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Mapping the human erythrocyte beta-spectrin dimer initiation site using recombinant peptides and correlation of its phasing with the alpha-actinin dimer site

, : Mapping the human erythrocyte beta-spectrin dimer initiation site using recombinant peptides and correlation of its phasing with the alpha-actinin dimer site. Journal of Biological Chemistry 271(12): 6636-6644

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, 1477514782). 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 (beta-1-4+) with this earlier starting point bound to full-length alpha-spectrin with a K-d 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 beta-1-4+ showed that the first motif was essential for dimerization since its deletion abolished binding, but beta-1+ alone could not associate with alpha-monomers. The first two motifs (beta-1-2+) represented the minimum lateral dimer assembly site with a K-d of about 230 nm for interaction with full-length alpha-spectrin or an alpha-spectrin nucleation site recombinant peptide, alpha-18-21. 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.


PMID: 8636080

DOI: 10.1074/jbc.271.12.6636

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