Structural insights into TSC complex assembly and GAP activity on Rheb

doi:10.21203/rs.3.rs-36453/v1

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Structural insights into TSC complex assembly and GAP activity on Rheb

https://doi.org/10.21203/rs.3.rs-36453/v1

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published 12 Jan, 2021

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Tuberous sclerosis complex (TSC) integrates upstream stimuli and regulates cell growth by controlling the activity of mTORC1. TSC 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 assembly and GAP activity.

Structural Biology

Tuberous sclerosis complex

GAP activity

Rheb

The mechanistic target of rapamycin complex 1 (mTORC1) is a master regulator of cell growth by phosphorylating a variety of substrates, as exemplified by ribosomal S6 kinase 1 (S6K1) and eukaryote initiation factor 4E binding protein (4EBP)^1,2. As a well-known tumor suppressor, the tuberous sclerosis complex (TSC) integrates cues of growth factors, energy status and various stress to maintain Rheb in GDP-bound state, and therefore keeps mTORC1 in check to limit undesirable cell growth^2–5. The TSC acts as a GTPase-activating protein (GAP) towards a small G-protein Ras homolog enriched in brain (Rheb) required for mTORC1 activation⁶. In GTP-bound state, Rheb directly binds to and activates mTORC1^7–11.

The TSC complex consists of Tuberous Sclerosis Complex 1 (TSC1), Tuberous Sclerosis Complex 2 (TSC2) and an auxiliary subunit Tre2-Bub2-Cdc16-1 domain family member 7 (TBC1D7)^4,5. Mutations of TSC1 or TSC2 genes cause tuberous sclerosis, an autosomal dominant genetic disease characterized by the development of histologically diverse hamartomas or benign tumors, including skin, brain, and kidneys^5,12. TSC patients are frequently associated with severe neurological manifestations, including epilepsy, intellectual disability and autism^5,13. Although the functions of TSC have been extensively studied for decades, there are only a few structures of isolated domains, including TSC1 peptide bound to TBC1D7^3,14, N-terminal domains of yeast TSC1¹⁵ and TSC2 (Chaetomium thermophilum)¹⁶. The lack of TSC complex structure has hampered understanding the mechanisms for complex assembly, GAP activity, and disease correlation.

Here we present the first cryo-EM structure of human TSC complex and elaborate on its characteristic assembly and GAP function. Our structure also provides a framework for understanding the regulation of TSC function in mTORC1 pathway and its pathological significance.

Structure determination. To obtain TSC structure, we overexpressed human TSC1, TSC2, and TBC1D7 in Expi293F cells and purified the complex to homogeneity (Supplementary Fig. 1a). The purified TSC complex showed relatively weak in vitro GAP activity against Rheb, consistent with the known poor in vitro activity^8,17 (Supplementary Fig. 1b). The structure was determined by cryo-EM single particle reconstruction and the cryo-EM map was refined to an overall resolution of 4.4 Å (Fig. 1b). The core and two wings were locally refined to 3.6 Å, 3.9 Å, and 4.1 Å resolution, respectively (Supplementary Figs. 2 and 3). The majority of structural model was unambiguously built ab initio aided by the structure of TSC1 fragment bound to TBC1D7¹⁴ and secondary structure analyses (Table 1 and Supplementary Movies 1–4). The TSC1 (residues 746–971), TSC2 (residues 127–1732), and TBC1D7 (residues 21–287) were modeled, in which a few regions were built using poly alanine due to the weak cryo-EM density (Supplementary Fig. 4).

Table 1

Statistics of cryo-EM data collection, refinement and validation statistics.
	TSC
Data collection and processing
Magnification	105,000x
Voltage (kV)	300
Electron exposure (e–/Å²)	50
Defocus range (µm)	1.0 to 3.5
Pixel size (Å)	1.356
Symmetry imposed	C1
Initial particle images (no.)	1,528,982
Final particle images (no.)	131,022
Map resolution (Å) Consensus reconstruction Focus wing-a reconstruction Focus core reconstruction Focus wing-b reconstruction FSC threshold	4.4 4.1 3.6 3.9 0.143
Map resolution range (Å) Consensus reconstruction Focus wing-a reconstruction Focus core reconstruction Focus wing-b reconstruction	4.0–20.0 4.0–20.0 3.0–10.0 3.0–10.0
Refinement
Initial model used (PDB code)	5EJC
Model resolution (Å) FSC threshold	4.5 0.5
Model resolution range (Å)	4.0-4.5
Map sharpening B factor (Å²)	-129.92
Model composition Non-hydrogen atoms Protein residues Ligands	23934 3089 0
B factors (Å²) Protein Ligand	141.96 ---
R.m.s. deviations Bond lengths (Å) Bond angles (°)	0.004 0.673
Validation MolProbity score Clashscore Poor rotamers (%)	2.42 24.52 0.12
Ramachandran plot Favored (%) Allowed (%) Disallowed (%)	90.30 9.70 0.00

Overall structure of human TSC. The complex consists of a central core and two wings (termed wing-a and wing-b) and the overall structure exhibits an elongated arch-shaped fold with approximate dimensions of ~ 390 × 133 × 88 Å³ (Fig. 1b). TSC1, TSC2, and TBC1D7 assemble the TSC complex with a 2:2:1 stoichiometry, generating an asymmetric modular organization. Although isolated domains adopt similar folds, the two TSC1 (termed TSC1a/1b) and two TSC2 (TSC2a/2b) reveal distinct conformations, respectively (Supplementary Fig. 5). The two TSC2 molecules form a pseudo-symmetric dimer through tail-to-tail interactions. The coiled-coil domains (CCs, residues 746–971) of TSC1a and TSC1b interwind in parallel and form an extended two-helix bundle (Fig. 1c and Supplementary Fig. 6a-d), which makes multiple contacts with TSC2 dimer and stabilizes the overall conformation of the complex. This parallel dimerization of TSC1 leads to an asymmetric formation of TSC1-TSC2 tetramer and recruitment of a single TBC1D7 molecule, generating a unique and characteristic modular organization (Fig. 1b and Supplementary Fig. 5f-g).

Each TSC2 consists of a HEAT repeat domain (HEAT), a dimerization domain (DD), followed by a C-terminal GAP catalytic domain (GAP) (Fig. 1a, b, d). The N-terminal twelve HEAT repeats (wing HEAT, wHEAT) flank out of the central core and are stabilized by TSC1. The following six HEAT repeats (core HEAT, cHEAT) associate with and are stabilized by the central GAP and DD domains. The cHEAT-DD-GAP of the two TSC2 molecules adopt pseudo-symmetric fold whereas the two wHEAT domains adopt distinct conformations due to differently associated TSC1 (Supplementary Fig. 5). TSC2a and TSC2b bind the N-terminal (N-CC) and C-terminal (C-CC) halves of the TSC1 CC dimer, respectively (Fig. 1b).

The TBC1D7 associates with the C-terminal helices (residues 937–971) of TSC1a/1b but has no direct contact with TSC2. The TBC1D7-TSC1a/1b module adopts a similar fold to the human TSC1-TBC1D7 crystal structure¹⁴ (Supplementary Fig. 6e), and is positioned far away from the central core, consistent with its auxiliary role in TSC assembly and function⁴. The observation agrees with immunoprecipitation results showing that TBC1D7 binds TSC1 but not TSC2 (Supplementary Fig. 6f).

TSC1 structure and its interaction with TSC2. The two CC domains of TSC1a and TSC1b are paired in parallel and form a two-turn left-handed supercoil (Fig. 1c). The pairwise coiled-coil involves extensive intermolecular contacts. The TSC1 homodimer interface is enriched in nonpolar residues, which make extensive hydrophobic contacts to support a stable TSC1 dimerization and its scaffolding function (Supplementary Fig. 6a-d). Sequence alignment indicates that the coiled-coil region of TSC1 is highly conserved across eukaryotic species, suggesting an evolutionarily conserved dimeric coiled-coil conformation (Supplementary Fig. 4b).

The TSC1 CC dimer adopts an arch-shaped architecture and packs against the ridge of TSC2 dimer (Figs. 1b, 2a). The CC dimer makes four major contacts with TSC2. (1) The central region of TSC1a (residues 800–890) sits on a “saddle” formed by TSC2 DD dimer (Fig. 2b, c). (2) The TSC1a-N-CC (residues 746–795) packs against the ridge of repeats HEAT7-HEAT12 of TSC2a (Fig. 2d). (3) The C-CC (residues 890–936) dimer of TSC1 packs against the ridge of the repeats HEAT8-HEAT12 of TSC2b (Fig. 2e). (4) The TSC1a-C-CC (residues 937–971) packs against the ridge of repeats HEAT3-HEAT7 of TSC2b (Fig. 2f), confirmed the known interaction between the N-terminus of TSC2 (residues 1-418) and TSC1^16,18,19. Consistent with the asymmetric complex formation, TSC1a plays a major role in binding TSC2 dimer.

Another asymmetric feature of the complex exists around the end of wing-a module. The cryo-EM map reveals repetitive helical region covering the N-terminal HEAT repeats of TSC2a but not TSC2b. This region is likely derived from the predicted N-terminal HEAT repeat domain of TSC1 (Fig. 1b and Supplementary Fig. 4b)¹⁵. Other TSC1 regions were invisible in our cryo-EM map due to flexibility, suggesting a nonessential role in TSC complex assembly. In our immunoprecipitation assay, the full-length and CC of TSC1 shows comparable binding to TSC2 (Supplementary Fig. 6g), consistent with the maintenance of TSC1-TSC2 upon deletion of TSC1 several N-terminal fragments²⁰.

Domain organization and dimerization of TSC2. The TSC2 monomer adopts a seahorse-shaped conformation, in which the wHEAT (HEAT1 to HEAT12) and cHEAT (HEAT13-HEAT18) together adopt a right-handed super helical fold (Fig. 1d). The isolated cHEAT and wHEAT domains adopt almost identical conformations, respectively, in the two TSC molecules. However, the whole HEAT domain of TSC2b tends to be more extended than that of TSC2a (Supplementary Fig. 5e-g). The two HEAT domains bind the TSC1 dimer in a distinct manner, suggesting that different features of N- and C-terminal portions of TSC1 CC dimer lead to distinct conformation of the two TSC2 molecules (Supplementary Fig. 5f).

The cHEAT-DD-GAP parts of two TSC2 molecules form an almost symmetrical core of the complex and the dimerization is mediated by two stably associated DD domains (Fig. 2a-c and Supplementary Fig. 5c-e). Each DD domain consists of a four-stranded antiparallel β-sheet (Dβ1-Dβ4) and five flanking α-helices (Dα1-Dα5) (Fig. 2c). The two β-sheets together form a saddle-shaped eight-stranded β-sheet. The Dα5 helix (residues 1472–1483) packs against the concave surface of the saddle and binds the other TSC2 molecule on HEAT18 and the following loop (residues 1024–1038) (Fig. 2a and Supplementary Fig. 7). The loop preceding helix Dα4 inserts into a hydrophobic pocket of the other TSC2 molecule located in a three-way junction formed by the cHEAT, DD, and GAP domains (Fig. 2a). The TSC2 dimerization is further supported by DD helices of the two TSC2 molecules, which sandwich the TSC1a CC domain. Around this region, the TSC2 dimer interface (~ 2805Å²) is larger than TSC1-TSC2 interface (~ 1761Å²), suggesting that TSC2 forms homodimer independent of TSC1 and the TSC2 dimer is required for generating a stable TSC1-TSC2 tetramer²¹.

The TSC2 GAP structure and its positioning in TSC complex. The two GAP catalytic domains are symmetrically cradled within the central core module and each GAP adopts a characteristic mixed α/β fold (Figs. 1b and 3a). A central seven-stranded β-sheet is stabilized by a long α helix (Gα5) from the concave side. The helix Gα3 (catalytic helix) and two loops (L1 and L2) pack against the convex surface of the β-sheet. Three intermodular contacts involve positioning of each GAP domain (Fig. 3a and Supplementary Fig. 8). (1) Two parallel α helices (GαN and GαC) form a GAP extension, which protrudes out of the catalytic core and binds the edge of the β-sheet of the DD domain and repeats HEAT17-HEAT18 (Supplementary Fig. 8b). (2) The helix Gα5, strand Gβ7 and its preceding loop, together pack against the concave surface of repeats HEAT13 to HEAT16 (Supplementary Fig. 8c). (3) The helix Gα1 (TSC2b) or Gα2 (TSC2a) and loop L1 bind TSC1 coiled-coil and the binding pattern is slightly different in two GAP domains. The GAP of TSC2a binds single CC (residues 800–830) of TSC1b whereas the GAP of TSC2b binds two CC strands (residues 860–895) of TSC1a/1b (Supplementary Fig. 8d). The lack of TSC1 largely decreased the GAP activity, indicating that TSC1 is required for assembly of fully active GAP domains in TSC complex (Supplementary Fig. 6h). The two GAP catalytic core adopt almost identical conformations and their catalytic pockets both open outwards, suggesting a similar manner of substrate recognition and catalysis (Fig. 2a and Supplementary Fig. 5d-e).

Catalytic mechanism of TSC2 GAP. The TSC2 GAP domain is highly conserved from yeast to human and shares considerable sequence homology to Rap1GAP (Supplementary Fig. 9a), suggesting that TSC2-stimulated GTP hydrolysis of Rheb follows the same mechanism as in Rap-Rap1GAP system^8,17,19. To investigate the mechanism of TSC2-stimulated GTP hydrolysis of Rheb, we superimposed TSC2 GAP structure and Rheb-GTP (PDB: 1XTS)²² with Rap-Rap1GAP structure (PDB: 3BRW)¹⁷ and the classical small G protein Ras-RasGAP (PDB: 1WQ1)²³ (Fig. 3b-d). Structural comparison confirms the predicted structural similarity between the GAP domains of TSC2 and Rap1GAP and reveals distinct fold of the associated domains, which may provide substrate specificity (Fig. 3b, c).

Previous structural and biochemical studies of small GTPases and their GAPs have proposed a generally conserved activation mechanism^24,25. All the GAP domains provide positively charged residues, neutralize negative charges generated during phosphoryl transfer reactions, and thus accelerate GTP hydrolysis^24,25. Ras-RasGAP represents the prototypic small GTPase-GAP system, in which a trans-arginine finger (R789 in RasGAP) and a cis-glutamine (Q61 in Ras) are critical for catalysis through stabilizing the γ-phosphate in the transition state^23,26 (Fig. 3e, f). The arginine finger is shared by GAPs of some other Ras superfamily members²⁵. As a representative exception, Rap1-RapGAP lacks the arginine finger, but instead, has an asparagine thumb (N290 in Rap1GAP), which stabilizes γ-phosphate and is essential for GAP activity^17,27 (Fig. 3g-h).

Superimposition of Rheb²² and TSC2 GAP domain structures to the Rap1-Rap1GAP complex structure¹⁷ suggests that Rheb binds TSC2 GAP in a manner similar to that in Rap1-Rap1GAP (Fig. 3b, c). The catalytic helix (Gα3, K¹⁶³⁸RHLGN¹⁶⁴³) of TSC2 faces toward the catalytic cavity formed by the switch I, switch II, and P-loop of the superimposed Rheb (Fig. 3i-j). The switch I is conserved among the small G proteins (Supplementary Fig. 9b). Residue N1643 of TSC2 is similarly positioned to N290 of Rap1GAP, suggesting a shared asparagine thumb of the two GAP domains. As an equivalent of residue Y32 of Rap1 and Y32 in Ras, residue Y35 of Rheb is positioned close to N1643 and may facilitate GTP hydrolysis. The TSC2 catalytic helix is located relatively closer to the Rheb compared to that in Rap1-RapGAP structure, suggesting the helix might rotate back upon binding Rheb and allow the reaction to occur (Fig. 3g, i).

Other residues of the catalytic helix may support catalytic helix conformation (Fig. 3i-j). Residues K1638 and R1639 (equivalent to K285 and R286 of Rap1GAP) face toward the putative Rheb. Residues H1640 and L1641 (equivalent to H287 and I288 of Rap1GAP) face toward the core of TSC2 GAP. The conformational stability of the equivalent catalytic helix of RasGAP is essential for its activity^6,23, suggesting that these catalytic helix residues may also be required for GAP activity (Fig. 3e, g, i).

We performed cell-based assay to investigate the GAP activity of TSC complex by detecting the phosphorylation of S6K1 at T389, which is well-accepted to represent the level of Rheb in GTP-bound form and TSC2 GAP activity^8,28 (Fig. 3k). The co-transfection of TSC1 and TSC2 largely decreased the level of phosphorylated-S6K1, indicating a robust GAP activity in cells (Fig. 3k, lanes 1–3). Alanine substitutions of K1638 or R1639 and tuberous sclerosis-associated mutation of K1638 showed weak to moderate defect on GAP activity (Fig. 3k, lanes 4–6), whereas alanine substitutions or tuberous sclerosis-associated mutations of H1640, L1641, or N1643 on TSC2, largely impaired the GAP activity (Fig. 3k, lanes 7–12), to a level comparable to that of lacking TSC complex (Fig. 3k, lane 2). The result is consistent with structural observation and supports the notion that TSC2 uses the asparagine thumb (N1643) to accelerate GTP hydrolysis of Rheb and residues K1638, H1640 and L1641 of the catalytic helix function in supporting the conformation of the asparagine thumb. These results are consistent with previous studies^10,29, in which residues K1638, H1640, N1643 are important for TSC2 GAP activity.

The Rheb recognition by TSC2 GAP domain. Structure superimposition suggests that Rheb is well-accommodated by the TSC2 GAP domain and has no clash with other domains. This putative Rheb-TSC2 binding pattern differs from that of Rap1-Rap1GAP and Ras-RasGAP due to characteristic features of TSC2 GAP, which may confer specificity toward Rheb. Besides the catalytic helix, the loops L1 and L2 are respectively positioned close to switch II and switch I of the superimposed Rheb, possibly generating two putative TSC2-Rheb contacts (Fig. 3b-j and Supplementary Fig. 8e). Previous study reported that L1594 and F1666 mutations decreased GAP activity²⁹. Our GAP assay shows that Alanine substitutions or disease-associated mutations of L1 (L1594, L1597), L2 (Q1665, F1666), and F1645 impaired its GAP activity, confirming their supportive roles in substrate recognition and/or catalysis (Fig. 3l, lanes 13–14 and 8–11).

The structure reveals a characteristic helix pair formed by GαN (residues 1525–1536) and GαC (residues 1739–1754) of TSC2 that was not observed in other GAP-GTPase systems. The helix pair is positioned near the helices α2 (residue Q72) and α3 (residues D105 and M106) of the superimposed Rheb and likely supports TSC2-Rheb interactions (Fig. 3f, h, j). Mutations R1529A, L1533A, and double mutation R1529A/L1533A on GαN and R1749A on GαC led to moderate to severe decrease in TSC2 GAP activity, suggesting their critical roles in supporting TSC2-Rheb contacts (Fig. 3l, lanes 4–7 and 12). Previous study also showed that R1749Q mutation (equivalent to R1707 in the short isoform used in this work) decreased the GAP activity to some extent¹⁹. During our manuscript preparation, Hansmann et al³⁰ reported the crystal structure of TSC2 GAP domain, which consists of the central core but lacks the helix pair. The isolated GAP domain structure is similar to that in TSC complex and the proposed mechanism of GAP activity on Rheb is consistent with our independent studies.

Mutations of TSC genes have been frequently observed in tuberous sclerosis and cancers and missense mutations occurred throughout the protein sequences³¹ (Fig. 4a and Supplementary Fig. 4). Notably, most of cancer derived mutations in TSC2 are enriched on the central core module, supporting the pathological significance of TSC complex in these diseases. Furthermore, the mutations in the wing modules are predominantly enriched on the ridges of HEAT domains of TSC2, consistent with their roles in mediating TSC1-TSC2 interactions and TSC conformational stability^19,32−36.

The surface electrostatic calculations of TSC2 structure reveals four predominant positively charged patches around the DD (D-patch) and GAP (G-patch) domains. The four patches are located on the bottom surface of the central core and close to the putative Rheb-binding pockets of the two GAP domains, suggesting a regulatory role related to its GAP function (Fig. 4b, c). It is tempting to speculate that these positively charged patches may involve charge-charge interactions and associate with negatively charged phosphorylated residues and/or lipids.

It is well documented that residues S939, S981, and T1462 of TSC2 are phosphorylated by the AKT kinase and these modifications promote TSC2 translocation from lysosomal membrane to the cytosol via binding of 14-3-3 protein, and therefore inhibit GAP activity on Rheb and activate mTORC1^37,38. Residues T1271 and S1387 are phosphorylated by the AMPK kinase under energy deficiency, which leads to activation of TSC and inhibition of mTOR activity³⁹. The MK2 kinase phosphorylates S1210 and modulates its interaction with 14-3-3^40,41. Although these residues were invisible due to the lack of corresponding cryo-EM density, their nearest modeled residues, S937, R1245, and G1494, are located around these positively charged patches (Fig. 4c), which may recruit and stabilize these abovementioned phosphorylated residues.

Inactivation of mTORC1 requires TSC2 lysosomal localization⁴². The size of typical lysosome ranges from 100 nm to 1000 nm⁴³. The characteristic arch-shaped architecture of TSC spans ~ 40 nm, suggesting that TSC may packs against the surface of lysosome and fits the membrane curvature (Fig. 4d). It is well known that TSC2 is recruited to lysosome membrane through nonexclusive pathways, such as binding C181 farnesylated Rheb⁴⁴, Rag GTPases⁴⁵, and polycystin-1⁴⁶. Structural superimposition indicates that the farnesylated Rheb has no clash with the positive patches of TSC, supporting its co-localization with TSC on lysosomal surface (Fig. 4b and Supplementary Fig. 8f). Lipid phosphorylation has been known to regulate membrane localization of proteins⁴⁷. TSC complex may bind phosphorylated lipid on lysosome membrane via its positive patches through charge-charge interactions, providing an alternative approach for its lysosomal localization.

Reagents. Flag M2 affinity agarose gel was from Raygene; Mono Q and Superose 6 were from GE Healthcare; Polyethylenimine (PEI) was from polysciences (23966); HEK293 cells were from Invitrogen. Inc. and culture medium were from Sino Biological Inc. Antibodies against phosphorylated-S6K (Thr 389) was from Cell Signaling Technology; Flag-HRP (A8592) from Sigma, Horseradish peroxidase-labeled anti-mouse and anti-rabbit secondary antibodies was from AbMart.

Protein expression and purification. The ORFs of human TSC1, TSC2 and TBC1D7 were sub-cloned into three modified pCAG vectors. The three plasmids were co-transfected to suspension Expi293F cells using Polyethylenimine (PEI). After culture at 37 °C, 5% CO2 for 3 days, cells were collected and lysed in 50 mM HEPES (pH 7.4), 300 mM NaCl, 0.2% CHAPS, 5 mM MgCL₂, 5 mM ATP, 10 mM NaF and 3 mM DTT at 4 °C for 30 min, and the insoluble fraction was removed by centrifugation at 16,000 rpm for 30 min. Supernatants were incubated with FLAG-M2 monoclonal antibody-agarose for 4 hours and washed extensively. The fusion proteins (FLAG-tagged TSC1, Myc-tagged TSC2 and Myc-tagged TBC1D7) were digested using PreScission protease overnight and the eluted proteins were further purified using ion exchange and gel filtration chromatography. The peak fractions were pooled for gradient fixation (Grafix)⁴⁸. The gradient was generated from a 10% glycerol light solution [10% (v/v) glycerol, 300 mM NaCl, 25 mM HEPES (pH 7.4), 1 mM TCEP], and a 30% glycerol heavy solution [30% (v/v) glycerol, 300 mM NaCl, 25 mM HEPES (pH 7.4), 1 mM TCEP, and 0.1% (v/v) glutaraldehyde]. Centrifugation was performed at 38,000 r.p.m. in a SW41Ti swinging bucket rotor for 18 h at 4 °C using Beckman L-100XP. Subsequently, peak fractions were collected and quenched with 100 mM Tris-HCl (pH 8.0). The cross-linked TSC complex was concentrated and dialyzed to 0.5 mg/ml for Cryo-EM grids.

Sample preparation. For negative staining EM grids preparation, 5 µL of TSC complex sample was applied onto glow-discharged copper grids supported by a continuous thin layer of carbon film for 60 s before negatively stained by 2% (w/v) uranyl formate solution at room temperature. The grids were prepared in the Ar/O₂ mixture for 15 s using a Gatan 950 Solarus plasma cleaning system with a power of 35W. The negatively stained grids were loaded onto a Thermo Fisher Scientific Talos L120C microscope equipped with a Ceta CCD camera and operating at 120 kV at a nominal magnification of 92,000 x, corresponding to a pixel size of 1.58 Å on the specimen.

For cryo-EM grids preparation, 4 µL of the sample at a concentration of ~ 0.5 mg/mL TSC complex was applied to freshly glow-discharged Quantifoil R1.2/1.3 holey carbon grids. After incubation of 5 s at a temperature of 4 °C and a humidity of 100%, the grids were blotted for 4 ~ 6 s in a Thermo Fisher Scientific Vitrobot Mark IV and plunge-frozen in liquid ethane at liquid nitrogen temperature. The grids were prepared in the H₂/O₂ mixture for 60 s using a Gatan 950 Solarus plasma cleaning system with a power of 5W. The ø 55/20 mm blotting paper is made by TED PELLA used for plunge freezing.

Data collection. The cryo-EM grids of TSC were loaded onto a Thermo Fisher Scientific Titan Krios transmission electron microscope equipped with a Gatan GIF Quantum energy filter (slit width 20 eV) and operating at 300 kV for data collection. All the cryo-EM images were automatically recorded by a post-GIF Gatan K2 Summit direct electron detector in the super-resolution counting mode using Serial-EM⁴⁹ with a nominal magnification of 105,000 x in the EFTEM mode, which yielded a super-resolution pixel size of 0.678 Å on the image plane, and with a defocus ranged from 1.0 to 3.5 µm. Each micrograph stack was dose-fractionated to 32 frames with a total electron dose of ~ 50 e⁻/ Å² and a total exposure time of 11.49 s. For the first dataset of TSC sample, 3,316 micrographs from a total of 3,605 micrographs were selected for further processing. As for the second dataset of TSC sample, 1,381 micrographs from a total of 1,546 micrographs were selected for further processing.

Image processing. For cryo-EM data, drift and beam-induced motion correction were applied on the super-resolution movie stacks using MotionCor2⁵⁰ and binned two fold to a calibrated pixel size of 1.356 Å/pix. The defocus values were estimated by Gctf⁵¹ from summed images without dose weighting. Other procedures of cryo-EM data processing were performed within RELION v3.0^52,53 using the dose-weighted micrographs.

For the first datasets of the TSC, a subset of ~ 10,000 particles were picked by Gautomatch (Zhang, unpublished) without reference and subjected to reference-free 2D classification. Some of the resulting 2D class averages were low-pass filtered to 15 Å and used as references for automatic particle picking of the whole datasets in RELION resulting in an initial set of 1,073,891 particles for reference-free 2D classification. 510,614 particles were selected from good 2D classes for the initial 3D classification, using a 60 Å low-pass filtered initial model from our previous cryo-EM reconstruction. After several rounds of 2D and 3D classification, 152,396 particles were 3D auto-refined and post-processed, yielding a reconstruction of TSC complex at 5.11 Å resolution. Also, for the second dataset of the TSC complex, a subset of ~ 10,000 particles were picked by Gautomatch (Zhang, unpublished) without reference and subjected to reference-free 2D classification. Some of the resulting 2D class averages were low-pass filtered to 20 Å and used as references for automatic particle picking of the whole datasets in RELION resulting in an initial set of 455,091 particles for reference-free 2D classification. 244,896 particles were selected from good 2D classes for the initial 3D classification, using a 60 Å low-pass filtered initial model from our previous cryo-EM reconstruction. After several rounds of 2D and 3D classification, 71,265 particles were 3D auto-refined and post-processed, yielding a reconstruction of TSC complex at 5.22 Å resolution. According to these reconstructions, TSC^dataset1 and TSC^dataset2 are the same sample. Thus, two datasets were merged to improve the map quality. After several rounds of 2D and 3D classification, 131,022 particles were 3D auto-refined and post-processed, yielding a reconstruction at 4.4 Å resolution. We used a local mask 3D refinement for the wing-a, core and wing-b region, 131,022 particles were local refined and post-processed, yielding a 4.1 Å reconstruction of TSC wing-a region, a 3.6 Å reconstruction of TSC core region, and a 3.9 Å reconstruction of TSC wing-b region, respectively.

All reported resolutions are based on the gold-standard Fourier shell correlation (FSC) = 0.143 criterion. The GSFSC curves were corrected for the effects of a soft mask with high-resolution noise substitution. All cryo-EM maps were sharpened by applying a negative B-factor estimated during post-processing in RELION. All the visualization and evaluation of the 3D volume map were performed within UCSF Chimera or UCSF ChimeraX⁵⁴, and the local resolution variations were calculated using RELION⁵².

Model building and structure refinement. The cryo-EM maps of the TSC wing-a region complex at 4.1 Å resolution, the TSC core region complex at 3.6 Å resolution and the TSC wing-b region complex at 3.9 Å resolution were used for model fitting. The structures of TSC1-TBC1D7 (PDB: 5EJC) was used as initial structural templates, which were docked into the cryo-EM maps by rigid-body fitting using UCSF Chimera⁵⁴. The structural models were further manually built de novo in COOT⁵⁵ and refined in real space using Phenix⁵⁶ with secondary structure and geometry restraints using the cryo-EM map. Overfitting of the model was monitored by refining the model in one of the two half maps from the gold-standard refinement approach and testing the refined model against the other map⁵⁷. Statistics of the map reconstruction and model refinement can be found in Table 1. The final models were evaluated using MolProbity⁵⁸. Map and model representations in the figures and movies were prepared by PyMOL (http://www.pymol.org), UCSF Chimera or UCSF ChimeraX⁵⁹.

In vitro GAP assay. GTPase-activating activity was determined with a calorimetric assay⁶⁰ measuring the formation of inorganic phosphate. TSC complex (1 µM) and Rheb (3 µM) samples were added in buffer containing 25 mM Hepes (pH 7.4), 200 mM NaCL, 1 mM GTP in 50 µl reaction mixtures and incubated at 37° for 3 hours. Reactions were terminated by the addition of 100 µl of malachite green/acid molybdate solution. After 20 min of color development, OD620 was determined.

In vivo GAP assay. The 293A cells were transfected with Flag-S6K1, Flag-Rheb, Flag-TSC1, Myc-TBC1D7, Flag-TSC2 WT and mutants using PEI. After 48 h, the cells were collected and lysed for 30 min. The supernatant was collected by centrifuge and boiled with SDS loading buffer. The sample was conducted for western blotting. The primary antibody was incubated overnight and washed three times with TBST, and incubated with secondary antibody for 1 h. After extensive rinsing with TBST for three times, ECL was detected.

Acknowledgments

We thank Center of Cryo-Electron Microscopy, Fudan University, Center of Cryo-Electron Microscopy, Peking University, Center for Biological Imaging of Institute of Biophysics (IBP) of Chinese Academy of Sciences, and National Center for Protein Science Shanghai (NCPSS) for the supports on cryo-EM data collection and data analyses.

This work was supported by grants from the National key R&D program of China (2016YFA0500700), the National Natural Science Foundation of China (31770781, 31830107, 31821002, 31425008), the Shanghai Municipal Science and Technology Major Project (2017SHZDZX01), Shanghai Municipal Science and Technology Commission (19JC1411500), the National Ten-Thousand Talent Program, the National Program for support of Top-Notch Young Professionals, and the Strategic Priority Research Program of the Chinese Academy of Sciences (grant no. XDB08000000).

Author Contributions

H. Y. prepared the samples for structural and biochemical analyses with help from X. C., J. L., H. Y., D. Y., and K. G.; Z. Y. performed EM analyses and model building with the help from N. L., J. C., and N. G.; Y. X. wrote the manuscript with the help from H. Y. and K. G.; Y. X. supervised the project.

Competing interests

Authors declare no competing interests.

Data and materials availability

Cryo-EM maps and atomic coordinates will be deposited upon acceptance of the manuscript.

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Structural insights into TSC complex assembly and GAP activity on Rheb

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