Nuclear magnetic resonance studies of mutations at the tetramerization region of human alpha spectrin

Abstract

AbstractMany spectrin mutations that destabilize tetramer formation and lead to hereditary hemolytic anemias are located at the N-terminal region of α-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, Spα1-156, consisting of the first 156 residues in the N-terminal region of α-spectrin (αN). The association of these α-spectrin peptides that have single amino acid replacements with a β-spectrin model peptide, consisting of the C-terminal region of β-spectrin (βC), 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 Spα1-156Arg45Thr and Spα1-156Arg45Ser, although these 2 mutant peptides associated with β-spectrin peptide with significantly differing affinities. The Spα1-156Arg28Ser peptide showed an affinity for the β-spectrin peptide comparable to that of Spα1-156Arg45Ser, 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 Spα1-156, 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 αβ-tetramerization site, rather than through conformational disruption, as has been suggested in prior literature.