Abstract
We have solved the crystal structure of a segment of nonerythroid alpha-spectrin (alphaII) consisting of the first 147 residues to a resolution of 2.3 A. We find that the structure of this segment is generally similar to a corresponding segment from erythroid alpha-spectrin (alphaI) but exhibits unique differences with functional significance. Specific features include the following: (i) an irregular and frayed first helix (Helix C'); (ii) a helical conformation in the junction region connecting Helix C' with the first structural domain (D1); (iii) a long A(1)B(1) loop in D1; and (iv) specific inter-helix hydrogen bonds/salt bridges that stabilize D1. Our findings suggest that the hydrogen bond networks contribute to structural domain stability, and thus rigidity, in alphaII, and the lack of such hydrogen bond networks in alphaI leads to flexibility in alphaI. We have previously shown the junction region connecting Helix C' to D1 to be unstructured in alphaI (Park, S., Caffrey, M. S., Johnson, M. E., and Fung, L. W. (2003) J. Biol. Chem. 278, 21837-21844) and now find it to be helical in alphaII, an important difference for alpha-spectrin association with beta-spectrin in forming tetramers. Homology modeling and molecular dynamics simulation studies of the structure of the tetramerization site, a triple helical bundle of partial domain helices, show that mutations in alpha-spectrin will affect Helix C' structural flexibility and/or the junction region conformation and may alter the equilibrium between spectrin dimers and tetramers in cells. Mutations leading to reduced levels of functional tetramers in cells may potentially lead to abnormal neuronal functions.
Highlights
Associate to form heterodimers (␣II or ␣IIII) (6)
We have provided indirect evidence that the conformation of the region connecting the first structural domain and the partial domain region of ␣-spectrin plays an important role in the interactions between ␣- and -spectrin leading to the formation of spectrin tetramers (7, 11, 13)
Using the structure of Helix CЈ, we developed a homology model of the ␣II Helix CЈ bundled with Helices AЈ and BЈ of -spectrin to form a three helix-bundle (AЈBЈCЈ) at the tetramerization site, followed by MD simulations
Summary
An N-terminal segment of brain ␣II-spectrin consisting of the first 147 residues (␣II-N1), similar to a well studied erythroid ␣-spectrin segment consisting of the first 156 residues (␣IN1) (14), was prepared from a similar recombinant protein with 149 residues (7), following standard procedures (8, 13, 17). Because PDBsum identifies protein-protein interactions as chain-chain interactions, we labeled each helix in our structure as a chain, including half of the loops connecting to the adjacent helices, in each PDB file to give inter-helical interactions From these inter-helical interactions, we examined clusters of atoms involved in NBC, which consist of hydrogen bonds, and salt bridges. We built an AЈBЈCЈ complex consisting of Helix CЈ of ␣II and Helices AЈ and BЈ of I or II, using homology modeling methods similar to those used for the ␣I and I complex (14, 17). Because we use the crystal structure of free (unbound) Helix CЈ for ␣II, we used the unbound Helix CЈ in the ␣II-t␣I model (14) for MD simulations (see below). Refinement statistics Resolution range No of reflections in working set No of reflections in test set Rcryst Rfree Wilson B Average B-factor (Å2) (protein) No of structures in asymmetric unit r.m.s.d. from ideal geometry Bond lengths Bond angles Ramachandran plot Allowed Generous Disallowed
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