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

Yuanli Song, Chloe Antoniou, Adnan Memic, Brian K. Kay, L. W.M. Fung

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

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 (βI-C1 and βII-C1, respectively) to explore the structural uniqueness of erythroid and nonerythroid β-spectrin isoforms. We have identified interacting scFvs, with clones "G5" and "A2" binding only to βI-C1, and clone "F11" binding only to βII-C1. The Kd values, estimated by competitive enzyme-linked immunosorbent assay, of these scFvs with their target spectrin proteins were 0.1-0.3 μM. A more quantitative Kd value from isothermal titration calorimetry experiments with the recombinant G5 and βI-C1 was 0.15 μM. The α-spectrin fragments (model proteins), αI-N1 and αII-N1, competed with the βI-C1, or βII-C1, binding scFvs, with inhibitory concentration (IC50) values of ∼50 μM for αI-N1, and ∼0.5 lM for αII-N1. Our predicted structures of βI-C1 and βII-C1 suggest that the Helix B' of the Cterminal partial domain of βI differs from that of βII. Consequently, an unstructured region downstream of Helix B' in βI may interact specifically with the unstructured, complementarity determining region H1 of G5 or A2 scFv. The corresponding region in βII was helical, and βII did not bind G5 scFv. Our results suggest that it is possible for cellular proteins to differentially associate with the C-termini of different β-spectrin isoforms to regulate α- and β-spectrin association to form functional spectrin tetramers, and may sort β-spectrin isoforms to their specific cellular localizations.

Original languageEnglish
Pages (from-to)867-879
Number of pages13
JournalProtein Science
Volume20
Issue number5
DOIs
Publication statusPublished - May 2011

Keywords

  • Affinity difference
  • Beta spectrin
  • Erythroid and nonerythroid
  • Scfv
  • Structural difference

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