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.
Department of Chemistry, Department of Biological Sciences, UNIST Central Research Facilities (UCRF), and Deparment of Biomedical Engineering, Ulsan National Institute of Science and Technology (UNIST) , Ulsan 44919, Korea.
Song-Yi
SY
Lee
Department of Chemistry, Department of Biological Sciences, UNIST Central Research Facilities (UCRF), and Deparment of Biomedical Engineering, Ulsan National Institute of Science and Technology (UNIST) , Ulsan 44919, Korea.
Hakbong
H
Lee
Department of Chemistry, Department of Biological Sciences, UNIST Central Research Facilities (UCRF), and Deparment of Biomedical Engineering, Ulsan National Institute of Science and Technology (UNIST) , Ulsan 44919, Korea.
Hye-Kyeong
HK
Lee
Department of Chemistry, Department of Biological Sciences, UNIST Central Research Facilities (UCRF), and Deparment of Biomedical Engineering, Ulsan National Institute of Science and Technology (UNIST), Ulsan 44919, Korea; Department of Chemistry, Department of Biological Sciences, UNIST Central Research Facilities (UCRF), and Deparment of Biomedical Engineering, Ulsan National Institute of Science and Technology (UNIST), Ulsan 44919, Korea.
Seung-Won
SW
Lee
Pohang Accelerator Laboratory, Pohang University of Science and Technology , Pohang, Kyungbuk 37673, Korea.
Sung Chul
SC
Ha
Department of Chemistry, Department of Biological Sciences, UNIST Central Research Facilities (UCRF), and Deparment of Biomedical Engineering, Ulsan National Institute of Science and Technology (UNIST) , Ulsan 44919, Korea.
Taejoon
T
Kwon
Department of Chemistry, Department of Biological Sciences, UNIST Central Research Facilities (UCRF), and Deparment of Biomedical Engineering, Ulsan National Institute of Science and Technology (UNIST) , Ulsan 44919, Korea.
Jeong Kon
JK
Seo
Department of Chemistry, Department of Biological Sciences, UNIST Central Research Facilities (UCRF), and Deparment of Biomedical Engineering, Ulsan National Institute of Science and Technology (UNIST) , Ulsan 44919, Korea.
Changwook
C
Lee
Department of Chemistry, Department of Biological Sciences, UNIST Central Research Facilities (UCRF), and Deparment of Biomedical Engineering, Ulsan National Institute of Science and Technology (UNIST) , Ulsan 44919, Korea.
Hyun-Woo
HW
Rhee