Three-dimensional structure of 30-kDa 4.1R and competition of MESA binding to 4.1 by p55. (A) The putative 24-residue MESA binding site of protein 4.1 is located within a 51-residue region encoded by exon 10 of 4.1R, located in the C-lobe of the protein molecule. The binding affinity between 4.1R and MESA was enhanced by sequences within the 16-kDa domain, although MESA did not bind to the 16-kDa domain alone. The binding residues within this 30-kDa region for phosphatidylserine (PS), p55, calmodulin (CaM), band 3, and GPC are also shown. The representation is drawn on the basis of the 3D crystal structure described by Han et al.¹²  (B) Competition of binding was demonstrated using GST-pull down assays, in which GST-4.1 80 kDa (coupled to glutathione Sepharose beads; Amersham Pharmacia Biotech, Piscataway, NJ) pre-incubated (30 minutes at room temperature) with increasing concentrations of p55 (ranging from 0.0 μM to 2.0 μM), before the addition of 1.0 μM His-tagged MESA(S)+19 (30 minutes at room temperature). Subsequently, beads were washed and collected, and the proteins resolved by SDS-PAGE (10% [wt/vol]). The binding of MESA to 4.1R was detected by Western blot using monoclonal anti-His antibody (Roche, Indianapolis, IN). The first lane contains 0.45 μg His-tagged MESA that was included as an immunoblot MESA-positive control. Competition of MESA for 4.1 by p55 was indicated by the decreasing levels of MESA detected in samples with increasing concentrations of p55 used in the preincubations.