A β_H-spectrin self-interaction domain forms antiparallel dimers that may facilitate non-canonical spectrin membrane...

Tetrameric spectrin proteins crosslink filamentous-actin (F-actin) into planar membrane-associated networks known as spectrin based membrane skeletons (SBMS). The importance of tetramer formation in SBMS formation is amply demonstrated by mutations in human αΙ-spectrin that break the H2H association leading to hereditary elliptocytosis (HE). Tetramer formation requires antiparallel αβ-spectrin or αβ_Η-spectrin dimerization via dimer nucleation sites, followed by (αβ)₂ or (αβ_H)₂ tetramer formation via the head-to-head (H2H) association of two dimers. The presence of a singular N-terminal F-actin binding site in each β chain, makes tetramer formation obligatory for F-actin crosslinking in all structurally defined SBMS. We previously showed that the α-spec^R22S mutation in Drosophila (homologous to the human αI^R28S HE allele), eliminates H2H binding in both conventional (αβ)₂ and heavy (αβ_H)₂ spectrin isoforms in vitro. However, α-spec^R22S mutant flies remain surprisingly unaffected. In (αβ)₂ tetramers the β chains do not overlap; however, β_H chains are much longer than and overlap in an (αβ_H)₂ tetramer, raising the possibility of direct β_H-β_H binding that could ameliorate the α-spec^R22S mutation. Here we use a combination of non-denaturing gel electrophoresis, dissociation isothermal titration calorimetry (ITC), small angle X-ray scattering (SAXS), and structural mass spectroscopy to identify and characterize a β_H self-interaction domain (βHSID) in the overlapping β_H segments 25-31 of Drosophila β_H._. The βHSID interaction is antiparallel and has a K_d = 187-213nm at 25^oC, similar to that of the H2H interaction in human (αΙβΙ)₂, making the βHSID physiologically relevant. This interaction suggests that (β_H)₂ dimers may crosslink F-actin to form non-canonical SBMS.