Proximity-Directed Labeling Reveals a New Rapamycin-Induced Heterodimer of FKBP25 and FRB in Live Cells

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. In this study,
we employed a new proximity biotin-labeling method using promiscuous
biotin ligase (pBirA) to identify unknown elements in the rapamycin-induced
interactome on the FK506-rapamycin binding (FRB) domain in living
cells. FKBP25 showed the strongest biotin labeling by FRB–pBirA
in the 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
there 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.