Identification of a Critical Ankyrin-binding Loop on the Cytoplasmic Domain of Erythrocyte Membrane Band 3 by Crystal Structure Analysis and Site-directed Mutagenesis

Abstract

The cytoplasmic domain of erythrocyte membrane band 3 (cdb3) serves as a center of membrane organization, interacting with such proteins as ankyrin, protein 4.1, protein 4.2, hemoglobin, several glycolytic enzymes, a tyrosine phosphatase, and a tyrosine kinase, p72syk . The crystallographic structure of the cdb3 dimer has revealed that residues 175-185 assume a ␤-hairpin loop similar to a putative ankyrin-binding motif at the cytoplasmic surface of the Na ؉ /K ؉ -ATPase. To test whether this hairpin loop constitutes an ankyrin-binding site on cdb3, we have deleted amino acids 175-185 and substituted the 11-residue loop with a Gly-Gly dipeptide that bridges the deletion without introducing strain into the structure. Although the deletion mutant undergoes the same native conformational changes exhibited by wild type cdb3 and binds other peripheral proteins normally, the mutant exhibits no affinity for ankyrin. This suggests that the exposed ␤-hairpin turn indeed constitutes a major ankyrin-binding site on cdb3. Other biochemical studies suggest that ankyrin also docks at the NH 2 terminus of band 3. Thus, antibodies to the NH 2 terminus of cdb3 block ankyrin binding to the cdb3, and ankyrin binding to cdb3 prevents p72 syk phosphorylation of cdb3 at its NH 2 terminus (predominantly at Tyr-8). However, a truncation mutant of cdb3 lacking the NH 2 -terminal 50 residues displays the same binding affinity as wild type cdb3. These data thus suggest that the NH 2 terminus of cdb3 is proximal to but not required for the cdb3-ankyrin interaction.Ankyrin mediates the attachment of a diverse set of membrane spanning proteins to spectrin-based membrane skeletons. Depending on the cell type, ankyrin may bridge between the ␤ subunit of spectrin and the anion exchanger (1), the Na ϩ /K ϩ -ATPase (2, 3), a voltage-dependent Na ϩ channel (4), or the Na ϩ /Ca 2ϩ exchanger (5). Cell adhesion molecules such as CD44 (6) and L1CAM family members (7,8), as well as calcium-release channels such as IP3 receptor (9) and ryanodine receptor (10) are also known to associate with ankyrin.Ankyrin is folded into three independent domains that include an 89-kDa NH 2 -terminal membrane-binding domain, followed by a 62-kDa spectrin-binding domain and a COOHterminal regulatory domain. The membrane-binding domain of ankyrin consists of 24 tandem repeats of a 33-amino acid motif known as the ankyrin repeat that is involved in protein recognition (11)(12)(13)(14). Because ankyrin interacts with a highly diverse group of membrane proteins, much effort has been devoted to identifying the structural features that mediate these interactions (15-18).The major linkage between the membrane bilayer and spectrin-based cortical skeleton in erythrocytes is mediated by ankyrin binding to band 3. Because previous studies (19 -22) aimed at mapping the docking site(s) of ankyrin on the cytoplasmic domain of erythrocyte membrane band 3 (cdb3) 1 were conducted without the benefit of the crystal structure of cdb3, these investigations of necessity led to inexact conc...

Acute pH-dependent Regulation of AE2-mediated Anion Exchange Involves Discrete Local Surfaces of the NH2-terminal Cytoplasmic Domain

“…This phenotype most likely arises by alteration of the hinge angle between the large globular domain and the dimerization arm (25), as revealed by the crystal structure of the hAE1 NH 2 -terminal cytoplasmic domain oligomer (10). We hypothesized that a similar pH-dependent movement around a homologous hinge region might contribute to pH-dependent regulation of AE2-mediated anion exchange.…”

Evidence of a Structural and Functional Ammonium Transporter RhBG·Anion Exchanger 1·Ankyrin-G Complex in Kidney Epithelial Cells

“…All three domains must be deleted or mutated for a complete inhibition of ankyrin-G binding. Crystallographic and other biophysical analyses suggested a different conformation of kAE1 cytoplasmic N terminus compared with that of eAE1 (16,17,21), which probably accounts for the very limited redundancy of ankyrin binding regions in the two proteins. Moreover, the three ankyrin species (R, B, and G) display 17% amino acid sequence divergence between their membrane binding domains (33), which should imply the involvement of different residues of ankyrin-G D3-D4 repeat subdomains in the interaction with kAE1 compared with ankyrin-R-eAE1 association (32).…”

“…First, the deletion of the D3-D4 ankyrin-R binding site in eAE1 protein (amino acids 175-185; Ref. 21) was achieved (amino acids 110 -120 in kAE1 numbering). The deleted 11 residues were substituted with a bridging Gly-Gly dipeptide, as originally done by Chang and Low (21) on eAE1.…”

An 11-amino acid β-hairpin loop in the cytoplasmic domain of band 3 is responsible for ankyrin binding in mouse erythrocytes

“…Erythrocytes from ankyrin-deficient (nb/nb) and band 3-deficient (Slc4a1 Ϫ/Ϫ ) mice exhibit a severe loss of mechanical stability, altered morphology, and dramatically reduced survival (20)(21)(22). We and others have recently solved the crystal structure of cdb3 (23) and the cdb3 binding domain of ankyrin (24), respectively. The crystal structures predict that an 11-aa ␤-hairpin loop in cdb3 (residues 188-198 in the mouse and 175-185 in human) binds to the D3D4 domains of ankyrin (24).…”

“…The crystal structures predict that an 11-aa ␤-hairpin loop in cdb3 (residues 188-198 in the mouse and 175-185 in human) binds to the D3D4 domains of ankyrin (24). Deletion of the ␤-hairpin loop specifically abolishes ankyrin binding to cdb3 in vitro, leading to the conclusion that ankyrin and band 3 interact specifically through this loop (23).…”