Conformational changes of helix C' of erythroid alpha spectrin upon binding beta spectrin

Erythroid spectrin is a major component of the membrane skeleton of erythrocytes that plays an important role in their deformability by maintaining their unique biconcave shape. Two heterodimers of alpha- and beta-spectrin associate to form functional tetramers. This association at the tetramerization site involves a pair of identical interactions between the N-terminal region of the alpha-spectrin subunit and the C-terminal region of the beta-spectrin subunit. The affinity of this association varies amongst spectrin isoforms, with the dissociation constant of intact non-erythroid spectrin being 15-fold higher than that of erythroid spectrin. Furthermore the regions involved in the association in erythroid spectrin are the locations for the majority of spectrin mutations that have been linked to hematological disorders.;Using model recombinant proteins, the regions flanking the single helix (Helix C') of the N-terminal end of alpha-spectrin were studied using site directed spin labeling electron paramagnetic resonance (SDSL-EPR). The results suggest that these two regions undergo a conformational change upon the association with beta-spectrin, likely becoming helical. Isothermal titration calorimetry (ITC) methods were used to study the association affinities of mutations in those regions as well as in Helix C' to further investigate the role of each amino acid residue in this region. Our results provide a detailed mechanism for the association that can be used to predict clinical severity.