Molecular Epitopes of the Ankyrin−Spectrin Interaction

Abstract

Isoforms of ankyrin and its binding partner spectrin are responsible for a number of interactions in a variety of human cells. Conflicting evidence, however, had identified two different, non-overlapping human erythroid ankyrin subdomains, Zu5 and 272, as the minimum binding region for β-spectrin. Complementary studies on the ankyrin-binding domain of spectrin have been somewhat more conclusive yet have not presented binding in terms of well-phased, integral numbers of spectrin repeats. Thus, the objective of this study was to clearly define and characterize the minimal ankyrin−spectrin binding epitopes. Circular dichroism (CD) wavelength spectra of the aforementioned ankyrin subdomains show that these fragments are 30−60% unstructured. In contrast, human erythroid β-spectrin repeats 13, 14, 15, and 16 (prepared in all combinations of two adjacent repeats) demonstrated proper folding and stability as determined by CD and tryptophan wavelength and heat denaturation scans. Native polyacrylamide gel electrophoresis (PAGE) gel shifts as well as affinity pull-down assays implicated Zu5 and β-spectrin repeats 14−15 as the minimum binding epitopes. These results were confirmed by analytical ultracentrifugation to sedimentation equilibrium by which a 1:1 complex was obtained if and only if Zu5 was mixed with β-spectrin constructs containing repeats 14 and 15 in tandem. Surface plasmon resonance yielded a K D of 15.2 nM for binding of β-spectrin fragments to the ankyrin subdomain Zu5, accounting for all of the binding observed between the intact molecules. Collectively, these results show the 14th and 15th β-spectrin repeats comprise the minimal, phased region of β-spectrin, which binds ankyrin at the Zu5 subdomain with high affinity.

Identification of cytoskeletal elements enclosing the ATP pools that fuel human red blood cell membrane cation pumps

“…The prime distinctions between the two complexes lie in the fact that the former contains actin, adducin, and protein 4.1, whereas the latter contains ankyrin. Our results clearly demonstrate that N 3 -ATP labels ankyrin in its ZU5 domain, which contains the binding site for β-spectrin (20) and lies adjacent to the ankyrin-binding site for band 3 (27,28). N 3 -ATP also labels β-spectrin in a flexible region near the site responsible for association of αβ-spectrin dimers to tetramers and not too distant from the ankyrin-binding site on β-spectrin (18)(19)(20).…”

“…Our results clearly demonstrate that N 3 -ATP labels ankyrin in its ZU5 domain, which contains the binding site for β-spectrin (20) and lies adjacent to the ankyrin-binding site for band 3 (27,28). N 3 -ATP also labels β-spectrin in a flexible region near the site responsible for association of αβ-spectrin dimers to tetramers and not too distant from the ankyrin-binding site on β-spectrin (18)(19)(20). In addition, N 3 -ATP labels band 3 near the junction of its cytoplasmic and membrane-spanning domains within a peptide recently found to include a second binding site for GAPDH.…”

“…The ATP labeling on β-spectrin is at the extreme COOH terminus, near the tetramerization site and just beyond the ankyrin-binding site (18,19). Ankyrin is labeled in its ZU-5 domain, within the spectrin-binding sequence of ankyrin (20,21). Band 3 is labeled specifically at a glycolytic enzyme-binding site that can associate with GAPDH, aldolase, phosphofructokinase, and lactate dehydrogenase.…”

Erythrocyte membrane changes of chorea-acanthocytosis are the result of altered Lyn kinase activity

“…An intercrossed search to identify whether these peptides could also serve as substrates for Src Tyr kinases restricted the Lyn-specific target sequences to 2 Tyr residues Tyr-307 and Tyr-1590. Inspection of the 3D structure indicated that the Tyr-307 is located inside the triple-helical bundle, whereas Tyr-1590 is in the linker region preceding repeat 14, at the interface of the ankyrin binding domain (repeats 14 and 15) [33][34][35] (Table 3). The phosphopeptide of ␤-adducin (Tyr-377) is a specific target of Lyn with little specificity for Src (Table 3) and is located in a highly conserved sequence of the neck domain of the protein.…”

The structure of the ankyrin-binding site of β-spectrin reveals how tandem spectrin-repeats generate unique ligand-binding properties

“…Kennedy et al 23 and Ipsaro et al 24 established that the ankyrin-binding site in ␤I-spectrin lay between codons 1768-1898, a sequence bridging the terminal third of the 14th repeat (␤I-14) and most of the 15th repeat (␤I-15). This region is well conserved in all classical ␤-spectrins (␤I-␤IV); its most unusual feature is an absence of highly conserved tryptophan and histidine residues in ␤I-15.…”