Crystal Structure of the Nonerythroid α-Spectrin Tetramerization Site Reveals Differences between Erythroid and Nonerythroid Spectrin Tetramer Formation

Abstract

We have solved the crystal structure of a segment of nonerythroid ␣-spectrin (␣II) consisting of the first 147 residues to a resolution of 2.3 Å . We find that the structure of this segment is generally similar to a corresponding segment from erythroid ␣-spectrin (␣I) 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 ␣II, and the lack of such hydrogen bond networks in ␣I leads to flexibility in ␣I. We have previously shown the junction region connecting Helix C to D1 to be unstructured in ␣I (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 ␣II, an important difference for ␣-spectrin association with ␤-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 ␣-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.Spectrin isoforms are proteins associated with the cytoplasmic surface of plasma membranes of most cells. Spectrin associates with other cytoskeletal proteins, such as ankyrin and protein 4.1, to establish and maintain a diverse set of specialized plasma membrane domains (1). Nonerythroid, or brain, spectrin (spectrin II) exhibits high sequence homology with erythroid spectrin (spectrin I), despite their different cellular physiological functions (1). Because of their sequence homology, it is possible in theory to apply most of the molecular information obtained from the well studied spectrin I (both ␣I 3 and ␤I subunits) to the less studied spectrin II (␣II and ␤II) or from one domain to another domain (2). Yet, the two spectrin isoforms exhibit quite different functional properties as follows: (i) the ability of the spectrin I network to deform or the spectrin II network to remain rigid, and (ii) the ability ␣␤ heterodimers to associate to form functional (␣␤) 2 tetramers with much higher affinity in spectrin II than in spectrin I. Tetramer formation in nonerythroid spectrin is essential in the regulatory step for neuritogenesis (3). ␣II-spectrin has recently been reported to be essential for stabilizing nascent sodium channel clusters (4), assembling the mature node of Ranvier (4), and regulating endothelial cell-cell contacts (5).The C terminus of ␣I or ␣II and the N terminus of ␤I or ␤II associate to f...

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].…”

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

“…Without the fusion protein tag on the bI protein, we have shown previously that the K d value for the bI-C1/aI-N1 complex was about 1 lM, 16,30,31 and that for the bI-C1/aII-N1 ranged from 4 to 12 nM. [15][16][17][18] Thus, it appears that the fusion protein tag in bI-C1, bII-C1, aI-N1, and aII-N1 did not affect the association of these proteins.…”

“…These IC 50 values also indicated that aII-N1 exhibits higher affinity than aI-N1 in association with bI-C1, in agreement with previous isothermal titration calorimetry (ITC) findings. 17 Following a published method (see ''Materials and Methods'' Section) to calculate K d values from corresponding IC 50 values, we obtained a K d value of 0.2 lM from the aI-N1 data and 0.1 lM from the aII-N1 data for the bI-C1/G5 complex. Similarly, for the bI-C1/A2 complex [ Fig.…”

“…The affinity of dimers to form tetramers is lower in erythroid cells than in nonerythroid cells, with the K d in the lM range for model erythroid proteins, and in the nM range for model nonerythroid proteins. [14][15][16][17] The spectrin isoforms exhibit high sequence identity and similarity. [16][17][18] We have shown that a small but key difference in the N-terminal junction region in aI-and aII-spectrin is primarily responsible for the large difference in spectrin tetramer formation in erythroid and nonerythroid spectrin.…”

“…[14][15][16][17] The spectrin isoforms exhibit high sequence identity and similarity. [16][17][18] We have shown that a small but key difference in the N-terminal junction region in aI-and aII-spectrin is primarily responsible for the large difference in spectrin tetramer formation in erythroid and nonerythroid spectrin. 17 The tetramerization sites for bI-and bII-spectrin not only exhibit 80% sequence similarity but also exhibit affinities similar to each other in their association with a-spectrin to form spectrin tetramers.…”