Studies of the erythrocyte spectrin tetramerization region

Sunghyouk Park, University of Illinois at Chicago
Shahila Mehboob, Loyola University of Chicago
Bing Hao Luo, Loyola University of Chicago
Michael Hurtuk, Loyola University of Chicago
M. E. Johnson, University of Illinois at Chicago
L. W.M. Fung, Loyola University of Chicago


Human erythrocyte spectrin dimers associate at the N-terminal region of α-spectrin (αN) and the C-terminal region of β-spectrin (βC) to form tetramers. We have prepared model peptides to study the tetramerization region. Based on phasing information obtained from enzyme digests, we prepared spectrin fragments consisting of the first 156 amino-acid residues and the first 368 amino-acid residues of α-spectrin (Spα1-156 and Spα1-368, respectively), and found that both peptides associate with a β-spectrin model peptide, with an affinity similar to that found in αβ dimer tetramerization. Spin label EPR studies show that the region consisting of residues 21-46 in α-spectrin is helical even in the absence of its β-partner. Multi-dimensional nuclear magnetic resonance studies of samples with and without a spin label attached to residue 154 show that Spα1-156 consists of four helices, with the first helix unassociated with the remaining three helices, which bundle to form a triple helical coiled coil bundle. A comparison of the structures of erythrocyte spectrin with other published structures of Drosophila and chicken brain spectrin is discussed. Circular dichroism studies show that the lone helix in Spα1-156 associates with helices in the β-peptide to form a coiled coil bundle. Based on NMR and CD results, we suggest that the helices in Spα1-156 exhibit a looser (frayed) conformation, and that the helices convert to a tighter conformation upon association with its β-partner. This suggestion does not rule out possible conversion of a non-structured conformation to a structured conformation in various parts of the molecule upon association. Spectrin mutations at residues 28 and 45 of α-spectrin have been found in patients with hereditary elliptocytosis. NMR studies were also carried out on Spα1-156R28S, Spα1-156R45S and Spα1-156R45T. A comparison of the structures of Spα1-156 and Spα1-156R28S, Spα1-156R45S and Spα1-156R45T is discussed.