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. 2023 Feb 7;120(7):e2213670120. doi: 10.1073/pnas.2213670120

Fig. 4.

Fig. 4.

MOA of EO on TFEB. (A) EO is recognized of by TFEB through the HLH hydrophobic helix bundle according to molecular docking and dynamics simulation. (B) Chemical structure of compound 3 which lacks the carboxyl group compared with EO. (C) Compound 3 showed significantly lower inhibition activity against TFEB-DNA interaction than EO in the fluorescence anisotropy assay. Error bars represent the SEMs of four repeats. (D) Pull-down analysis of the interactions between EO and His-tagged TFEB WT, MutCom_1, MutCom_2, MutCom_3, and MutCom_4, using EO-Biotin immobilized on streptavidin beads. The precipitated TFEB proteins were analyzed by anti-His antibody. (E) Pull-down analysis of the interactions between EO and His-tagged TFEB WT, MutCom_1, R248A, R254A, R271A, and K274A. A representative result was shown with long exposure time (4 s) and short exposure time (0.5 s). (F) The CLEAR DNA is modeled onto the TFEB crystal structure using MITF-DNA complex (protein data bank code: 6KWI) as a reference. EO binding on the bottom surface of HLH region induces significant steric hindrance preventing further DNA binding.