Structure of the complete human TSC:WIPI3 lysosomal recruitment complex

<p>Tuberous sclerosis complex (TSC) turns off cell growth in response to energy stress by inhibiting the master kinase mechanistic target of rapamycin complex (mTORC1). TSC hydrolyzes RAS homolog-mTORC1 binding (RHEB) from its GTP-bound to GDP-bound state, preventing the allosteric activation of mTORC1. Loss-offunction TSC mutations hyperactivate mTORC1 resulting in the common genetic disorder TSC characterized by excess cell growth and tumor formation. Here we overcome a high degree of continuous conformational heterogeneity to determine the 2.9 Å cryo-electron microscopy (cryo-EM) structure of the complete human TSC in complex with the lysosomal recruitment factor WIPI3. TSC forms an elongated 40 nm wing-like structure with a core HEAT-repeat scaffold formed by a TSC2 dimer joined centrally by the juxtaposition of two catalytic domains. The TSC1 coil-coil dimer runs across the TSC2 surface, forming a previously undetected N-terminal TSC1 dimer that clamps onto the core scaffold on a single TSC wing. Structural and biochemical analysis reveals a novel phosphatidylinositol phosphate (PIP)-binding pocket in the TSC1 dimer interface that specifically binds singularly phosphorylated PIPs. WD repeat domain phosphoinositide-interacting-protein-3 (WIPI3) binds to the extreme tip of the complex through a conserved motif in TSC1, providing a second membrane anchor point for TSC lysosomal recruitment. The TSC:WIPI3 complex helps explain how TSC lysosomal recruitment proteins coordinate with endolysosomal phosphoinositide-signaling networks to regulate TSC localization, RHEB hydrolysis, and mTORC1 inhibition. More broadly, the high-resolution structure of the complete human TSC identifies novel mutational hotspots that unravel crucial new mechanisms of TSC dysregulation in disease.</p>