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 alpha-spectrin that occur upon binding to beta-spectrin, and it reports the first structure of the beta-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.

Figure 1

Schematic of human erythrocyte spectrin. (A) Both the α (top) and β (bottom) isoforms of erythroid spectrin are composed of many tandem 3-helix bundle motifs (each helix is depicted as a cylinder). In the case of α-spectrin, 20 full repeats and 1 partial repeat at the N-terminus (light blue) make up the majority of the molecule. For historical reasons, the SH3 domain, located between the B and C helix of repeat α9, is assigned as the α10 motif. Similarly, β-spectrin possesses 16 full repeats and 1 partial repeat at the C-terminus (yellow). Selected regions of each spectrin isoform with a particular structure or function are colored. (B) Spectrin assembly in red cells begins with dimerization of α and β chains that is nucleated by specialized dimerization repeats (red). Subsequent tetramer formation occurs through head-to-head interaction of the tetramerization domains. (C) An enlarged schematic of the molecular components presented in this study that includes α-spectrin repeats 0-1 and β-spectrin repeats 16-17.

Figure 2

Structural features of the assembled tetramerization site. (A) Crystallographic structure determination of the assembled tetramerization site showed a complex akin to many 3-repeat spectrin structures. As can be shown from the ribbon diagram (α-helices are depicted as coils), the interaction surface is between α-spectrin (blue) partial repeat 0 and β-spectrin (yellow) partial repeat 17. (B) The helical wheel diagram of the interacting helices shows a clustering of hydrophobic residues (yellow) at the core of the 3-helix bundle. Each helical wheel depicts the radial positions of side chains upon projection down the helical axis. The helices are roughly positioned according to their placement in the structure. Residues are labeled according to single-letter code with each being color coded on the basis of properties of the side chains (positively-charged = blue, negatively charged = pink, polar = purple, hydrophobic = yellow, other = white/beige). (C) A cut-away view of the ribbon diagram near the core of the interacting surface; the orientation of the helices is similar to that in panel B.

Figure 3

Interactions along the spectrin tetramerization domain interface are extensive. (A) The interacting surfaces of the tetramerization domain span nearly the entire composite spectrin repeat (α-spectrin shown in blue, β-spectrin shown in yellow). The interface of the complex is opened to the reader such that the interacting surface on each molecule is viewable. Specific side chains that make contact in the crystal structure are shown as sticks and labeled. (B) The molecular surfaces of α0-1 (blue, surface that interacts with β16-17 in green) and β16-17 (yellow, surface that interacts with α0-1 in red) indicate the extent of binding along these 2 molecules. (C) The electrostatic surfaces of α0 and β17 show some charge complementarity, the electrostatic map was contoured at 15 ± k_BT/e. Views in panels B and C are similar to those in panel A, with both of the interacting surfaces turned to face the reader.

Figure 4

Comparison with other spectrin structures shows that the composite 3-helix bundle recapitulates a spectrin repeat. (A) Comparison of human erythroid spectrin repeats α0-1 from the crystal structure of the complex (α0-1, blue) to the structure of α0-1 in solution (purple) and to a crystal structure of human brain α0-1 spectrin (green) indicates stabilization of the helical linker between repeats 0 and 1 in the head-to-head α/β complex. In contrast to the NMR structure and crystal structure, the entirety of the repeat α0 is helical when in complex with β16-17. (B) Superposition of the structure of the tetramerization domain (blue and yellow) with the 3-repeat structure of human brain β-spectrin repeats 14-16 (red) demonstrate that the composite repeat highly resembles an intact spectrin repeat.

Figure 5

The spectrin tetramerization domains show multiple, highly conserved surfaces. (A) The interacting surfaces of α0-1 and β16-17 show regions of relatively high conservation. This is especially notable in comparing repeat β16 (mostly blue) to the partial repeat β17 (mostly red). As in previous figures, the interacting surfaces of both molecules are opened to face the reader. (B) Conservation analysis of the assembled tetramerization domain displays one surface with markedly greater conservation than the other, which may present a biologically relevant interaction surface. The conservation maps presented were generated using sequences and alignments derived by the Consurf database. An additional analysis with manually selected and aligned sequences yielded similar findings.

Figure 6

Locations of α0 domain HE/HPP mutations in the tetramer complex. Mutations that have been associated with HE or HPP are indicated as space-filling spheres on the ribbon diagram. Most of the mutations map directly to interacting residues. The relationship to our previously reported binding affinities with the use of isothermal titration calorimetry are color coded by the use of the previously defined binding affinity categories: red = severe (marginal or no detected binding); orange = moderate (∼100-fold weaker than wild type); yellow = modest (∼10-fold weaker than wild type); green = no effect (wild-type binding). (A) Mutations of positively charged residues. (B) Mutations of uncharged residues.

Figure 7

Locations of β17 domain HE/HPP mutations in the tetramer complex. Mutations that have been associated with HE or HPP are indicated as space-filling spheres on the ribbon diagram. Where known, the relationship to reported binding affinities are color coded by the use of binding affinity categories similar to those used in Gaetani et al: red = severe (marginal or no detected binding); orange = moderate (∼ 100-fold weaker than wild type); yellow = modest (∼ 10-fold weaker than wild type); green = no effect (wild-type binding); gray = not characterized. (A) Mutations that disrupt the local structure of the β17 domain. (B) Mutations predicted to disrupt the local structure of the β17 domain. (C) Mutations predicted to disrupt association of the β17 domain with the α0 domain.