A PARTIAL STRUCTURAL REPEAT FORMS THE HETERODIMER SELF-ASSOCIATION SITE OF ALL BETA-SPECTRINS

The self-polymerization of alphabeta-spectrin heterodimers to form tet ramers and higher oligomers is central to its role as a membrane stabi lizer and organizer. Mutations near the amino terminus of alphaISIGMA1 -spectrin or the COOH terminus of betaISIGMA1-spectrin often lead to p rofound impairment of heterodimer polymerization and to hemolytic dise ase of varying severity. Previous studies using an 80-kDa univalent fr agment of alphaISIGMA1-spectrin have established that the amino-termin al segment of alphaISIGMA1-spectrin mediates the association of the al pha subunit with either intact heterodimers or with isolated beta-spec trin (betaISIMGA1). However, the nature of the self-association site i n beta-spectrin has remained unclear. In the present study, native bet a-spectrin and recombinant beta-spectrin peptides representing COOH-te rminal portions of two alternative transcripts of the gene on chromoso me 2 (betaISIGMA1 or ''erythrocyte'' spectrin and betaISIGMA2 or ''mus cle'' spectrin), and one transcript of the gene on chromosome 14 (beta IISIGMA1 or ''beta(G)-fodrin'') have been examined for their ability t o bind either intact alphabeta-spectrin or the alphaI-spectrin 80-kDa univalent fragment. Deletion of the nonhomologous beta-spectrin sequen ce downstream of repeat 17 (spectrin domain III) had no discernible ef fect on binding. Truncations proximal to codon 2085 of betaISIGMA1-spe ctrin demonstrated a precipitous loss of activity, accounted for by a loss of both binding affinity and capacity. Further truncations to rep eat 16 (codon 1979) restored binding activity to levels approximating that of the intact molecule. Repeat 16/17 and 17/16 chimeras displayed reduced binding activity. Collectively, these data indicate that the beta-subunit self-association site is highly sensitive to conformation , involves widespread interactions within the 17th repeat unit, is lar gely independent of sequences in domain III, and can be recreated by t he deletion of all residues distal to the COOH end (codon 1979) of the 16th and presumably other spectrin sequence repeat units. All beta-sp ectrins appear to use this binding motif, regardless of the nature of the nonhomologous sequence in domain III.