Nuclear magnetic resonance studies of mutations at the tetramerization region of human alpha spectrin.
Many spectrin mutations that destabilize tetramer formation and lead to hereditary hemolytic anemias are located at the N-terminal region of alpha-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 alpha 1-156, consisting of the first 156 residues in the N-terminal region of alpha-spectrin (alpha N). The association of these alpha-spectrin peptides that have single amino acid replacements with a beta-spectrin model peptide, consisting of the C-terminal region of beta-spectrin (beta 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 alpha 1-156Arg45Thr and Sp alpha 1-156Arg45Ser, although these 2 mutant peptides associated with beta-spectrin peptide with significantly differing affinities. The Sp alpha 1-156Arg28Ser peptide showed an affinity for the beta-spectrin peptide comparable to that of Sp alpha 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 alpha 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 alpha beta-tetramerization site, rather than through conformational disruption, as has been suggested in prior literature.
Park
Sunghyouk
S
Center for Pharmaceutical Biotechnology, University of Illinois at Chicago, 60607, USA.
Johnson
Michael E
ME
Fung
Leslie W-M
LW
D019943:Amino Acid Substitution; D001665:Binding Sites / Q000235:genetics; D019281:Dimerization; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D020836:Protein Structure, Quaternary; D013049:Spectrin / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
D013049:Spectrin