The L49F mutation in alpha erythroid spectrin induces local disorder in the tetramer association region: Fluorescence and molecular dynamics studies of free and bound alpha spectrin

Song, Yuanli; Pipalia, Nina H.; Fung, Leslie W.‐M.

doi:10.1002/pro.202

Cited by 6 publications

(9 citation statements)

References 33 publications

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“…We compared different force fields for MD simulations of the spectrin system and found similar results (17). The particle mesh Ewald method (33) was used for long range electrostatic interactions.…”

Section: Simulationmentioning

confidence: 68%

“…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 (␣I-N1) (14), was prepared from a similar recombinant protein with 149 residues (7), following standard procedures (8,13,17). This segment is predicted to include a partial domain that is responsible for associating with ␣-spectrin to form tetramers, followed by the first full structural domain of brain ␣-spectrin (7,8).…”

Section: Recombinant Proteinsmentioning

confidence: 99%

“…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). In these models, the sequence of ␤I at the region predicted to be Helices AЈ and BЈ (34) is aligned with that of ␣-spectrin Helices A 1 and B 1 to determine the boundaries of Helices AЈ and BЈ.…”

Section: Homology Modelingmentioning

confidence: 99%

“…We have previously published two different models of AЈBЈCЈ of ␣I␤I with t␣I, with Helix CЈ terminating either at residue 45, as in free Helix CЈ (14), or at residue 52, as in bound Helix CЈ (17). In this study, because we use the crystal structure of free (unbound) Helix CЈ for ␣II, we used the unbound Helix CЈ in the ␣I␤I-t␣I model (14) for MD simulations (see below).…”

Section: Homology Modelingmentioning

confidence: 99%

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

Mehboob

Song

Witek

et al. 2010

Journal of Biological Chemistry

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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...

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Section: Simulationmentioning

confidence: 68%

Section: Recombinant Proteinsmentioning

confidence: 99%

Section: Homology Modelingmentioning

confidence: 99%

Section: Homology Modelingmentioning

confidence: 99%

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

Mehboob

Song

Witek

et al. 2010

Journal of Biological Chemistry

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“…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].…”

Section: Discussionmentioning

confidence: 99%

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

Sevinc

Fung

2011

Cellular and Molecular Biology Letters

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With yeast two-hybrid methods, we used a C-terminal fragment (residues 1697-2145) of non-erythroid beta spectrin (βII-C), including the region involved in the association with alpha spectrin to form tetramers, as the bait to screen a human brain cDNA library to identify proteins interacting with βII-C. We applied stringent selection steps to eliminate false positives and identified 17 proteins that interacted with βII-C (IPβII-C s). The proteins include a fragment (residues 38-284) of “THAP domain containing, apoptosis associated protein 3, isoform CRA g”, “glioma tumor suppressor candidate region gene 2” (residues 1-478), a fragment (residues 74-442) of septin 8 isoform c, a fragment (residues 704-953) of “coatomer protein complex, subunit beta 1, a fragment (residues 146-614) of zinc-finger protein 251, and a fragment (residues 284-435) of syntaxin binding protein 1. We used yeast three-hybrid system to determine the effects of these βII-C interacting proteins as well as of 7 proteins previously identified to interact with the tetramerization region of non-erythroid alpha spectrin (IPαII-N s) [1] on spectrin tetramer formation. The results showed that 3 IPβII-C s were able to bind βII-C even in the presence of αII-N, and 4 IPαII-N s were able to bind αII-N in the presence of βII-C. We also found that the syntaxin binding protein 1 fragment abolished αII-N and βII-C interaction, suggesting that this protein may inhibit or regulate non-erythroid spectrin tetramer formation.

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

et al. 2011

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We have screened a human immunoglobulin single-chain variable fragment (scFv) phage library against the C-terminal tetramerization regions of erythroid and nonerythroid beta spectrin (bI-C1 and bII-C1, respectively) to explore the structural uniqueness of erythroid and nonerythroid b-spectrin isoforms. We have identified interacting scFvs, with clones ''G5'' and ''A2'' binding only to bI-C1, and clone ''F11'' binding only to bII-C1. The K d values, estimated by competitive enzyme-linked immunosorbent assay, of these scFvs with their target spectrin proteins were 0.1-0.3 lM. A more quantitative K d value from isothermal titration calorimetry experiments with the recombinant G5 and bI-C1 was 0.15 lM. The a-spectrin fragments (model proteins), aI-N1 and aII-N1, competed with the bI-C1, or bII-C1, binding scFvs, with inhibitory concentration (IC 50 ) values of~50 lM for aI-N1, and 0.5 lM for aII-N1. Our predicted structures of bI-C1 and bII-C1 suggest that the Helix B 0 of the Cterminal partial domain of bI differs from that of bII. Consequently, an unstructured region downstream of Helix B 0 in bI may interact specifically with the unstructured, complementarity determining region H1 of G5 or A2 scFv. The corresponding region in bII was helical, and bII did not bind G5 scFv. Our results suggest that it is possible for cellular proteins to differentially associate with the C-termini of different b-spectrin isoforms to regulate a-and b-spectrin association to form functional spectrin tetramers, and may sort b-spectrin isoforms to their specific cellular localizations.Keywords: beta spectrin; erythroid and nonerythroid; scFv; structural difference; affinity difference Abbreviations: aI-N1, a recombinant protein of aI-spectrin segment with residues 1-156; aI-spectrin, erythroid a-spectrin; aII-N1, a GST fusion protein of aII-spectrin segment with residues 1-147; aII-spectrin, nonerythroid a-spectrin; bI-C1, a GST fusion protein of bI-spectrin segment with residues 1898-2083; bI-C1D, a GST fusion protein of bI-spectrin segment with residues 1898-2070, that is, the residues 2071-2083 in bI-C1 were deleted; bI-spectrin, erythroid b-spectrin; bII-C1, a GST fusion protein of bII-spectrin segment with residues 1906-2093; bII-spectrin, nonerythroid b-spectrin; CD, circular dichroism; CDRs, complementarity determining regions; ELISA, enzyme-linked immunosorbent assay; EPR, electron paramagnetic resonance; GST, glutathione S-transferase; HRP, horseradish peroxidase; ITC, isothermal titration calorimetry; PBS, phosphate buffered saline at pH 7.4; rG5, recombinant G5, a single-chain variable fragment found to bind bI-C1; scFv, single-chain variable fragment.Yuanli Song's current address is

show abstract

The L49F mutation in alpha erythroid spectrin induces local disorder in the tetramer association region: Fluorescence and molecular dynamics studies of free and bound alpha spectrin

Cited by 6 publications

References 33 publications

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

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

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

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

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