. Data points represent mean ± S.D., n = 2. (B) Comparison of the circular dichroism spectra of wild-type and mutant cdb3. The CD spectrum of wild-type and mutant cdb3 was monitored in 50 mM sodium phosphate, 50 mM sodium borate, 70 mM NaCl, pH 7.4 at 24°C using a Jasco Model J810 CD spectropolarimeter.

Figure 7

Structure of the cdb3 dimer with the adducin (red, blue, and yellow) and ankyrin (orange) binding sites shown on the white subunit. Glutamates 252 and 254 which are critical for adducin binding are colored in blue and yellow, respectively. A structurally identical unlabeled subunit of cdb3 is shown in green.