Crystal structure and functional interpretation of the erythrocyte spectrin tetramerization domain complex

Abstract

As the principal component of the membrane skeleton, spectrin confers integrity and flexibility to red cell membranes. Although this network involves many interactions, the most common hemolytic anemia mutations that disrupt erythrocyte morphology affect the spectrin tetramerization domains. Although much is known clinically about the resulting conditions (hereditary elliptocytosis and pyropoikilocytosis), the detailed structural basis for spectrin tetramerization and its disruption by hereditary anemia mutations remains elusive. Thus, to provide further insights into spectrin assembly and tetramer site mutations, a crystal structure of the spectrin tetramerization domain complex has been determined. Architecturally, this complex shows striking resemblance to multirepeat spectrin fragments, with the interacting tetramer site region forming a central, composite repeat. This structure identifies conformational changes in ␣-spectrin that occur upon binding to ␤-spectrin, and it reports the first structure of the ␤-spectrin tetramerization domain. Analysis of the interaction surfaces indicates an extensive interface dominated by hydrophobic contacts and supplemented by electrostatic complementarity. Analysis of evolutionarily conserved residues suggests additional surfaces that may form important interactions. Finally, mapping of hereditary anemia-related mutations onto the structure demonstrate that most, but not all, local hereditary anemia mutations map to the interacting domains. The potential molecular effects of these mutations are described. (Blood. 2010;115(23):4843-4852)

Non-erythroid beta spectrin interacting proteins and their effects on spectrin tetramerization

“…This interaction involves hydrophobic residue clustering, salt bridges and hydrogen bonds [25 - 29]. Despite high sequence homology and three-dimensional structural similarity, dissociation constant measurements using model proteins of different spectrin fragments show two orders of magnitude difference in the N-terminal α-spectrin and C-terminal β-spectrin association affinity between erythroid and non-erythroid spectrin [30, 31], in good agreement with earlier studies using intact spectrin [32].…”

“…Tetramerization is an important process for spectrin isoforms, and involves helical bundling of three helices, one from the α- and two from the β-spectrin [14, 25, 28]. The bundled complexes exhibit different K d values, with the non-erythroid complex αII-N/βII-C about 10 nM and the erythroid complex about 1 μM [31].…”

Apparent structural differences at the tetramerization region of erythroid and nonerythroid beta spectrin as discriminated by phage displayed scFvs

“…For the tetramerization regions, many studies have focused on the N-terminal region of aI-and aII-spectrin, [15][16][17][18]30,31,42,48 but only a few have studied the Cterminal region of b-spectrin. [32][33][34]41,49 Spin label EPR studies provide the first experimental information on a local region of Helix B 0 in free bI-spectrin (not associated with aI-spectrin), with residues before 2071P in helical conformation (Helix B 0 ), and residues after 2071P in an unstructured conformation. 33,34 The predicted structure of free Helix B 0 of bI in this study also showed that 42 and bound 15,30,36 forms of a-spectrin have been observed experimentally, with the N-terminal junction region in aI-spectrin (residues 46-52) undergoing conformational changes, from unstructured to helical conformation, upon binding to the C-terminal fragment of b1-spectrin.…”

“…33,34 The predicted structure of free Helix B 0 of bI in this study also showed that 42 and bound 15,30,36 forms of a-spectrin have been observed experimentally, with the N-terminal junction region in aI-spectrin (residues 46-52) undergoing conformational changes, from unstructured to helical conformation, upon binding to the C-terminal fragment of b1-spectrin. 30,32 The conformation of the bI-Helix B 0 bound to G5 was predicted to consist of residues 2044-2068, a conformation different from the free form and a form in the presence of aI [ Fig. 2(C)].…”

“…32 However, the structure of the free form is not known except that Helix B 0 in the partial domain ends at residue 2070, 33 and the region downstream of residue 2070 is unstructured, as shown by spin label electron paramagnetic resonance (EPR) studies. 34 Therefore, we used homology modeling methods to predict the free form of bI-C1 and bII-C1.…”