Determination of Structural Models of the Complex between the Cytoplasmic Domain of Erythrocyte Band 3 and Ankyrin-R Repeats 13–24

Abstract

The adaptor protein ankyrin-R interacts via its membrane binding domain with the cytoplasmic domain of the anion exchange protein (AE1) and via its spectrin binding domain with the spectrin-based membrane skeleton in human erythrocytes. This set of interactions provides a bridge between the lipid bilayer and the membrane skeleton, thereby stabilizing the membrane. Crystal structures for the dimeric cytoplasmic domain of AE1 (cdb3) and for a 12-ankyrin repeat segment (repeats 13-24) from the membrane binding domain of ankyrin-R (AnkD34) have been reported. However, structural data on how these proteins assemble to form a stable complex have not been reported. In the current studies, site-directed spin labeling, in combination with electron paramagnetic resonance (EPR) and double electron-electron resonance, has been utilized to map the binding interfaces of the two proteins in the complex and to obtain inter-protein distance constraints. These data have been utilized to construct a family of structural models that are consistent with the full range of experimental data. These models indicate that an extensive area on the peripheral domain of cdb3 binds to ankyrin repeats 18 -20 on the top loop surface of AnkD34 primarily through hydrophobic interactions. This is a previously uncharacterized surface for binding of cdb3 to AnkD34. Because a second dimer of cdb3 is known to bind to ankyrin repeats 7-12 of the membrane binding domain of ankyrin-R, the current models have significant implications regarding the structural nature of a tetrameric form of AE1 that is hypothesized to be involved in binding to full-length ankyrin-R in the erythrocyte membrane.Human erythrocytes exhibit an unusual biconcave disc shape and remarkable plasma membrane mechanical stability and deformability, all of which are necessary for their survival in the circulatory system. It is now well established that the unusual cell shape and membrane mechanical properties are due in large part to the presence of an extensive membrane skeleton, composed primarily of the proteins spectrin and actin, that lines the inner membrane surface and to specific bridging interactions between this membrane skeleton and intrinsic membrane proteins in the lipid bilayer. The spectrin-actin skeleton associates with the membrane bilayer via two types of contacts, one involving short actin protofilaments and protein 4.1, which interact with the cytoplasmic domain of glycophorin C, and the second involving the bridging protein ankyrin-R and protein 4.2, which interact with the cytoplasmic domain of the anion exchange protein (AE1) 3 also known as band 3. Alterations in this second class of interactions often result in spherical erythrocytes with decreased cell size and increased fragility, a condition known clinically as hereditary spherocytosis. Hereditary spherocytosis is a spectrum of inherited diseases, occurring in one family out of 2,000 -3,000, which present clinically as varying degrees of hemolytic anemia resulting from hemolysis of the spherical erythrocytes ...

Anatomy of the red cell membrane skeleton: unanswered questions

“…The fact that a stable subpopulation of band 3 tetramers can be isolated from nonionic detergent extracts of ghosts 75 argues for the former. But the structure of a band 3 tetramer binding to the full membrane domain has not been solved, and at least 1 recent paper argues that band 3 dimers must bind independently, 76 so the question remains. The ankyrin-spectrin binding site is well defined.…”

Analysis of the Mobilities of Band 3 Populations Associated with Ankyrin Protein and Junctional Complexes in Intact Murine Erythrocytes

“…Because lipid bilayers were already known to be intrinsically unstable (3,4), research rapidly focused on identifying linkages between the fragile bilayer and the more stable spectrin-based membrane skeleton that might stabilize the bilayer. A protein complex composed of the membrane-spanning protein, band 3 (AE1), linked to spectrin via ankyrin, was the first bridge shown to perform this linking function (5)(6)(7)(8)(9)(10). Evidence for the critical role of the band 3-ankyrin bridge came not only from studies of membranes in which the bridge was artificially ruptured (11)(12)(13) and from characterization of band 3-knock-out mice (14,15), but also from observations that natural mutations in any of the bridging components commonly led to an inherited membrane instability termed hereditary spherocytosis (16 -19).…”

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

“…One of these stretches (residues 110 -120) is identical to a major interacting loop (residues 175-185) of the cytoplasmic N terminus of eAE1 with ankyrin-R (21) but appears to be a secondary docking site for ankyrin-G in our two-hybrid experiments because its deletion alone has no effect on the binding efficiency. The two other interacting regions of kAE1 (residues 241-319 and 328 -331) do not encompass any of the determinants defined for eAE1 (from amino acids 70 -302 or 5-237 in eAE1 or kAE1 numberings, respectively) (31,32). All three domains must be deleted or mutated for a complete inhibition of ankyrin-G binding.…”