Recently, promiscuous biotin ligase (pBirA) was shown to biotin-label spatial localized proteome such as nuclear pore complex in living physiological condition. In this study, we expand to use pBirA to map spatiotemporal rapamycin-inducible interactome. Mammalian target of rapamycin (mTOR) signaling is a core pathway in cellular metabolism, and control of the mTOR pathway by rapamycin shows potential for the treatment of metabolic diseases.
FKBP12 is the most well-known interacting protein to FRB domain of mTOR in presence of rapamycin. However, other rapamycin-inducible interacting protein on mTOR has not been characterized well. Thus, we tested whether FRB-pBirA could label other proteins in presence of rapamycin in living mammalian cells. After reaction, the biotinylated proteins were purified and digested, and labeled peptides were analyzed by mass spectrometry. From this analysis, surprisingly, we found FKBP25 was strongly biotinylated by FRB-pBirA in presence of rapamycin. Immunoprecipitation and immunofluorescence experiments confirmed that endogenous FKBP25 has a rapamycin-induced physical interaction with the FRB domain. Furthermore, the crystal structure of the ternary complex of FRB-rapamycin-FKBP25 was determined at 1.67-Å resolution. In this crystal structure we found that the conformational changes of FRB generate a hole where is a methionine-rich space and covalent metalloid coordination was observed at C2085 of FRB located at the bottom of the hole. Our results imply that FKBP25 might have a unique physiological role related to metallomics in mTOR signaling.
From this experiment, we proposed that in vivo proteome mapping tools could be expanded to be used as an efficient method to identify drug target protein identification in living cells.