Structural basis for FLCN RagC GAP activation in TFEB substrate-selective mTORC1 regulation

mTORC1 regulates cell growth and catabolism in response to fluctuations in nutrients through phosphorylation of key substrates. The tumor suppressor FLCN is a RagC GTPase activating protein (GAP) that regulates mTORC1 phosphorylation of TFEB, controlling lysosome biogenesis and autophagy. Here, we determined the cryo-EM structure of the active FLCN complex (AFC) containing FLCN, FNIP2, the N-terminal tail of SLC38A9, the RagAGDP:RagCGDP.BeFx- GTPase dimer, and the Ragulator scaffold. Relative to the inactive lysosomal FLCN complex (LFC) structure, FLCN reorients by 90°, breaks its contacts with RagA, and makes new contacts with RagC that position its Arg164 finger for catalysis. Disruption of the AFC-specific interfaces of FLCN and FNIP2 with RagC eliminated GAP activity in vitro and led to nuclear retention of TFE3, with no effect on mTORC1 phosphorylation of S6K or 4E-BP1. The structure thus provides a roadmap to discover TFEB-selective mTORC1 antagonists. One-Sentence Summary The cryo-EM structure of the active FLCN RagC GAP complex provides a structural basis for TFEB/TFE3 substrate-selective targeting of mTORC1. ### 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. R.Z. is a cofounder and shareholder of Frontier Medicines and receives research funding from Genentech.