Mapping the Human Erythrocyte -Spectrin Dimer Initiation Site Using Recombinant Peptides and Correlation of Its Phasing with the -Actinin Dimer Site (*)

Human erythroid spectrin dimer assembly is initiated by the association of a specific region near the N-terminal of β-spectrin with a complementary region near the C-terminal of α-spectrin (Speicher, D. W., Weglarz, L., and DeSilva, T. M.(1992) J. Biol. Chem. 267, 14775-14782). Both spectrin subunits consist primarily of tandem, 106-residue long, homologous, triple-helical motifs. In this study, the minimal region of β-spectrin required for association with α-spectrin was determined using recombinant peptides. The start site (phasing) for construction of dimerization competent β-spectrin peptides was particularly critical. The beginning of the first homologous motif for both β-spectrin and the related dimerization site of α-actinin is approximately 8 residues earlier than most spectrin motifs. A four-motif β-spectrin peptide (β1-4) with this earlier starting point bound to full-length α-spectrin with a K of about 10 nM, while deletion of these first 8 residues reduced binding nearly 10-fold. N- and C-terminal truncations of one or more motifs from β1-4 showed that the first motif was essential for dimerization since its deletion abolished binding, but β1 alone could not associate with α-monomers. The first two motifs (β1-2) represented the minimum lateral dimer assembly site with a K of about 230 nM for interaction with full-length α-spectrin or an α-spectrin nucleation site recombinant peptide, α18-21. Each additional motif increased the dimerization affinity by approximately 5-fold. In addition to this strong inter-subunit dimer association, interactions between the helices of a single triple-helical motif are frequently strong enough to maintain a noncovalent complex after internal protease cleavage similar to the interactions thought to be involved in tetramer formation. Analysis of hydrodynamic radii of recombinant peptides containing differing numbers of motifs showed that a single motif had a Stokes radius of 2.35 nm, while each additional motif added only 0.85 nm to the Stokes radius. This is the first direct demonstration that spectrin's flexibility arises from regions between each triple helical motif rather than from within the segment itself and suggests that current models of inter-motif connections may need to be revised.