Structural basis for mTORC1-dependent regulation of the lysosomal and autophagic transcription factor TFEB

The transcription factor TFEB is a master regulator of lysosomal biogenesis and autophagy. The phosphorylation of TFEB by the mechanistic target of rapamycin complex 1 (mTORC1) is unique in its mTORC1 substrate recruitment mechanism, which is strictly dependent on the amino-acid-mediated activation of the RagC GAP FLCN. TFEB lacks the TOR signaling (TOS) motif responsible for the recruitment of other mTORC1 substrates. We used cryo-electron microscopy (cryo-EM) to determine the structure of TFEB as presented to mTORC1 for phosphorylation. Two full Rag-Ragulator complexes present each molecule of TFEB to the mTOR active site. One Rag-Ragulator complex is bound to Raptor in the canonical mode seen previously in the absence of TFEB. A second Rag-Ragulator complex (non-canonical) docks onto the first via a RagC GDP-dependent contact with the second Ragulator complex. The non-canonical Rag dimer binds the first helix of TFEB in a RagCGDP-dependent aspartate clamp in the cleft between the Rag G domains. Mutation of the clamp drives TFEB constitutively into the nucleus whilst having no effect on mTORC1 localization. The remainder of the 108-amino acid TFEB docking domain winds around Raptor and then back to RagA. This structure presents the phosphorylatable Ser residues of TFEB to the mTORC1 active site in a suitable geometry for their phosphorylation. The double use of RagC GDP contacts in both Rag dimers explains the strong dependence of TFEB phosphorylation on FLCN and the RagC GDP state. ### Competing Interest Statement J.H.H. is a co-founder and shareholder of Casma Therapeutics and receives research funding from Casma Therapeutics, Genentech, and Hoffmann-La Roche. A. B. is a cofounder and shareholder of Casma Therapeutics and advisory board member of Next Generation Diagnostics, Avilar Therapeutics, and Coave.