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

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 head to head (H2H) association leading to hereditary elliptocytosis (HE). Tetramer formation requires antiparallel αβ-spectrin or αβΗ-spectrin dimerization via dimer nucleation sites, followed by (αβ)2 or (αβH)2 tetramer formation via the 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-specR22S mutation in Drosophila (homologous to the human αIR28S HE allele), eliminates H2H binding in both conventional (αβ)2 and heavy (αβH)2 spectrin isoforms in vitro. However, α-specR22S mutant flies remain surprisingly unaffected. In (αβ)2 tetramers, the β chains do not overlap; however, βH chains are much longer and overlap in an (αβH)2 tetramer, raising the possibility of direct βH-βH binding that could ameliorate the α-specR22S 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 βH self-interaction domain (βHSID) in the overlapping βH segments 25 to 31 of Drosophila βH. The βHSID interaction is antiparallel and has a Kd = 187-213 nm at 25 °C, similar to that of the H2H interaction in human (αΙβΙ)2, making the βHSID physiologically relevant. This interaction suggests that (βH)2 dimers may crosslink F-actin to form non-canonical SBMS.