Tailored placement of a turn-forming PA tag into the structured domain of a protein to probe its conformational state

Yuki Fujii, Yukiko Matsunaga, Takao Arimori, Yu Kitago, Satoshi Ogasawara, Mika K. Kaneko, Yukinari Kato, Junichi Takagi

Research output: Contribution to journalArticlepeer-review

24 Citations (Scopus)

Abstract

Placement of a tag sequence is usually limited to either terminal end of the target protein, reducing the potential of epitope tags for various labeling applications. The PA tag is a dodecapeptide (GVAMPGAEDDVV) that is recognized by a high-affinity antibody NZ-1. We determined the crystal structure of the PA-tag-NZ-1 complex and found that NZ-1 recognizes a central segment of the PA tag peptide in a tight β-turn configuration, suggesting that it is compatible with the insertion into a loop. This possibility was tested and confirmed using multiple integrin subunits and semaphorin. More specifically, the PA tag can be inserted at multiple locations within the integrin α IIb subunit (encoded by ITGA2B) of the fibrinogen receptor a IIb β 3 integrin (of which the β 3 subunit is encoded by ITGB3) without affecting the structural and functional integrity, while maintaining its high affinity for NZ-1. The large choice of the sites for 'epitope grafting' enabled the placement of the PA tag at a location whose accessibility is modulated during the biological action of the receptor. Thus, we succeeded in converting a general anti-tag antibody into a special anti-integrin antibody that can be classified as a ligand-induced binding site antibody.

Original languageEnglish
Pages (from-to)1512-1522
Number of pages11
JournalJournal of cell science
Volume129
Issue number7
DOIs
Publication statusPublished - 2016 Apr 1

Keywords

  • Epitope tag
  • Integrin
  • Reporter antibody
  • Site-specific labeling
  • β-turn

ASJC Scopus subject areas

  • Cell Biology

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