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 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 recognized a central segment of the PA tag peptide in a tight β-turn configuration, suggesting its compatibility 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 αIIb subunit of the fibrinogen receptor αIIbβ3 integrin without affecting the structural and functional integrity, while maintaining its high affinity toward 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 reporter/activator anti-β1 integrin antibody that can be classified as a ligand-induced binding site antibody.