TOOLS AND TECHNIQUES Tailored placement of a turn-forming PA tag into the structured domain of a protein to probe its conformational state Yuki Fujii 1,2 , Yukiko Matsunaga 1 , Takao Arimori 1 , Yu Kitago 1 , Satoshi Ogasawara 2 , Mika K. Kaneko 2 , Yukinari Kato 2 and Junichi Takagi 1, * 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 α 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. KEY WORDS: Epitope tag, Integrin, Site-specific labeling, β-turn, Reporter antibody INTRODUCTION Monoclonal antibodies show exquisite specificity toward their respective antigen by displaying a binding pocket with high chemical and geometrical complementarity to the antigen surface. The pocket is lined by a set of spatially arranged residues in the complementarity-determining region (CDR) loops of the antibody variable region. On the antigen, the epitope residues also need to be correctly arranged in three dimensions, rather than just being closely positioned in a primary sequence. As the conformation of a peptide segment in the structured protein is usually very different from that of free oligopeptide with the same sequence in solution (Dyson et al., 1988), it can be difficult to obtain monoclonal antibodies that are capable of recognizing native protein with high affinity by using a synthetic linear peptide as the immunizing or screening antigen (Spangler, 1991). For antibodies raised against small peptide fragments of proteins in order to generate reactivity with the cognate sequence of the intact protein, it is generally believed that they have to be targeted to highly mobile segments (Tainer et al., 1984). However, anti-peptide monoclonal antibodies are indifferent from other anti-protein monoclonal antibodies in their requirement for correct three-dimensional (3D) arrangement of epitope residues, other than the fact that all crucial residues are provided from a single stretch of primary sequence. Meanwhile, numerous monoclonal antibodies recognizing short linear peptide with high affinity and/or specificity have been successfully obtained. Some such peptide and anti-peptide monoclonal antibody pairs have been incorporated into research tools collectively called ‘epitope tag system’, with which one can detect, label, capture and even purify tagged target proteins very easily (Wood, 2014). Use of such a system is vital to much biomedical research, especially when good antibodies against the target molecule are unavailable. In addition to the utility as an immunological detection agent, monoclonal antibodies can sometimes be used as probe or reporter for certain functional states of a protein, particularly when the protein undergoes a large conformational change during its biological action (Dennison et al., 2014; Humphries et al., 2003; Irannejad et al., 2013; Walker et al., 2004). If the 3D structure of the protein is known, it is theoretically possible to place a short peptide tag at a desired position and to use a monoclonal antibody against the tag to determine the function of the protein, as well as to modulate the function of the protein. However, this is not practically possible because the tag insertion into the middle of a target protein can often destroy its local conformation or alter its function. For this reason, affinity tags are fused to either the N- or C-terminus of a target protein in most applications. Even when the tag sequence insertion is not expected to affect the function of target protein, such attempts are rare, because the inserted peptide itself does not usually work as a tag any more owing to the limited accessibility of the antibody and/or the altered conformation of the tag. However, we sometimes come across cases where protein termini are important for the function, or are buried and not accessible to a binding molecule or agent, in which case we have to seek ways to attach tags somewhere else. There are several studies reporting successful insertion of tag sequence into the middle of a protein domain by targeting a flexible loop region that does not have secondary structure. In these reports, however, either very long loops were chosen as the insertion point (Dinculescu et al., 2002; Morlacchi et al., 2012; Smith et al., 2004) or the inserted tag was flanked by long linker sequences (Facey and Kuhn, 2003; Kendall and Senogles, 2006), limiting general applicability of this approach. Therefore, there are unmet needs for the development of a general tagging system that is compatible with the insertion or ‘grafting’ into a structured protein domain for various cell biology and biochemical applications. Recently, we have reported the successful construction of a novel epitope tag system, by combining a 12-residue peptide (PA tag) derived from human podoplanin and a high affinity monoclonal antibody (NZ-1) against it (Fujii et al., 2014). The binding affinity Received 29 June 2015; Accepted 10 February 2016 1 Laboratory of Protein Synthesis and Expression, Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan. 2 Department of Regional Innovation, Tohoku University Graduate School of Medicine, 2-1 Seiryo- machi, Aoba-ku, Sendai 980-8575, Japan. *Author for correspondence (takagi@protein.osaka-u.ac.jp) 1512 © 2016. Published by The Company of Biologists Ltd | Journal of Cell Science (2016) 129, 1512-1522 doi:10.1242/jcs.176685 Journal of Cell Science