title: Tailored placement of a turn-forming PA tag into the structured domain of a protein to probe its conformational state.
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 αIIb subunit (encoded by ITGA2B) of the fibrinogen receptor αIIbβ3 integrin (of which the β3 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.
authors.affiliation: Laboratory of Protein Synthesis and Expression, Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan Department of Regional Innovation, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Fujii
authors.affiliation: Laboratory of Protein Synthesis and Expression, Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Yukiko
authors.initials: Y
authors.lastname: Matsunaga
authors.affiliation: Laboratory of Protein Synthesis and Expression, Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Takao
authors.initials: T
authors.lastname: Arimori
authors.affiliation: Laboratory of Protein Synthesis and Expression, Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Yu
authors.initials: Y
authors.lastname: Kitago
authors.affiliation: Department of Regional Innovation, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Ogasawara
authors.affiliation: Department of Regional Innovation, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan.
authors.forename: Mika K
authors.initials: MK
authors.lastname: Kaneko
authors.affiliation: Department of Regional Innovation, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan.
authors.forename: Yukinari
authors.initials: Y
authors.lastname: Kato
authors.affiliation: Laboratory of Protein Synthesis and Expression, Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan takagi@protein.osaka-u.ac.jp.
authors.forename: Junichi
authors.initials: J
authors.lastname: Takagi
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology*; D001666:Binding Sites, Antibody / Q000276:immunology*; D002460:Cell Line; D057809:HEK293 Cells; D006801:Humans; D039421:Integrin alpha2 / Q000235:genetics / Q000378:metabolism*; D039661:Integrin beta3 / Q000235:genetics / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000276:immunology; D011487:Protein Conformation; D039961:Semaphorins / Q000235:genetics / Q000378:metabolism; D000098853:Podoplanin
chemical_list: D000911:Antibodies, Monoclonal; D039421:Integrin alpha2; D039661:Integrin beta3; D008562:Membrane Glycoproteins; D039961:Semaphorins; C056564:ITGA2B protein, human; C061248:ITGB3 protein, human; C117468:PDPN protein, human; D000098853:Podoplanin
keywords: Epitope tag; Integrin; Reporter antibody; Site-specific labeling; β-turn