Architecture of the Tuberous Sclerosis Protein Complex

Abstract

The Tuberous Sclerosis Complex (TSC) protein complex (TSCC), comprising of three subunits TSC1, TSC2, and TBC1D7, is widely recognised as a key integration hub for cell growth and intracellular stress signals upstream of the mammalian target of rapamycin complex 1 (mTORC1). The TSCC negatively regulates mTORC1 by acting as a GTPase-activating protein (GAP) towards the small GTPase Rheb. Both human TSC1 and TSC2 are important tumor suppressors, and their mutations underlie the tuberous sclerosis disease. We used single-particle cryo-EM to reveal the organisation and architecture of the complete human TSCC. We show that TSCC forms an elongated scorpion-like structure, consisting of a "body" in the middle, and a "pincer" and a "tail" at each side. The "body" is composed of a flexible TSC2 HEAT repeat dimer, along the inner surface of which runs the TSC1 coiled-coil backbone, breaking the symmetry of the dimer. Each end of the body is structurally distinct, representing the N- and C-termini of TSC1; a "pincer" is formed by the highly flexible N-terminal TSC1 core domains and a barbed "tail" makes up the TSC1 coiled-coil-TBC1D7 junction. The TSC2 GAP domain is found abutting the centre of the body on each side of the dimerisation interface, poised to bind a pair of Rheb molecules at a similar separation to the pair in activated mTORC1. Our architectural dissection reveals the mode of association and topology of the complex, casts light on the recruitment of Rheb to the TSCC, and also hints at functional higher order oligomerisation, which has previously been predicted to be important for Rheb-signalling suppression.

Polymeric assembly of endogenous Tuberous Sclerosis Protein Complex

“…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).…”

“…We found that recombinant human pTSC co-purifies with heat shock protein 70 (Hsp70), as observed in other studies[7,13]. However, no density could be found for the heat shock protein in the recent cryoEM maps indicating that the heat shock proteins most likely binds misassembled or partially unfolded pTSC.…”

“…Both TSC2 and TBC1D7 were detected with a clear western blot signal in the S3 fraction before and after passage through the F ab -column, indicating that the F ab 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].…”

“…It is also possible that their formation is differentially regulated in rat and pig species upon fasting of the animals. The recent cryoEM models of recombinant human pTSC suggests a head-to-tail oligomerization potential[7]. This hypothesis was supported by a concentration dependent observation of a head-to-tail association of individual pTSC complexes in the cryoEM micrographs and shifting of the elution profile of the recombinant protein toward large molecular weights by increasing concentration[7].…”

“…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.…”