Structural Basis of the Interaction between Tuberous Sclerosis Complex 1 (TSC1) and Tre2-Bub2-Cdc16 Domain Family Member 7 (TBC1D7)

Abstract

Mutations in TSC1 or TSC2 cause tuberous sclerosis complex (TSC), an autosomal dominant disorder characterized by the occurrence of benign tumors in various vital organs and tissues. TSC1 and TSC2, the TSC1 and TSC2 gene products, form the TSC protein complex that senses specific cellular growth conditions to control mTORC1 signaling. TBC1D7 is the third subunit of the TSC complex, and helps to stabilize the TSC1-TSC2 complex through its direct interaction with TSC1. Homozygous inactivation of TBC1D7 causes intellectual disability and megaencephaly. Here we report the crystal structure of a TSC1-TBC1D7 complex and biochemical characterization of the TSC1-TBC1D7 interaction. TBC1D7 interacts with the C-terminal region of the predicted coiled-coil domain of TSC1. The TSC1-TBC1D7 interface is largely hydrophobic, involving the α4 helix of TBC1D7. Each TBC1D7 molecule interacts simultaneously with two parallel TSC1 helices from two TSC1 molecules, suggesting that TBC1D7 may stabilize the TSC complex by tethering the C-terminal ends of two TSC1 coiled-coils.

Keywords: TBC1D7; TSC1; crystal structure; isothermal titration calorimetry (ITC); mTOR complex (mTORC); protein complex; tuberous sclerosis complex (TSC).

FIGURE 1.

Biochemical characterization of the TSC1-TBC1D7 interaction. a, mapping of the TBC1D7 binding site on TSC1 by GST pulldown assay. Human TSC1(939–992) is sufficient for TBC1D7 binding. The bands showing TBC1D7(19–293) pulled down are marked with red stars. The molecular mass markers above and below the GST-TSC1 and TBC1D7 bands are 45 and 31 kDa, respectively. b, ITC analysis. Homodimerization of TSC1(939–992) promoted by GST fusion enhances its interaction with TBC1D7. The flexible N-terminal tail of TBC1D7 is dispensable for the TSC1(939–992)-TBC1D7 interaction. c, SEC-MALS characterization of TSC1 fragments. The molar ratio of TSC1-TBC1D7 complexes appears to be 2:1. TSC1 fragments instead of TBC1D7 were added in excess for a better separation from the complex peak. All experiments in this figure were performed at least twice. All results were highly reproducible.

FIGURE 2.

Overall structure of a TSC1-TBC1D7 complex. Two orthogonal schematic views of TSC1-TBC1D7 are shown. TBC1D7 and two TSC1 molecules are colored in cyan, pink, and yellow, respectively.

FIGURE 3.

Details of the TSC1-TBC1D7 interface. a, electrostatic potential of the surface of TBC1D7. The core TSC1-binding surface of TBC1D7 is largely hydrophobic. b, hydrophobic interactions stabilizing the TSC1-TBC1D7 interaction. c, polar interactions on the “front” of the TSC1-TBC1D7 interface. D, polar interactions on the “back” of the TSC1-TBC1D7 interface. For clarity, the front interactions and hydrophobic interactions are not shown. Key interface residues (TSC1: Ile⁹⁵⁴ and Phe⁹⁵⁸ in the TSC1-A helix, Ile⁹⁶²′ and Tyr⁹⁶⁶′ in the TSC1-B helix; TBC1D7: Val⁹⁴, Val⁹⁵, Arg⁸¹, Gln⁸⁴, and Arg⁹⁶) are marked with red ovals.

FIGURE 4.

Sequence alignment of TSC1 and TBC1D7 from different organisms. Conserved residues are boxed in red and similar residues are highlighted in red. Residues are framed in blue if more than 70% of its residues are similar according to physicochemical properties. For TBC1D7, helices are marked with squiggles, turns with TT letters, and interface residues with red stars. For the two TSC1 helices interacting with TBC1D7, red stars in the upper row represent TSC1-A residues involved in TBC1D7 interaction, red hashtags in the lower row indicate TSC1-B residues interacting with TBC1D7. For counting convenience, small black dots are added in every 10 residues on top of sequences.

FIGURE 5.

GST pulldown analysis of the TSC1-TBC1D7 interaction. a and b, in vitro GST pulldown using purified proteins. SDS-PAGE gels were stained with Coomassie Brilliant Blue. Three independent GST pulldown experiments were performed, and all results were highly reproducible. a, wild-type GST-TBC1D7(1–293) pulls down untagged wild-type TSC1(939–992) and TSC1(939–992) mutants. b, wild-type GST-TBC1D7(1–293) and GST-TBC1D7(1–293) mutants pull down untagged wild-type TSC1(939–992). c and d, semi-quantification of the in vitro GST pulldown analysis. Purified wt/mutant TSC1 and TBC1D7 proteins were used for the pulldown analysis and the SDS-PAGE gels were stained using Oriole Fluorescent Gel Stain (Bio-Rad). A plot of relative densitometry values of the TSC1 is shown. Error bars indicate the mean ± 1 S.D. from three separate experiments.

FIGURE 6.

IP analysis of the TSC1-TBC1D7 interaction. C-terminal myc-tagged wild-type TSC1, N-terminal HA-tagged wild-type TBC1D7, or the TSC1 and TBC1D7 variants were coexpressed with TSC2 in HEK 293T cells. TSC complexes were isolated by IP, either with anti-myc affinity beads (specific for exogenous TSC1) or anti-HA affinity beads (specific for exogenous TBC1D7). Four separate transfection experiments were performed. In 3 experiments, IPs were washed 4 times with 20 volumes of lysis buffer. All 3 experiments gave very similar results. In a subsequent experiment, IPs were washed 4 times with lysis buffer and twice with lysis buffer containing 1 m NaCl. a and b, immunoblots showing co-IP of TSC2 and the TBC1D7 variants with immunoprecipitated TSC1. Immunoprecipitates were either washed with lysis buffer only (a), or with high salt buffer (b). c and d, immunoblots showing co-IP of TSC2 and TSC1 with immunoprecipitated TBC1D7. Immunoprecipitates were washed with lysis buffer only (c) or high salt lysis buffer (d), as in A and B. e-h, quantification of the signals on the immunoblots from 3 separate co-IP experiments (no high salt washes). The relative signals of the co-immunoprecipitated proteins are shown relative to the wild-type control, after adjusting for the signal of the immunoprecipitated protein (either TBC1D7 or TSC1). Error bars correspond to the mean ± S.E.; values significantly reduced compared with the wild-type controls (p < 0.05 Student's paired t test) are indicated with an asterisk. e, co-IP of TBC1D7 with TSC1. f, co-IP of TSC2 with TSC1. g, co-IP of TSC1 with TBC1D7. H, co-IP of TSC2 with TBC1D7.

FIGURE 7.

AUC analysis of the TSC1-TBC1D7 interaction. TSC1, TBC1D7, and mixtures of TBC1D7 and TSC1 with two different molar ratios were measured by analytical ultracentrifugation analysis using the sedimentation velocity method. TSC1 and TBC1D7 alone formed monomers. When TSC1 was in excess, the TSC1-TBC1D7 complex formed predominantly a 1:2 heterotrimer. In contrast, when TBC1D7 was in excess, 2:2 heterotetramers were also detected. All AUC measurements were performed at least twice, and all results were highly reproducible.

FIGURE 8.

Structural comparison of the TSC1-TBC1D7 complex with a TBC-domain/Rab-GTPase complex. a, the TSC1-TBC1D7 complex structure superimposed with that of a Rab-TBC complex between Rab33 and the TBC domain of Gyp1p (PDB code 2G77). Colors of the individual molecules are indicated. b, a close-up view of the GAP active site. GDP-aluminum fluoride, which mimics the transition state of GTP, and the two catalytic residues (Arg³⁴³ and Gln³⁷⁸) are shown for the Rab-TBC complex. TBC1D7 lacks these two critical residues and has a very different structure to other TBC domains.