Initiation of Spectrin Dimerization Involves Complementary Electrostatic Interactions between Paired Triple-helical Bundles

Abstract

The spectrin heterodimer is formed by the antiparallel lateral association of an ␣ and a ␤ subunit, each of which comprises largely a series of homologous triplehelical motifs. Initiation of dimer assembly involves strong binding between complementary motifs near the actin-binding end of the dimer. In this study, the mechanism of lateral spectrin association at this dimer nucleation site was investigated using the analytical ultracentrifuge to analyze heterodimers formed from recombinant peptides containing two or four homologous motifs from each subunit (␣20 -21/␤1-2; ␣18 -21/␤1-4). Both the two-motif and four-motif dimer associations were weakened substantially with increasing salt concentration, indicating that electrostatic interactions are important for the dimer initiation process. Modeling of the electrostatic potential on the surface of the ␣20 and ␤2 motifs showed that the side of the motifs comprising the A and B helices is the most favorable for association, with an area of positive electrostatic potential on the AB face of the ␤2 motif opposite negative potential on the AB face of the ␣20 motif and vise versa. Protease protection analysis of the ␣20 -21/␤1-2 dimer showed that multiple trypsin and proteinase K sites in the A helices of the ␤2 and ␣21 motifs become buried upon dimer formation. Together, these data support a model where complementary long range electrostatic interactions on the AB faces of the triple-helical motifs in the dimer nucleation site initiate the correct pairing of motifs, i.e. ␣21-␤1 and ␣20-␤2. After initial docking of these complementary triple-helical motifs, this association is probably stabilized by subsequent formation of stronger hydrophobic interactions in a complex involving the A helices of both subunits and possibly most of the AB faces. The ␤ subunit A helix in particular appears to be buried in the dimer interface.Members of the spectrin family of membrane skeleton proteins are widely expressed in vertebrates as well as in lower organisms. In erythrocytes, spectrin is the major component of the membrane skeleton, a network of spectrin oligomers crosslinked with short actin filaments that is bound to the membrane and provides cell membrane stability. The most common form of spectrin on intact cell membranes is the tetramer, which is formed by the "head-to-head" association of two elongated heterodimers, each comprising an ␣ subunit and a ␤ subunit with molecular masses of 280 kDa (1) and 246 kDa (2), respectively. Both the ␣ and ␤ subunits consist largely of a series of homologous motifs, each approximately 106 residues in length (3). The tertiary structure of these motifs is a triplehelical bundle, as determined by x-ray crystallography (4) and NMR spectroscopy (5). Head-to-head tetramers form by binding of complementary partial motifs at the ends of the ␣ and ␤ subunits to form a complete triple-helical bundle (6 -8).The antiparallel lateral association of the ␣ and ␤ subunits to form a heterodimer is initiated near the actin-binding end of the molecule at a dim...

Important residue (G46) in erythroid spectrin tetramer formation

“…The C-terminal region of the erythroid α-(SpαI) and the N-terminal region of the β-(SpβI) subunits associate to form an αβ hetero-dimer [4,5]. Two such dimers associate at the opposite end, the N-terminal region of SpαI and the C-terminal region of SpβI, to form a functional tetramer.…”

Influence of Lateral Association on Forced Unfolding of Antiparallel Spectrin Heterodimers

“…The dissociation constant (K d ) for the ␤ 1-4 and ␣ 18 -21 dimer complex was measured to be ϳ10 nM compared with the ϳ10 pM reported for ␣-actinin dimers (14,15). Furthermore, the affinity of ␤ 1-2 for ␣ 21-20 was found to be much stronger than that of ␤ 3-4 for ␣ 19 -18 (13) thus dividing the lateral dimer complex into a strong end and a weak end (Fig. 1C).…”

“…The two chains of the heterodimer may come apart during unfolding, because dimeric interactions rely heavily on complementary electrostatic interactions within the triple-helical dimer interface (13). These might be easily severed with modest shearing force well below that experienced at the molecular level by the red cell in the circulation (26).…”

“…Although other AFM studies have elaborated RNA hairpin interactions (16) and insulin monomer-monomer association/dissociation interactions (17), none to date have explicitly examined the inhibiting effects, if any, of protein dimerization on domain unfolding. Spectrin dimerization is predicated on the typical range of protein interactions: hydrophobic sequestration, hydrogen bonding, and most importantly electrostatic interactions between paired triple-helical bundles (13). Here we probe these dimeric interactions through their effects on flexibility and unfolding.…”

“…The ␣-actinin homodimer and an eight-repeat rod segment of dystrophin were also studied. A variety of solution studies on spectrin dimerization have been conducted in the past (7,13). The dissociation constant (K d ) for the ␤ 1-4 and ␣ 18 -21 dimer complex was measured to be ϳ10 nM compared with the ϳ10 pM reported for ␣-actinin dimers (14,15).…”