title: Identification of contact sites between ankyrin and band 3 in the human erythrocyte membrane.
abstract: The red cell membrane is stabilized by a spectrin/actin-based cortical cytoskeleton connected to the phospholipid bilayer via multiple protein bridges. By virtue of its interaction with ankyrin and adducin, the anion transporter, band 3 (AE1), contributes prominently to these bridges. In a previous study, we demonstrated that an exposed loop comprising residues 175-185 of the cytoplasmic domain of band 3 (cdB3) constitutes a critical docking site for ankyrin on band 3. In this paper, we demonstrate that an adjacent loop, comprising residues 63-73 of cdB3, is also essential for ankyrin binding. Data that support this hypothesis include the following. (1) Deletion or mutation of residues within the latter loop abrogates ankyrin binding without affecting cdB3 structure or its other functions. (2) Association of cdB3 with ankyrin is inhibited by competition with the loop peptide. (3) Resealing of the loop peptide into erythrocyte ghosts alters membrane morphology and stability. To characterize cdB3-ankyrin interaction further, we identified their interfacial contact sites using molecular docking software and the crystal structures of D(3)D(4)-ankyrin and cdB3. The best fit for the interaction reveals multiple salt bridges and hydrophobic contacts between the two proteins. The most important ion pair interactions are (i) cdB3 K69-ankyrin E645, (ii) cdB3 E72-ankyrin K611, and (iii) cdB3 D183-ankyrin N601 and Q634. Mutation of these four residues on ankyrin yielded an ankyrin with a native CD spectrum but little or no affinity for cdB3. These data define the docking interface between cdB3 and ankyrin in greater detail.
authors.affiliation: Department of Chemistry, Purdue University, 560 Oval Drive, West Lafayette, IN 47907, USA.
authors.forename: Jesse L
authors.initials: JL
authors.lastname: Grey
authors.forename: Gayani C
authors.initials: GC
authors.lastname: Kodippili
authors.forename: Katya
authors.initials: K
authors.lastname: Simon
authors.forename: Philip S
authors.initials: PS
authors.lastname: Low
mesh_terms: D001457:Anion Exchange Protein 1, Erythrocyte / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017487:Ankyrins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D004910:Erythrocyte Membrane / Q000737:chemistry / Q000378:metabolism*; D004912:Erythrocytes / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation
chemical_list: D001457:Anion Exchange Protein 1, Erythrocyte; D017487:Ankyrins