Many spectrin mutations that destabilize tetramer formation and lead to hereditary hemolytic anemias are located at the Nterminal region of ␣-spectrin, with the Arg28 position considered to be a mutation hot spot. We have introduced mutations at positions 28 and 45 into a model peptide, Sp␣1-156, consisting of the first 156 residues in the N-terminal region of ␣-spectrin (␣N). The association of these ␣-spectrin peptides that have single amino acid replacements with a -spectrin model peptide, consisting of the Cterminal region of -spectrin (C), was determined, and structural changes due to amino acid replacements were monitored by nuclear magnetic resonance (NMR). We found evidence for similar and very localized structural changes in Sp␣1-156Arg45Thr and Sp␣1-156Arg45Ser, although these 2 mutant peptides associated with -spectrin peptide with significantly differing affinities. The Sp␣1-156Arg28Ser peptide showed an affinity for the -spectrin peptide comparable to that of Sp␣1-156Arg45Ser, but it exhibited substantial and widespread spectral changes. Our results suggest that both Arg45 replacements induce only minor structural perturbations in the first helix of Sp␣1-156, but the Arg28Ser replacement affects both the first helix and the following structural domain. Our results also indicate that the mechanism for reduced spectrin tetramerization is through mutation-induced changes in molecular recognition at the ␣-tetramerization site, rather than through conformational disruption, as has been suggested in prior literature.
IntroductionThe ␣ and  subunits of human erythrocyte spectrin both consist of multiple homologous sequence motifs, with each motif presumably folding into 3 helices, which bundle to form a structural domain similar to the structures determined for Drosophila spectrin 1 and chicken brain spectrin. 2,3 Both ␣-and -spectrin associate at the N-terminal end of the -subunit and the C-terminal end of the ␣-subunit (dimer nucleation site) with high affinity (nM dissociation constant [K d ] values) to give ␣ heterodimers. 4,5 It has been suggested that spectrin dimers then associate to form spectrin tetramers, with an association site at the other end of the dimers, involving 2 sets of identical, low-affinity (M K d ) interactions between the N-terminal region of the ␣-subunit (␣N) of one ␣ dimer and the C-terminal region of the -subunit (C) in another ␣ dimer to give an (␣) 2 tetramer. 6,7 Sequence homology studies predict that about 30 residues in this ␣N region, prior to the first structural domain, fold into a helical conformation; likewise, about 60 residues in the C region, following the last structural domain, are predicted to fold into 2 helices. 8,9 These one-and 2-helix regions are termed partial domains for ␣-and -spectrin, respectively. It is assumed that the dimer to tetramer formation involves association of these partial domains to form a triple helical bundle similar to the structural domains. 7,8 Spectrin tetramers in the human erythrocyte play a critical role in ma...