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Human erythroid spectrin dimer assembly is initiated by the association of a specific region near the N-terminal of beta-spectrin with a complementary region near the C-terminal of alpha-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 beta-spectrin required for association with alpha-spectrin was determined using recombinant peptides. The start site (phasing) for construction of dimerization competent beta-spectrin peptides was particularly critical. The beginning of the first homologous motif for both beta-spectrin and the related dimerization site of alpha-actinin is approximately 8 residues earlier than most spectrin motifs. A four-motif beta-spectrin peptide (beta1-4+) with this earlier starting point bound to full-length alpha-spectrin with a Kd 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 beta1-4+ showed that the first motif was essential for dimerization since its deletion abolished binding, but beta1+ alone could not associate with alpha-monomers. The first two motifs (beta1 2+) represented the minimum lateral dimer assembly site with a Kd of about 230 nM for interaction with full-length alpha-spectrin or an alpha-spectrin nucleation site recombinant peptide, alpha18-21. 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.

Spectrin, a heterodimer composed of alpha and beta subunits, interacts with itself head-to-head to form tetramers in the erythrocyte membrane cytoskeleton. The NH2-terminal region of alpha-spectrin, encompassing the alpha I 80-kDa domain, was expressed in Escherichia coli. In addition to the correctly initiated polypeptide, four smaller polypeptides were produced by initiation at internal codons. Only the full-length polypeptide was able to bind to spectrin dimers, beta monomers, and to a recombinant polypeptide containing the COOH terminus of beta-spectrin. The head-to-head interaction with beta-spectrin was also retained by a recombinant polypeptide containing the NH2-terminal 158 amino acids of the alpha subunit. Deletion of the first 27 or 49 NH2-terminal amino acids abolished binding of this polypeptide to the beta monomer. The phasing used to design these recombinant polypeptides was based on a conformational model recently refined by Speicher et al. (Speicher, D. W., DeSilva, T. M., Speicher, K. D., Ursitti, J. A., Hembach, P., and Weglarz, L. (1993) J. Biol. Chem. 268, 4227-4235), where the structural unit begins and terminates around residue 30 of the repeat unit. The binding properties, mobility on gel filtration, and circular dichroism data of the recombinant polypeptides indicated that most polypeptides were able to assume their native conformation.

The human erythrocyte alpha-spectrin gene which spans 80 kbp has been cloned from human genomic DNA as overlapping lambda recombinants. The exon-intron junctions were identified and the exons mapped. The gene is encoded by 52 exons whose sizes range from 684 bp to the smallest of 18 bp. The donor and acceptor splice site sequences match the splice site consensus sequences, with the exception of one splice site where a donor sequence begins with -GC. The size and location of exons do not correlate with the 106-amino-acid repeat, except in three locations where the surrounding codons are conserved as well. The lack of correspondence between exons and 106-amino-acid repeat is interpreted to reflect the appearance of a spectrin-like gene from a minigene early in the evolution of eukaryotes. Since current evidence indicates that introns were present in genes before the divergence of prokaryotes and eukaryotes, it is possible that the original distribution of introns within the minigene has been lost by the random deletion of introns from the spectrin gene.

Overlapping human erythroid alpha-spectrin cDNA clones were isolated from lambda gt11 libraries constructed from cDNAs of human fetal liver and erythroid bone marrow. The composite 8001-base pair (bp) cDNA nucleotide sequence contains 187-bp 5'- and 528-bp 3'-untranslated regions and has a single long open reading frame of 7287 bp that encodes a polypeptide of 2429 residues. As previously described (Speicher, D. W., and Marchesi, V. T. (1984) Nature 311, 177-180), spectrin is composed largely of homologous 106-amino acid repeat units. From the amino acid sequence deduced from the cDNA, alpha-spectrin can be divided into 22 segments. Segments 1-9 and 12-19 are homologous and can therefore be considered repeats; the average number of identical residues in pairwise comparisons of these repeats is 22 out of 106, or 21%. Of these 17 repeats, 11 are exactly 106 amino acids in length, whereas five others differ from this length by a single residue. Segments 11, 20, and 21, although less homologous, appear to be related to the more highly conserved repeat units. The very N-terminal 22 residues, segment 10, which is atypical both in length and sequence, and the C-terminal 150 residues in segment 22 appear to be unrelated to the conserved repeat units. The sequence of the erythroid alpha-spectrin polypeptide chain is compared to that of human alpha-fodrin and chicken alpha-actinin to which it is related. alpha-Spectrin is more distantly related to dystrophin.