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

• Published in: ACS central science Vol. 2; no. 8; pp. 506 - 516
• Main Authors: Lee, Song-Yi, Lee, Hakbong, Lee, Hye-Kyeong, Lee, Seung-Won, Ha, Sung Chul, Kwon, Taejoon, Seo, Jeong Kon, Lee, Changwook, Rhee, Hyun-Woo
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 24.08.2016
• ISSN: 2374-7943; 2374-7951
• DOI: 10.1021/acscentsci.6b00137

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.