Structural insights into TSC complex assembly and GAP activity on Rheb

Abstract

Tuberous sclerosis complex (TSC) integrates upstream stimuli and regulates cell growth by controlling the activity of mTORC1. TSC complex functions as a GTPase-activating protein (GAP) towards small GTPase Rheb and inhibits Rheb-mediated activation of mTORC1. Mutations in TSC genes cause tuberous sclerosis. In this study, the near-atomic resolution structure of human TSC complex reveals an arch-shaped architecture, with a 2:2:1 stoichiometry of TSC1, TSC2, and TBC1D7. This asymmetric complex consists of two interweaved TSC1 coiled-coil and one TBC1D7 that spans over the tail-to-tail TSC2 dimer. The two TSC2 GAP domains are symmetrically cradled within the core module formed by TSC2 dimerization domain and central coiled-coil of TSC1. Structural and biochemical analyses reveal TSC2 GAP-Rheb complimentary interactions and suggest a catalytic mechanism, by which an asparagine thumb (N1643) stabilizes γ-phosphate of GTP and accelerate GTP hydrolysis of Rheb. Our study reveals mechanisms of TSC complex assembly and GAP activity.

Polymeric assembly of endogenous Tuberous Sclerosis Protein Complex

“…Recombinant human pTSC forms flexible 40 nm filamentous structures: Consistent with results from two other groups [7,8], we detected ~10 × 40 nm highly flexible particles in NSEM analysis of human recombinant pTSC overexpressed under the control of CMV promoters in human cells (Fig. 1).…”

“…Direct association of TBC1D7 with TSC1 is also supported by immunoprecipitation studies showing that the 35kDa accessory protein binds TSC1 but not TSC2 [8]. TSC1 was detected at approximately 180kDa in the rat elution fractions using a rabbit monoclonal antibody that binds a synthetic peptide from human TSC1 around residue Val640 (Fig.…”

“…Both TSC2 and TBC1D7 were detected with a clear western blot signal in the S3 fraction before and after passage through the Fab-column, indicating that the Fab was capturing only a small fraction of the complexes from S3. Presence of the TBC1D7 in the elution fractions indicates the presence of TSC1 because the small 35 kDa accessory protein interacts directly with TSC1 in pTSC and not TSC2 as observed in the cryoEM structure of the human complex [7,8]. This mode of interaction is supported by X-ray crystallographic studies of truncated TSC1 with TBC1D7 [13,17].…”

“…Two recent studies have reported near-complete cryoEM density maps of recombinant human pTSC at near-atomic resolutions [7,8]. Both studies report a rod-shaped architecture that is highly flexible.…”

TSC1 binding to lysosomal PIPs is required for TSC complex translocation and mTORC1 regulation

“…On the basis of the observation of two pentameric ring structures stacked on top of each other in the crystal, it was suggested that the TSC complex might form pentamers in cells. Recently, cryoelectron microscopy (cryo-EM) analyses of the human TSC1/TSC2/TBC1D7 complex at sub-atomic resolution have been published (Ramlaul et al, 2021;Yang et al, 2021). The reconstructions reveal the overall architecture of the complex, but only a small C-terminal portion of TSC1 could be modeled.…”