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.
Introduction
Tuberous sclerosis complex (TSC) is an autosomal dominant disease characterised by benign tumours in multiple organs (Henske et al, 2016). It is caused by mutations in either of the genes TSC1 or TSC2, which encode the 130 kDa TSC1/hamartin and the 200 kDa TSC2/tuberin tumour suppressor proteins respectively. TSC1 contains an N-terminal α-helical ‘core’ domain and a coiled-coil at the C-terminus which is required for binding TSC2 (Nellist et al, 1999; Santiago Lima et al, 2014; van Slegtenhorst et al, 1998). TSC2 contains a long α-solenoid domain at the N-terminus and a C-terminal GTPase activating protein (GAP) domain, which is the sole catalytically active domain in the complex. Together with a small third subunit TBC1D7 (Dibble et al, 2012), TSC1 and TSC2 assemble to form the TSC protein complex (TSCC).
TSCC signalling restricts cell growth by negatively regulating mTORC1, the central coordinator of metabolism (González & Hall, 2017; Ramlaul & Aylett, 2018). Directly upstream of mTORC1, Rheb, a small GTPase localized to lysosomes through C-terminal farnesylation (Clark et al, 1997), stimulates mTORC1 kinase activity when GTP-bound (Yang et al, 2017). The TSCC stimulates Rheb GTPase activity, accumulating the GTPase in the inactive, GDP-bound, state to suppress mTORC1 (Huang & Manning, 2008). Spatial regulation of TSCC between the cytoplasm and lysosome is known to be pivotal for its function as a Rheb-GAP, with the current understanding being that TSCC translocates to the lysosome surface to catalytically and sterically inhibit mTORC1 by binding to Rheb and sequestering it (Menon et al, 2014; Demetriades et al, 2016). This translocation is reversed on TSC2 phosphorylation by AKT (Menon et al, 2014), and other kinases, which are thought to regulate localisation through an unknown mechanism involving 14-3-3 binding (Shumway et al, 2003; Cai et al, 2006), as well as TSCC breakdown by ubiquitination-targeted TSC2 degradation (Benvenuto et al, 2000; Chong-Kopera et al, 2006).
The architecture of the TSCC remains completely unknown, although small fragments of the complex have been structurally characterised. The core domain of S. pombe TSC1 (Sun et al, 2013), an N-terminal α-solenoid fragment of C. thermophilum TSC2 (Zech et al, 2016), and most recently the CtTSC2GAP domain (Hansmann et al, 2020) have been resolved crystallographically. Furthermore, two co-crystal structures of TBC1D7 interacting with C-terminal coiled-coil fragments of TSC1 have been determined (Gai et al, 2016; Qin et al, 2016). In this study, we have used cryogenic electron microscopy (cryo-EM) to examine the molecular architecture of the full-length, human holo-TSCC.
Results
Structure of TSCC
We cloned human TSC1, TSC2, and TBC1D7 for expression in human embryonic kidney cells, and Rheb for expression in Escherichia coli. A clone yielding an internal deletion of TSC1(Δ400-600) was used for SEC-MALLS to minimise inter-complex interactions. While both TBC1D7 and TSC1 could be expressed and purified independently, TSC2 could not be produced in the absence of TSC1, being degraded in cell (Supp. Fig. 1), consistent with the role of TSC1 in preventing TSC2 degradation (Benvenuto et al, 2000; Chong-Kopera et al, 2006).
We retrieved the complete human TSCC from cell lysate using FLAG-tagging, and purified the TSCC from most remaining contaminants by size-exclusion chromatography (SEC) (Fig. 1A). Purified TSCC exhibited its physiological GAP activity towards Rheb (Fig. 1B). SEC of the full-length TSCC yielded a broad peak with an estimated mass of 5,200 kDa, due to inter-TSCC lateral interactions (Supp. Fig. 2), whereas SEC at lower concentrations, or TSC1(Δ400-600) TSCC, yielded a defined peak with an estimated molecular weight by multi-angle LASER light scattering of 690-710 kDa (Fig. 1C), roughly corresponding to a composition of 2:2:2-TSC1:TSC2:TBC1D7.
We initially investigated the molecular architecture of the TSCC by negative staining. We observed extremely flexible, independent particles, however three defined ordered regions could be isolated, and two-dimensional averaging of these regions provided a complete picture (Fig. 1D). The TSCC was extraordinarily elongated (∼400 Å) and exhibited a characteristic “scorpion” shape, with a bulkier central “body”, flexible “pincer”-like appendage at one end, and a barbed “tail” at the other. Cryo-EM of TSCC at low concentrations revealed identical particles (Fig. 1E), whereas at higher concentrations we observed web-like networks which appear to be formed of head-to-tail TSCC particles (Supp. Fig. 2). Once again, complete TSCC particles were too flexible to average beyond low resolution (Fig. 2A). We isolated the same regions; both the “pincer” and “tail” proved to be strongly preferentially oriented and flexible, the “pincer” refining only to low resolution (11 Å), and the “tail” to low-intermediate resolution (9 Å) (Fig. 2A). The body of the TSCC exhibited pseudo-C2 symmetry, and was refined in C2 initially to 4.2 Å, before symmetry was relaxed for a final C1 structure at 4.5 Å (Fig 2A).
The resolution of the “body” was high enough to trace chains and identify all secondary structural elements, but too low to definitively assign sequence. The published structure of the TSC2 GAP domain (Hansmann et al, 2020) fitted unambiguously into density adjoining a central α-solenoid on each side of the origin of C2 pseudo-symmetry (Fig. 2A, Supp. Fig. 3). At the juncture of the two α-solenoids, we observed a dimerisation interface comprising two back-to-back β-sheets (Fig. 2A). With the exception of the GAP domain, the only β-elements remaining predicted within the sequence of any TSC protein are at the C-terminus of the α-solenoid of TSC2 (Supp. Fig. 3), which is consistent with the TSC2 α-solenoids running outwards from C-terminus to N-terminus from the dimerisation site. Because of the extensive regions of predicted unstructured sequence within this region, it is entirely possible that there are multiple topologies for the dimerisation interface and the two TSC2 molecules cannot be clearly delineated. The C2 symmetry of TSC2 is broken by two helices running directly across the top of the dimerisation site. This helical density forms a weakly connected “backbone” running over both GAP domains, and along the TSC2 α-solenoid outwards to both the “pincer” and “tail”. We assign this continuous helical coiled-coil as that from the C-terminal regions of TSC1, implying that the two ends are its N- and C-terminus respectively. The “pincer” density is uninterpretable, however the TSC1-TBC1D7 structure (Qin et al, 2016) unambiguously fits the density corresponding to the “barb” lying on the α-solenoid of the “tail” (Fig. 2A, Supp. Fig. 4), confirming the orientation of the TSC1 dimer, and implying that the “pincer” is made up of the TSC1 HEAT-domains.
Under the reasonable assumption that the GAP-Rheb interaction will mirror that of the published Rap-GAP complex (Scrima et al, 2008), we have docked Rheb accordingly. The natural docking yields no clashes with the current structure, and implies a further interaction with the two helices adjoining the GAP domain (Fig. 2B). We note that the Rheb farnesylation sites would both be situated on the same side of the TSCC, consistent with this being the correct orientation for lysosomal binding.
Tumorigenic mutations in TSC2
TSC2 is an important tumor suppressor protein, mutations of whose encoding gene are highly correlated with tuberous sclerosis. We examined the tumorigeic missense mutations of TSC2 (Table 1) frequently listed in the Tuberous sclerosis database (http://chromium.lovd.nl/LOVD2/TSC/home.php?select_db=TSC2). Half of these TSC2 residues with tumorigeic missense mutations (27 of 54 residues total) occur on the N-terminal α-solenoid domain, most of which clustering together (such as residues A84, P91, and E92; residues C244, M286, G294, E337, A357, and R367; residues G440, L448, A460, R462, L466, and L493; residues A583, H597, Y598, A607, R611, R622, and M649; residues L826, L847, R905, and L916; Suppl. Fig. 3A). Given that the N-terminal domain (NTD) of TSC2 is found to closely contact the GAP domain of TSC2 (Fig. 2), these mutation-clustered regions might represent the GAP-contacting sites on TSC2-NTD. In tsc2 mutant cancer cells harbouring these TSC2 missense mutations, the association between TSC2-NTD and TSC2-GAP might be disrupted, leading to diminished GAP activity toward Rheb and abnormally elevated mTORC1 activity.
In addition, the C-terminal GAP domain of TSC2 represents the region possessing the second highest number of tumorigeic missense-mutated amino acids, with more than 30% of all residues listed (17 of 54 in Table 1). A lot of these disease-related residues are highly conserved across a vast variety of organisms from human to yeast, such as H1620, G1642, N1643, and R1743 (Suppl. Fig. 3A). Presumably, these residues play key roles for TSC2-GAP’s enzymatic activity. Interestingly, H1620, G1642, and N1643 on TSC2-GAP correspond to H266, G289, and N290 of RapGAP, which directly contact or are very close to Rap in the Rap-RapGAP crystal structure (Scrima et al., 2008, Supp. Fig. 5). These residues might be responsible for interaction with Rheb, and their mutations in tuberous sclerosis patients could subvert the GAP activity of TSC2.
Discussion
We show that the TSCC forms an elongated, flexible architecture, comprising two copies of each of TSC1, TSC2, and TBC1D7. The orientation of Rheb implied by the GAP domains (Fig. 2B) matches the slight curvature of the complex, and the lysosomal membrane will therefore lie on the opposite side of the TSCC from the TSC1 backbone. The super-structure observed forming at higher concentrations (Supp. Fig. 2) may well play a part in retaining TSCC at the lysosome and reducing the off-rate once Rheb signalling has been suppressed as previously predicted (Menon et al, 2014). Further structural investigation of these inter-TSCC interactions, likely mediated by the TSC1 termini (Nellist et al, 1999; Hoogeveen-Westerveld et al, 2010), is required to understand the higher-order organisation of TSCC and its role in mTORC1 regulation.
The C2 symmetry of each of the dimeric TSC proteins is broken by the presence of the other, that of TSC1 by the curvature of its coiled-coil along the TSC2 α-solenoids, and that of TSC2 by the involvement of TSC1 in its dimerisation. We have confirmed once again that while TSC1 can fold independently of TSC2, the reverse does not occur (Benvenuto et al, 2000; Chong-Kopera et al, 2006; Woodford et al, 2017). Our architecture suggests a structural explanation for this observation, and indicating direct TSC1 involvement in the TSC2 dimerisation interface. The previously observed breakdown of TSC2, following ubiquitination in the absence of TSC1 (Benvenuto et al, 2000; Chong-Kopera et al, 2006), would be expected when structural elements cannot fold appropriately in the absence of their partner. This would also constrain the presence of functional TSC2 to subcellular regions containing TSC1 dimers.
In complete agreement with the conclusions of the Manning group (Menon et al, 2014), the presence of the TSCC will both catalytically and sterically prevent Rheb–mTORC1 interactions (Fig. 2B), rotating the Rheb pair in relation to its position when interacting with mTORC1. We believe that one of the most interesting observations from our results is that TSC2 binds Rheb as such a pair, as does mTORC1. Despite the fact that they are completely different in architecture and approach from different directions, the TSCC GAP domains are poised to bind two copies of Rheb at an almost identical separation to that resolved for Rheb in the structure of activated mTORC1 (Fig. 2B). While it is possible that this is entirely happenstance, this would also be expected were Rheb bound by each partner as part of a greater, at least dimeric, complex on the lysosomal surface.
Our improved architectural understanding of the TSCC provides a starting point for the investigation of the molecular mechanisms by which TSCC directly regulates Rheb, and poses new questions on the nature of the superstructures formed by TSCC complexes, their partners, and the involvement of such quaternary structures in mTORC1 regulation.
Materials and methods
Protein expression and purification
pRK7 plasmids subcloned with FLAG-tagged full-length (FL) human TSC1 (1164 amino acids, UniProtKB/Swiss-Prot accession number Q92574- 1) and FLAG-tagged FL human TSC2 (1807 amino acids, UniProtKB/Swiss-Prot accession number P49815- 1) were purchased from Addgene, and pRK7 was subcloned with FLAG-tagged human TBC1D7 (293 amino acids, GenBank accession number AAH07054). FL TSC1-TSC2-TBC1D7 (TSCCFL) plasmids, or TSC1Δ400-600-TSC2-TBC1D7 (TSCC1Δ) plasmids, were co-transfected into human embryonic kidney (HEK) Expi293F cells (Thermo Fisher Scientific, Waltham, MA, USA). Two days after transfection, the harvested Expi293F cells were lysed by three cycles of freeze-thaw in lysis buffer (20 mM Tris, pH 8.0, 300 mM NaCl, 2 mM TCEP, 0.5 mM PMSF, 1 μg/ml aprotinin, and 1 μg/ml leupeptin), and TSCC was purified from the cell lysate by M2 anti-FLAG affinity chromatography (Sigma) followed by size exclusion chromatography using a Superose 6 column (GE Healthcare) pre-equilibrated with buffer containing 20 mM Tris, pH 8.0, 300 mM NaCl, and 2 mM TCEP. The identity of each TSCC component was verified by ESI-MS (Mass Spectrometry Facility, University of St. Andrews).
The cDNA of residues 1-169 of human Rheb (184 amino acid isoform, GenBank accession number EAW53989) was subcloned into the pET28a vector (Novagen), with an N-terminal 6×His-tag. Rheb was overexpressed in E.coli strain BL21(DE3). After lysis of the bacteria with a cell homogenizer (JNBIO) and clarification, the lysate was subjected to Ni2+-NTA affinity chromatography (Qiagen) followed by size exclusion chromatography using a Superdex 200 column (GE Healthcare) pre-equilibrated with buffer containing 20 mM Tris, pH 8.0, 200 mM NaCl, and 2 mM DTT.
GTPase activity endpoint assay
The GTPase activity of Rheb was assayed using the QuantiChrom ATPase/GTPase assay kit (BioAssay Systems), in which the amount of released inorganic phosphate was measured through a chromogenic reaction with malachite green. In the assays 75 nM RheB, either alone or mixed with 227.5 nM TSCCFL, were added to the reaction buffer (40 mM Tris, pH 8.0, 80 mM NaCl, 8 mM MgOAc, 1 mM EDTA and 14 mM GTP) at 28 °C for 40 min. A further 200 μL of assay kit reagent was then added, and the reaction was incubated for 20 min, before a microplate reading at OD 620 nm was measured. Spontaneous GTP hydrolysis was calculated by measuring background absorbance in the absence of Rheb and sample values were normalised by subtraction of the background. Each experiment was repeated three times.
Size exclusion chromatography−multi-angle laser light scattering (SEC-MALLS)
Both full length TSCC and TSCC1Δ (consisting of TSC1 deleting residues 400-600, TSC2, and TBC1D7) were analysed by SEC-MALLS using an Infinity liquid chromatography system (Agilent Technologies), linked to a Dawn Heleos multi-angle light scattering detector (Wyatt Technology) and Optilab T-rEX refractive index detector (Wyatt Technology). The sample was injected onto a Superose 6 10/300 size exclusion column (GE Healthcare) pre-equilibrated overnight with buffer containing 25 mM K-HEPES, pH 7.6, 250 mM KCl, 0.5 mM EDTA, 1 mM TCEP and trace amounts of NaN3, using 0.2 mL/min flow rate at room temperature. In-line UV absorbance, light scattering and refractive index measurements were analysed using the ASTRA software package (Wyatt Technology) to determine molar mass estimates.
Sample preparation for cryo-EM studies
TSCC, after the above purification steps, was loaded onto a Superose 6 10/300 size exclusion chromatography column (GE Healthcare) pre-equilibrated with a preparation buffer containing 25 mM K-HEPES, pH 7.6, 175 mM KCl or 150 mM LiCl, 1 mM TCEP, and 0.5 µM EDTA. TSCC eluted as a single peak with a slight shoulder at lower retention volume. The integrity of the complex was confirmed by SDS-PAGE of both the peak and shoulder fractions. Main peak fractions were combined and concentrated to 0.1–0.2 mg/mL using Amicon 100 kDa molecular weight cut-off (MWCO) centrifugal filters and used for grid preparation.
Generation of an initial TSCC reference density
A sample of concentrated wild-type TSCCFL was applied to a carbon-coated holey carbon grid (R1.2/1.3, Quantifoil) and stained with 2% (w/v) uranyl acetate. A total of 224 micrographs were collected using an FEI Tecnai T12 electron microscope (Thermo Fisher Scientific, Waltham, MA, USA) at a magnification of 81,000-fold, an acceleration voltage of 120 kV, and a total dose of 50 e- /Å2 over a defocus range of −0.5 to −2.0 μm. A dataset of 9 597 particles was selected manually using BOXER. The parameters of the contrast transfer function were determined using CTFFIND4. Particles were 2D-classified into 100 classes in two dimensions using RELION and sixteen well-defined classes were selected for initial three-dimensional reconstruction. Initial models were created using the initial model functions in EMAN2, refined in three dimensions at low resolution using SPIDER, then filtered to 60 Å and used as an initial reference for automatic refinement in RELION. The resulting initial model at a resolution of 26 Å, with independent volume Fourier Shell Correlation (FSC) of 0.143, was used for further refinement.
TSCC cryo-EM sample preparation
Samples of concentrated TSCCFL were adsorbed to a thin film of graphene oxide deposited upon the surface of holey carbon copper grids (R2/1, 300 mesh, Quantifoil). Grids were blotted for 2-3 seconds before plunge freezing in liquid ethane using a Vitrobot Mark IV (Thermo Fisher Scientific, Waltham, MA, USA) at 4 °C and 100% humidity.
TSCC cryo-EM data collection
Data were collected for TSCC on a Titan Krios (Thermo Fisher Scientific, Waltham, MA, USA) at the Electron Bioimaging Centre (eBIC, Diamond Light Source), equipped with a K2 Summit direct electron detector (GATAN, San Diego, USA) and operated at 300 kV, 37000-fold magnification and with an applied defocus range of −0.75 to −3.25 μm. Frames were recorded automatically using EPU, resulting in 5387 images of 3838 by 3710 pixels with a pixel size of 1.35 Å on the object scale. Images were recorded in two successive datasets (of 1880 and 3507 images, respectively) as either 40 or 60 separate frames in electron counting mode, comprising a total exposure of 52.3 or 80.2 e-Å-2, respectively.
TSCC cryo-EM data processing
Frames were aligned, summed and weighted by dose according to the method of Grant and Grigorieff using MotionCor2 (Zheng et al, 2017) to obtain a final image. Poor-quality micrographs were rejected based on diminished estimated maximum resolution on CTF estimation using CTFFIND4 (Rohou & Grigorieff, 2015) and visually based on irregularity of the observed Thon rings. Particles were selected using BATCHBOXER (Tang et al, 2007), and refinement thereafter performed using RELION3 (Scheres, 2012; Zivanov et al, 2018).
Two-dimensional reference-free alignment was performed on ∼1,500,000 initially boxed particles to exclude those that did not yield high-resolution class averages and to identify the principal ordered regions of the TSCC molecule. Of these, ∼300,000 showed resolution extending to high-resolution and were retained for further refinement.
TSCC proved to be highly preferentially oriented on the grid, however it was possible to identify 2D classes for each of the “body”, “pincer” and “tail” regions. Iterated re-centring, two-dimensional refinement, and re-boxing using the neural network particle picker Topaz (Bepler et al, 2018) was performed from the “body” region outwards in order to maximise recovery of the “pincer” and “tail” views and provide a complete description and definitive topology for all three regions.
Particles belonging to the “body” frequently displayed C2 symmetry in 2D class averages, and this particle subset was refined in three dimensions using this symmetry restraint. After several iterations of re-picking particles using Topaz (Bepler et al, 2018) and refinement, the final gold-standard refinement of the “body”, including 59 524 particles, reached 4.2 Å at an independent FSC =0.143. The symmetry was subsequently relaxed to C1 and refined (gold-standard) to 4.6 Å resolution at an independent FSC = 0.143. Reconstructions were also performed for the “pincer” and “tail” regions, however these suffered from persistent highly preferred orientation and conformational flexibility, with gold-standard refinements reaching 11 Å and 9 Å resolution, respectively, at an independent FSC = 0.143.
Author contributions
G.W. and C.H.S.A. conceived of the project. W.F., K.R., and H.L. purified the protein, performed initial negative staining analysis, and carried out the GAP assay. K.R. and N.M.G. carried out and analysed SEC-MALLS experiments. C.H.S.A., K.R., and W.F. prepared samples and grids for cryo-EM, collected electron micrographs and calculated single-particle reconstructions. All authors were involved in the interpretation of results and the drafting of the manuscript.
Competing financial interests
The authors declare that they have no competing financial interests.
Data and materials availability
The cryo-EM density map corresponding {###} of the HsTSCC complex has been deposited in the EM Databank under accession codes EMD-{####}.
Acknowledgements
The authors would like to thank Xinqiu Guo at Instrumental Analysis Centre of Shanghai Jiao Tong University for assistance with electron microscopy experiments. G.W. thanks Dr. Sheng Wang, Dr. Xinqi Gong, Lijun Yan, Hongpeng Wang, and Ailiang He for their contribution. We are indebted to the Imperial Centre for Structural Biology and the UK national electron Bio-imaging Centre at Diamond Light Source (proposal EM14769, funded by the Wellcome Trust, MRC and BBSRC) for access to electron microscopy equipment and in particular to Paul Simpson for technical support. The authors would also like to thank Marc Morgan for help with SEC-MALLS analysis, the St. Andrews Proteomics facility for mass spectrometric protein identification and Nadezhda Aleksandrova for GTPase assay preparation. G.W. is supported by National Natural Science Foundation of China (grant numbers 31670106 and 31872627), Shanghai Jiao Tong University Scientific and Technological Innovation Fund, and the National Key R&D Program of China (YS2020YFA090044). C.H.S.A. is supported by a Sir Henry Dale Fellowship jointly funded by the Wellcome Trust and the Royal Society (206212/Z/17/Z).