Mapping the Human Erythrocyte β-Spectrin Dimer Initiation Site Using Recombinant Peptides and Correlation of Its Phasing with the α-Actinin Dimer Site

Abstract

Human erythroid spectrin dimer assembly is initiated by the association of a specific region near the N-terminal of ␤-spectrin with a complementary region near the C-terminal of ␣-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 ␤-spectrin required for association with ␣-spectrin was determined using recombinant peptides. The start site (phasing) for construction of dimerization competent ␤-spectrin peptides was particularly critical. The beginning of the first homologous motif for both ␤-spectrin and the related dimerization site of ␣-actinin is approximately 8 residues earlier than most spectrin motifs. A four-motif ␤-spectrin peptide (␤1-4 ؉ ) with this earlier starting point bound to full-length ␣-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 ␤1-4 ؉ showed that the first motif was essential for dimerization since its deletion abolished binding, but ␤1؉ alone could not associate with ␣-monomers. The first two motifs (␤1-2 ؉ ) represented the minimum lateral dimer assembly site with a K d of about 230 nM for interaction with full-length ␣-spectrin or an ␣-spectrin nucleation site recombinant peptide, ␣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.The membrane skeleton of the human erythrocyte consists of a network of proteins that associates with the inner surface of the cell membrane and imparts remarkable structural integrity and flexibility to circulating erythrocytes. Spectrin is the major structural component of this specialized submembranous protein network. The basic functional unit of spectrin is a heterodimer formed by side-to-side, antiparallel association of a 280-kDa ␣ subunit with a 246-kDa ␤ subunit. Spectrin dimers associate head-to-head to form tetramers, the predominant form of spectrin in the membrane skeleton. These tetramers cross-link short actin oligomers, an association modulated by band 4

Crystal structure and functional interpretation of the erythrocyte spectrin tetramerization domain complex

“…In the case of the erythrocyte membrane skeleton, spectrin assembly begins with a lateral heterodimeric association of ␣ and ␤ subunits (Figure 1B top) that is nucleated by specialized dimerization repeats located near the C-terminus of ␣-spectrin and the N-terminus of ␤-spectrin. [10][11][12] Subsequently, head-to-head interactions between 2 ␣/␤ dimers lead to the formation of a spectrin heterotetramer ( Figure 1B bottom), the predominant form of the molecule in the erythroid membrane skeleton. Biochemical, biophysical, and clinical data have implicated regions near the N-terminus of ␣-spectrin and C-terminus of ␤-spectrin as being responsible for tetramer formation.…”

Plasmodium falciparum Erythrocyte Membrane Protein 3 (PfEMP3) Destabilizes Erythrocyte Membrane Skeleton

“…However, the function of PfEMP3-skeleton interaction has not been explored. Spectrin, the major component of erythrocyte membrane skeleton, is composed of an ␣-chain and a ␤-chain that associate side to side in an antiparallel orientation to form ␣␤-heterodimers (15). Spectrin heterodimers then self-associate head to head to form spectrin tetramers (16).…”

Interactions of the α-Spectrin N-terminal Region with β-Spectrin

“…The initial, antiparallel, side-by-side association of ␣-and ␤-spectrin to form ␣␤ dimers occurs at the amino terminus of ␤-spectrin and the carboxyl terminus of ␣-spectrin, with high affinity (K d ϳ 10 nM) (20). This site is referred to as the dimer nucleation site.…”