, Stephan Kiontke , Uwe Mueller§, Andrea Oeckinghaus ... · by Rag GTPases upon amino acid starvation (20). In contrast, insulin-dependent AKT phosphorylation of TSC2 at S939 and

Crystal structure of the TSC2 N-terminus

1

Structure of the tuberous sclerosis complex 2 (TSC2) N-terminus provides insight into complex assembly

and tuberous sclerosis pathogenesis

Reinhard Zech‡, Stephan Kiontke‡, Uwe Mueller§, Andrea Oeckinghaus¶ & Daniel Kümmel‡

From the ‡Structural Biology Section, FB5 Biology/Chemistry, University of Osnabrück, 49076

Osnabrück, Germany, §Macromolecular Crystallography (BESSY-MX), Helmholtz Zentrum Berlin für

Materialien und Energie, 12489 Berlin, Germany, and ¶Institute of Molecular Tumor Biology (IMTB),

Medical Faculty of the WWU Münster, 48149 Münster Germany

Running title: Crystal structure of the TSC2 N-terminus

To whom correspondence should be addressed: Dr. Daniel Kümmel, FB5 Biology/Chemistry, University

of Osnabrück, Barbarastr. 13, 49076 Osnabrück, Germany; Telephone: +49 541 9693423; FAX: +40 541

9692884; Email: [email protected]

Keywords: protein structure, X-ray crystallography, protein-protein interaction, signaling, tuberous

sclerosis complex (TSC), tuberin, hamartin

ABSTRACT

Tuberous sclerosis complex (TSC) is caused by

mutations in the TSC1 and TSC2 tumor suppressor

genes. The gene products hamartin and tuberin

form the TSC complex that acts as GTPase

activating protein for Rheb and negatively

regulates the mammalian target of rapamycin

complex 1 (mTORC1). Tuberin contains a

RapGAP homology domain responsible for

inactivation of Rheb, but functions of other protein

domains remain elusive. Here we show that the

TSC2 N-terminus interacts with the TSC1 C-

terminus to mediate complex formation. The

structure of the TSC2 N-terminal domain from

Chaetomium thermophilum and a homology model

of the human tuberin N-terminus are presented.

We characterize the molecular requirements for

TSC1-TSC2 interactions and analyze pathological

point mutations in tuberin. Many mutations are

structural and produce improperly folded protein,

explaining their effect in pathology, but we

identify one point mutant that abrogates complex

formation without affecting protein structure. We

provide the first structural information on

TSC2/tuberin with novel insight into the molecular

function.

INTRODUCTION

In eukaryotic cells growth factor signaling is

responsible to adjust proliferation and metabolism

to environmental and developmental cues. Growth

factor receptors regulate, among other targets,

activation of the mTORC1 (mammalian target of

rapamycin) kinase complex, considered the master

regulator of cellular growth (1). This pathway

involves the protein kinase B/AKT, the TSC

(tuberous sclerosis) complex and the small GTPase

Rheb (2). Under resting conditions, Rheb is kept

inactive by TSC, which represents the cognate

GTPase activating protein (GAP) (3). Rheb

undergoes the classical GTPase conversion and

cycles between an active GTP-bound and an

inactive GDP-bound state. Active Rheb is required

for full activation of mTORC1 at the lysosomal

membrane (4). Upon stimulation of growth factor

signaling and the downstream kinase AKT, TSC

gets inactivated by phosphorylation, thus leading

to increasing levels of active Rheb and mTORC1.

The TSC complex consists of the 130 kDa

subunit hamartin (encoded by TSC1) (5), the 200

kDa protein tuberin (TSC2 gene product) (6) and

the recently identified TBC1D7 (7, 8), which form

high oligomeric assemblies in cells (9) and have a

similar architecture as the RalGAP complexes (10,

11). Tuberin contains a RapGAP-like domain

which catalyzes the hydrolysis of Rheb●GTP to

Rheb●GDP (12). Besides the GAP domain,

hamartin and tuberin show no sequence homology

to other known proteins, but the N-terminus of

tuberin has been shown to mediate interaction with

harmartin (13–16). TBC1D7 is a RabGAP domain

containing protein with specificity for Rab17

http://www.jbc.org/cgi/doi/10.1074/jbc.M116.732446The latest version is at JBC Papers in Press. Published on August 4, 2016 as Manuscript M116.732446

Copyright 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

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involved in trafficking from endosomes to cilia

(17). In the context of the TSC complex, TBC1D7

stabilizes the hamartin dimerization, the interaction

with tuberin and increases the GAP activity of the

complex (7, 18, 19). Activity of TSC is in part

regulated by its recruitment to Rheb on the

lysosome, which has been reported to be promoted

by Rag GTPases upon amino acid starvation (20).

In contrast, insulin-dependent AKT

phosphorylation of TSC2 at S939 and S981 is

responsible for interactions between TSC2 and 14-

3-3 scaffold proteins, leading to a translocation of

the TSC complex from the lysosomal membrane to

the cytosol and thus physically away from its

target Rheb (21, 22). Structural information on the

TSC complex is limited, but recently, the crystal

structure of the N-terminal HEAT repeat domain

of TSC1 from Schizosaccharomyces pombe

(TSC1-N) has been reported (23). TSC1-N forms a

dimer in solution and a decameric assembly in the

crystal, suggesting an involvement in

oligomerization of TSC. Furthermore, TSC1-N has

been proposed to interact with membranes. In

addition, the complex structure of TBC1D7 with a

short coiled-coil fragment of hamartin revealed

how TBC1D7 stabilizes hamartin dimerization (18,

19).

Mutations in the TSC1 and TSC2 genes lead to

tuberous sclerosis (TSC), a genetic disorder that

affects 0.01-0.02% of all births and is

characterized by the formation of benign tumors in

skin, brain, heart, kidneys and multiple other

organs (24). Further clinical manifestations are

epilepsy, learning difficulties and behavioral

problems (25). Mutations that cause TSC are

spread over the entire sequences of both TSC1 and

TSC2 (26–29), but mechanistic understanding of

the functional consequences is precluded by the

lack of biochemical and structural information.

Also, the diverse spectrum of pathological

phenotypes depending on different mutations

remains elusive.

Orthologues of TSC1 and TSC2 exist in a range

of eukaryotes (30), including the thermophilic

fungus Chaetomium thermophilum, which we used

for recombinant production of TSC proteins. These

proteins show increased solubility and stability as

compared to mammalian homologues and are thus

better suited for structural studies (31). We find

that the TSC2 N-terminus (TSC2-N) forms a

HEAT repeat domain and interacts with the C-

terminal half of TSC1 (TSC1-C). The structure

helps to explain the effect of pathogenic missense

mutations in tuberin (we will use TSC1/2 and

hamartin/tuberin to distinguish between C.

thermophilum and human proteins in the

following), and we identify a point mutation that

specifically blocks TSC complex formation

without destroying protein folding. Our structural

and biochemical characterization provides

important molecular insight into TSC complex

function and also represents a step forward in

understanding tuberous sclerosis pathogenesis.

RESULTS

TSC2 N-terminus interacts with TSC1 C-

terminus - Initial trials for recombinant expression

of hamartin and tuberin in bacteria revealed

problems with protein solubility and stability of

the tested constructs. We then turned to TSC1 and

TSC2 from the thermophilic fungus Chaetomium

thermophilum which have an identical domain

organization as their mammalian counterparts (Fig.

1a) and show sequence conservation over the

entire length (Fig. S-1). Studies on CtTSC1 and

CtTSC2 can therefore serve as a model for

hamatrin and tuberin.

In our efforts to characterize the structure and

assembly of the TSC complex, we focused on the

N-terminus of TSC2/tuberin which had been

implicated in mediating the interaction with

hamartin (13, 14, 16). We initially expressed a

construct comprising residues 70-800 of TSC2 that

was designed based on primary sequence analysis

(32). In a limited proteolysis experiment with this

protein (Fig. 2a), we identified a stable and soluble

proteolytic fragment of TSC2 comprising residues

70-530 (referred to as TSC2-N in the following,

Fig. 1a,). When co-expressed recombinantly in E.

coli (Fig. 1b), a stable complex of full-length

TSC1 (TSC1-fl, residues 1-820) and TSC2-N

could be co-purified with affinity chromatography,

showing that TSC2-N confers interaction with

TSC1. In contrast, no complex between TSC1-N

(1-415) and TSC2-N was observed. Note that

much more TSC2-N can be purified if no TSC1 is

bound, likely due to reduced solubility of the

complex compared to the isolated protein. We also

observe that TSC1-fl, which is sensitive to C-

terminal degradation when expressed alone, is

stabilized by co-expression with TSC2-N. This is

indirect evidence for TSC2-N binding to the C-

terminus of TSC1.


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Because with recombinant complex purification

we were limited in the choice of fragments by the

insolubility of some constructs, we further mapped

the TSC1-TSC2 interaction in a cell culture

system. HEK293 cells were transiently transfected

with Flag-TSC1 and HA-TSC2 constructs. Both

full-length TSC2 (TSC2-fl) and TSC2N 70-530

bound to TSC1-fl with comparable efficiency (Fig.

1c). This confirms that the identified fragment

represents the TSC1 interaction domain of TSC2.

We next determined the interaction domain in

TSC1 required for TSC2 association (Fig. 1d).

Like hamartin, TSC1 from C. thermophilum

contains a predicted N-terminal HEAT repeat

domain and a C-terminal coiled-coil domain which

are connected by a putative disordered linker. No

interaction was detected between TSC1-N and full-

length TSC2, but TSC1-C with and without the

linker region (415-820 and 501-820, respectively)

bound robustly. Additionally, we performed co-

immunoprecipitations with human tuberin and

hamartin and also observe that the N-terminus of

tuberin and the C-terminus of hamartin are

sufficient for complex formation (Fig. 1e). Thus,

the interactions mapped are conserved for C.

thermophilum proteins and the human

homologues, suggesting identical complex

architectures.

Crystal structure of TSC2 N-terminus (TSC2-N)

- The mapped TSC1-binding domain TSC2-N

expressed well in E. coli and could be purified,

yielding homogenous monomeric protein (Fig. 2b).

We obtained crystals that diffracted anisotropically

to 2.3 Å, and selenomethionine-substituted crystals

were grown for phase determination. The model

was build and refined against native data to 2.5 Å

(Table 1). The asymmetric unit contains four

molecules with only small structural variations:

molecules A and B show an rmsd of 0.016 Å, C

and D of 0.014 Å; and the rmsd between A or B

versus C or D is 1.14 Å. The final models contain

residues 88-527 with variable loop regions missing

in the individual copies of TSC2-N due to the

differential packing. Combining the information

from the different copies of the asymmetric unit

we could generate a composite model lacking only

the loop regions 232-242, 436-438, and 467-474

(Fig. 2c,d). TSC2-N forms an α-solenoid,

consisting of 19 helices that are arranged in nine

HEAT repeats (HR) and an additional C-terminal

helix. Two subdomains can be defined that are

separated by a short loop between HR 6 and 7,

leading to a 75° tilt of the repeats with respect to

each other. HR 4 and 9 are interrupted by

extensive loop sequences, the latter one making

crystal packing contact. Search with the Dali

server (33) revealed the homology to other HEAT

repeat proteins (Fig. 2e), including importin α3 (z-

score 18.0, rmsd 10 Å), a karyopherin involved in

transport through the nuclear pore complex (34)

and the core domain of TSC1 (z-score 11.1, rmsd

7.5 Å) (23), and the armadillo (ARM)-repeat

protein HspBP1 (z-score 14.2, rmsd 3.0 Å) (35).

Homology modeling of the tuberin N-terminus

and analysis of pathogenic mutations - The

structure of TSC2-N from C. thermophilum can

serve as model to study the function of its human

homologue tuberin. We performed a multiple

sequence alignment with TSC2 from C.

thermophilum, S. pombe, D. melanogaster and H.

sapiens (Fig. 3a, Supplementary Fig. 1) The

sequence identity between TSC2-N and tuberin-N

is only ~18%, but a strong similarity can be

detected on the level of secondary structure. A

prediction for the N-terminus of tuberin (32, 36)

shows that - like the TSC2-N domain from C.

thermophilum – it is probably composed of 19 α-

helices. In a multiple sequence alignment, the

predicted helices in tuberin N-terminus (tuberin-N,

residues 40-415) match very well with the

secondary structure elements observed in TSC2-N

(Fig. 3a). Based on this, we generated a model for

tuberin-N with SWISS-MODEL/DeepView (37,

38) (Fig. 3b,c). Tuberin-N was also modeled with

nine HEAT repeats and an extra C-terminal helix,

but lacking the extensive loop regions in HR 4 and

9.

Membrane interaction of TSC2-N - Active TSC

is located on lysosomes, but its mechanism of

membrane recruitment remains elusive. We

generated an electrostatic potential map (39) of

TSC2-N to identify potential membrane interaction

interfaces, which often are represented by

positively charged patches (Fig. 4a). Charges on

TSC2-N are rather evenly distributed, with only

one slightly basic patch at helix α14 that is also

present at an equivalent position in tuberin-N (Fig

4b). We did not observe a specific interaction of

TSC2-N with a particular lipid in a protein lipid

overlay assay (Fig. 4c). However, in a liposome

sedimentation assay, TSC2-N bound specifically to

a vacuolar lipid mix, but not to liposomes

composed of neutral lipids (Fig. 4d, e). The

vacuolar SNARE Vam7, which binds to


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phosphoinositol-3-phosphate (PI(3)P) via a PX

domain, served as a positive control, and TSC2-N

binding to liposomes was inefficient in

comparison. This indicates that TSC2-N has a

weak affinity for negatively charged membranes.

Analysis of the TSC2-N surface - To identify

putative functionally important surface patches on

TSC2-N we mapped conserved residues on the

structure (Fig. 5a). Most conserved residues are

located in the interior of the protein and involved

in stabilizing the domain fold. In addition, an

accumulation of conserved residues can be found

at the base of HEAT repeats 3-5, highlighting a

potential functional interface (Fig. 5a). To test if

this interface might represent the binding site for

TSC1 on TSC2-N we designed three sets of

mutations of conserved surface residues (set 1:

K263E, F264A, F307A; set2: N194A, D260A;

set3: P159T, R166A). There was no negative

influence on the overall structure and stability of

these mutants in comparison to wild-type TSC2-N

detectable by circular dichroism spectroscopy (CD

Fig. 5b) or differential scanning fluorimetry (DSF,

Fig. 5c). The co-purification of TSC1 was

impaired with the set 3 mutant (Fig. 5 d, e),

suggesting that these residues play a role in

mediating the contact between TSC1 and TSC2.

Characterization of pathogenic point mutations

in tuberin-N - More than 2200 unique mutations in

the TSC2 gene are reported in the Tuberous

sclerosis database of the Leiden Open Variation

Database (LOVD,

http://chromium.lovd.nl/LOVD2/TSC) (29). We

searched the database for missense point mutations

reported or concluded as pathogenic or probably

pathogenic and mapping to the region covered by

our structure. Deletion, insertion, frame shifts and

stop mutations that likely lead to misfolded protein

were excluded. The search yielded a list of 33

mutations (Table 2), which can be mapped on the

model of tuberin-N and classified by their likely

effect on protein structure (Fig. 6a). Six of the

identified mutations (shown in green) do map to

the surface of tuberin-N and should not affect

folding. Such mutations can be suspected to

interfere with molecular functions of tuberin, like

its interaction with hamartin. In contrast, eleven

changes (labeled red) are of residues located in α-

helices to proline. Such substitutions will be helix

breaking and might destroy the secondary structure

and, leading to destabilized or misfolded protein.

Another class of structural mutants could affect

intramolecular packing (labeled yellow): exchange

of residues that constitute the hydrophobic core of

tuberin-N to amino acids with side chains of

different shape or polarity also leads to

destabilization and/or partial unfolding of the

domain. Although amino acid changes in the

protein core can be tolerated to some extent, we

suggest that the disease-causing effect of these

structural mutations could be explained by

improper folding of tuberin-N and, consequently,

dysfunctional protein.

To support our classification of pathogenic

mutations, we generated substitutions in C.

thermophilum TSC2-N equivalent to the

pathogenic surface mutations in tuberin-N (Table

2). These include the surface mutants L122G,

S341W, R361A, R420A, and

V464P/T465A/L466A. In addition to V464 to

proline, corresponding to Q373 in tuberin, we

changed the subsequent highly conserved residues

T465 and L466 to alanine because they might also

be functionally important. We also tested a subset

seven mutations (L192R, N265I, S341P, G381E,

A411P, L428R, L497P) that were classified as

structural and are conserved between C.

thermophilum and humans. Five of the tested

structural mutants were insoluble, aggregated or

degraded during purification. (Table 2). The

variants L192R and N265I could be purified but

showed reduced stability and a lower melting point

in DSF (Fig. 6b). In contrast, the melting

temperature of all surface mutants was comparable

to wild-type TSC2-N (Fig. 6b) and CD spectra also

did not indicate any severe structural defects (Fig.

6c), suggesting that their structures are unaffected

by the mutations.

Because a structural defect of the surface

mutants likely is not the cause their pathogenicity,

we asked if binding to TSC1 might be impaired.

Using our co-purification assay, we tested these

mutants for their ability to interact with TSC1-fl

(Fig. 6d, e). Indeed, L122G showed a binding

defect, but the remaining mutants bound TSC1 like

wild-type.

To confirm that the human pathogenic point

mutation E75G - which is equivalent to L122G in

C. thermophilum TSC2 - indeed disrupts the

complex we tested binding of hamartin to tuberin

in co-immunoprecipitation studies (Fig. 7a, b). The

interaction of tuberin E75G to hamartin was

impaired when compared to the wild-type protein.

Also, the mutation R261P, which we had classified


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as structural and where we showed that the

equivalent substitution S341P destroys TSC2-N

structure, had reduced interaction with hamartin. In

contrast, the surface mutation tuberin R261W did

not affect TSC1 association, consistent with the

results for the corresponding mutant S341W in C.

thermophilum TSC2.

Thus, despite their low sequence identity, the

interaction site between TSC1 and TSC2 is

conserved for tuberin and hamartin. When we map

the location of mutations that affect TSC complex

assembly in the structure of TSC2-N, strikingly,

both the pathogenic mutation and set 3 residues are

found in the same region of TSC2-N at the base of

HEAT repeats 1 to 3 (Fig. 7c). Thus, we identified

the binding site for TSC1 on TSC2, and pinpoint a

pathogenic surface mutation to this region.

DISCUSSION

The crystallographic analysis of the N-terminal

domain of TSC2 from C. thermophilum provides

the first structural information of the TSC2 subunit

of the TSC complex. Our work reveals that this

domain adopts a HEAT repeat fold that also served

as a template to generate a model of the N-

terminus of human tuberin.

In the active state, TSC has to be recruited to its

substrate Rheb onto the lysosomal membrane. A

possible targeting mechanism is the interaction

with specific membrane lipids. In the case of

lysosomes, phosphoinositol-3,5-bisphosphate

(PI(3,5)P2) is considered a marker lipid and PH

(pleckstrin homology) domains have been reported

as PI(3,5)P2-binding module (40, 41). TSC2-N

does not show structural homology to PH domains

but might represent a novel PI(3,5)P2 interaction

domain. We therefore performed a protein lipid

overlay assays with TSC2-N but could not detect

any specific interaction of TSC2-N with a panel of

15 different lipid species (Fig. 4c). However,

TSC2-N bound weakly to lipososmes of a vacuolar

lipid composition containing 2 mol% PI(3)P, 1

mol% PI(3,5)P2, and 4.4 mol% PS. We identified

basic patches at equivalent positions on TSC2-N

and the model of tuberin-N, and this region could

mediate the interaction with positively charged

membranes. However, since the interaction is

rather weak and the domain does not show

specificity for an organelle marker lipid we expect

that additional factors will play a role in membrane

recruitment of the TSC complex. Lipid specificity

would likely be mediated by a different domain of

TSC, or membrane targeting could involve

protein-protein interactions with a receptor on the

lysosomal surface, which warrants further

investigation. In this scenario, TSC2-N membrane

binding could contribute to lysosomal attachment,

which likely requires multiple TSC domains.

We further determined that TSC2-N represents

an interaction domain for the assembly of the TSC

complex. In both co-immunoprecipitation from

cell culture and co-expression studies in E. coli,

TSC2-N was sufficient to form a stable complex

with TSC1. Vice versa, the C-terminal domain, but

not the N-terminus of TSC1, bound to TSC2,

demonstrating that TSC1-C and TSC2-N represent

the key domains responsible for TSC assembly.

Importantly, we confirmed the same interaction

domains in the human orthologues hamartin (C-

terminus) and tuberin (N-terminus). Consistent

with our findings, previous studies with tuberin

and hamartin using yeast two-hybrid analysis (13,

14) also suggested that N-terminal regions of

TSC2 are involved in binding TSC1. There was,

however, discrepancy over which elements of

hamartin mediate complex assembly, suggesting

either the C-terminus (13), a fragment from the N-

terminal half of hamartin (302-430) (14), or

binding sites in both regions (16). Our study,

probing for direct interactions and reconstituting a

TSC subcomplex, shows that TSC1-C and

hamartin-C are sufficient to form a complex with

TSC2/tuberin (Fig. 1). We were unable to detect

any stable interaction between TSC2-N and TSC1-

N in co-immunoprecipitation, co-expression or

reconstitution experiments with individually

purified proteins. However, since the TSC

complex has been described as a higher oligomeric

assembly, it may be stabilized by additional

secondary interaction sites. Because our

experiments were done at expression levels above

the endogenous concentrations, these additional

sites may be dispensable in vitro but could be of

relevance in a physiological setting.

Based on our structure of TSC2-N and the

model of tuberin-N, we ought to analyze

pathogenic TSC mutations that map to this region.

We classified mutations that map to the interior of

the protein or proline mutations in secondary

structure elements as structural and suggest that

their pathogenicity might be explained by reduced

protein stability or folding defects. We confirmed

for seven positions that are conserved in TSC2 and

tuberin that protein stability of these mutants is


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indeed impaired. Some conservative substitutions

in the protein core might be tolerated, and we

found for the core residue mutation L320F (L403F

in C. thermophilum) described as polymorphism

(42), that the structure was indeed unaffected (Fig.

6c). However, our findings back the notion that for

patient mutations in the protein core, the

pathogenicity could be explained by problems with

protein structure. Further support comes from the

finding that the structural mutation R261P in

tuberin also showed a functional defect and

impaired binding to hamartin in co-

immunoprecipitation assays.

In contrast, surface mutations on TSC2 did not

affect protein structure. Their pathogenicity could

therefore be caused by impaired molecular

functions, like TSC1 binding. The pathogenic

mutation L122G does not affect protein structure

but specifically leads to loss of TSC1 binding,

while other mutations (e.g. S341W) had no effect.

The equivalent mutations in tuberin, E75G and

R261W, respectively, had the same differential

effect on complex assembly (Fig. 7a).

Consistently, a functional assessment of TSC2

variants had shown that mTORC1 activity is

elevated for the mutation E75G, but comparable to

wild-type for R261W (27, 28).

Surprisingly, the residue of the pathogenic

mutation E75G in the human protein is not

conserved and the chemical character of the

equivalent position L122 is different in C.

thermophilum. The fact that we nevertheless

observe that the effect on complex assembly is

preserved could be explained by the mutation to

glycine, which does not affect the overall structure

but could locally destabilize the loop region with

detrimental consequences on TSC1 binding.

Previously, pathogenic and non-pathogenic

mutations in tuberin had been tested for their

influence on the interaction with hamartin (14).

The pathogenic mutations G294E and the in-frame

deletion I365del led to loss of binding, and our

tuberin model suggests that these changes likely

produce unfolded protein and therefore have an

indirect effect. G294 is located in helix α14

pointing towards the interior of the protein, and

substitution with glutamate will disrupt the fold.

I365 is in the middle of helix α17 and part of the

hydrophopic core. Its deletion will result in a

destabilizing shift in helix register. The non-

pathogenic mutations R261W, M286V, and

R367Q were reported as not effecting hamartin

binding and, consistently, are surface exposed

residues in our model. R261W was described to

not cause tuberous sclerosis (43) but was identified

in patients with pulmonary

lymphangioleiomyomatosis (LAM) (44). Since the

mutation R261W is located on the surface of

tuberin, does not affect recruitment of hamartin

(Fig. 7a, (14)) and does not alter mTORC1 activity

(27), it could play a role in the interaction with an

unknown ligand that is involved in the

development of LAM.

We also identified a patch on the surface of

TSC2-N that is required for interaction with TSC1

based on sequence conservation. This patch of set

3 mutations - like L122G - maps to the linker

regions between HEAT repeats 1/2 and 2/3. The

residues are located within 15 Å, identifying this

area as one molecular surface that mediates the

interaction with TSC1. However, because the

interacting part of TSC1 is predicted to form a

likely extended coiled-coil structure, it is possible

that the actual interface is rather extensive.

Taken together, we conclude that our tuberin-N

model is fundamentally correct and we find that

the insight obtained from the mutational studies of

C. thermophilum proteins is highly relevant to

understand pathological mutations in the human

proteins. Structural studies on tuberin and hamartin

have been problematic in the past. Working with

proteins from C. thermophilum proves to be a

useful approach to obtain structural information on

these medically relevant proteins from a better

suited organism. While further investigations will

be required, our results clearly demonstrate that

structural studies on the TSC complex can guide

characterizing the diverse and variable spectrum of

symptoms observed in tuberous sclerosis.

EXPERIMENTAL PROCEDURES

Cloning and Mutagenesis - Constructs of TSC1

and TSC2 were amplified from codon optimized

synthetic genes (Genscript, USA) using Q5

Polymerase (NEB). DNA fragments were cloned

into a modified pCDF (Novagen, USA) vector for

expression with an N-terminal GST (Gluthathion-

S-Transferase) tag and PreScission proteases

cleavage site or a modified pET28 (Novagen,

USA) with N-terminal His6-SUMO (small

ubiquitin-like modifier) tag. For cell culture

expression, pCDNA3 vectors with 3xFLAG or HA

tag were used. Mutations were generated

according to the megaprime protocol (45).


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Interaction studies - For mammalian

expression, constructs were cloned into pCDNA3

vectors with 3xFLAG or HA tag and introduced in

HEK293 (human embryonic kidney 293) cells

through calcium phosphate-mediated transfection.

After 24h hours, transfected HEK293 cells were

lysed in CoIP buffer (25 mM HEPES pH7.5, 150

mM NaCl, 0.2 % NP-40, 10 % glycerol, 1 mM

DTT [dithiothreitol] plus protease inhibitors) and

protein complexes precipitated for 4 hours with

anti-flag antibody (M2, Sigma, USA) and protein

A/G agarose. Immunopreciptitates and lysate

samples were run on SDS-PAGE and proteins

were detected after immunoblotting with anti-flag

(M2, Sigma, USA) or anti-HA (Y-11, Santa Cruz,

USA) antibodies.

To probe for complex formation of

recombinant proteins, His6-SUMO-TSC1-fl (full-

length) and GST-TSC2-N wild-type or mutants

were co-expressed in E.coli Rosetta. Cells were

lysed in lysis buffer and the soluble supernatant

was incubated with GSH (glutathione)-agarose

(Thermo scientific). After washing, complexes

were eluted off the beads by the addition of SUMO

protease and PreScission. Equal amounts of eluted

TSC2-N were loaded on SDS-PAGE gels and

proteins visualized with coomassie staining.

Western blots and coomassie stained gels were

scanned with an Odyssey system (LI-COR) and

quantified with Image Studio Light. Significance

analysis was performed by t-tests (* p ≤ 0.05, ** p

≤ 0.01, *** p ≤ 0.001).

Protein Expression and Purification - Proteins

were expressed alone or in combination in E. coli

Rosetta (DE3) (Novagen, USA) after cold shock

overnight at 16°C. Selenomethionine substituted

TSC2-N was produced with the same conditions

using feedback inhibition of methionine synthesis

(46). Cells were resuspended in lysis buffer (50

mM NaH2PO4, pH 7.5, 300-500 mM NaCl, 10 mM

imidazole) supplemented with EDTA free protease

inhibitor, DNAse and Lysozyme 0.3 mg mL-1

and

lysis was performed with a microfluidizer

(M110L, Microfluidics) or sonication (Branson

Sonifier 250). His-tagged proteins were captured

via gravity flow chromatography using Ni-NTA

resin, GST-tagged proteins were purified by

applying supernatant to glutathione super flow

agarose (Thermo scientific). The matrix was

washed with 100 mL lysis buffer. Protein bound to

Ni-NTA agarose were additionally washed with

lysis buffer containing increasing imidazole

concentration (20 and 50 mM). GST-tagged

proteins were incubated overnight at 4°C with

PreScission-, His6-SUMO-tagged proteins with

SUMO proteases to cleave off the tags. Target

proteins were eluted and concentrated for gel

filtration chromatography. TSC2-N was purified

with a Superdex 200 HiLoad 16/600 column (GE

Healthcare, USA) equilibrated with gelfiltration

buffer (25 mM HEPES, 500 mM NaCl, 1 mM

TCEP, 5 % glycerol, pH 7.0) and peak fractions

were pooled.

Limited Proteolysis - The construct TSC2 70-

800 (0.8 mg mL-1

) was incubated with trypsin at

different ratios (1:1600; 1:800; 1:400; 1:160; 1:80;

1:40 w/w) in 25 mM HEPES, 300 mM NaCl, 1

mM DTT, pH 7.0 and 5% glycerol for 1 h at 37°C.

The reaction was stopped by adding SDS loading

dye and boiling the samples for 3 minutes, and

samples were analyzed via SDS-PAGE. A stable

degradation fragment was cut out of the gel,

destained and digested with trypsin (sequencing

grade, Promega) overnight at 37°C. Peptides were

identified with the Mascot search engine tool after

applying HPLC and ESI-ETD-Ion trap analysis

(Bruker).

Crystallization and Structure Determination -

Initial crystallization conditions were identified

with sitting drop vapor diffusion experiments using

a semi-automated dispensing system (Gryphon,

Art Robinson, USA). Best crystals were obtained

after several rounds of microseeding (47) with a

protein concentration of 6 mg mL-1

and 100 mM

Tris (pH 7.0), 14 % PEG 6000, 15 % glycerol, 200

mM MgCl2 as reservoir solution at 20°C.

Selenomethionine (SeMet) derivative crystals were

also obtained by micro seeding. Prior to data

collection, crystals were briefly soaked in a cryo-

protection condition containing 24 % instead of 14

% PEG 6000 and flash cooled in liquid nitrogen.

A native dataset of TSC2-N was measured at

ESRF ID23-1 (Grenoble, France) and anomalous

data was collected at BESSY II BL14.1

(Helmholtz-Zentrum Berlin, Germany) (48). The

collected data was processed using XDSAPP (49,

50) and SCALA (51). Initial phases were

determined with Phenix/AutoSol (52), followed by

automated model building using ARP/wARP (53).

The final model was obtained through iterative

cycles of model building in COOT (54) and

refinement against native data using REFMAC5

(55, 56). Structure factors and the final model have

been deposited to the protein data base (PDBID:


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5HIU). A homology model of the human tuberin

N-terminus was generated based on a ClustalW

(57) sequence alignment with a DeepView project

(38) in SWISS-MODEL (37). All images of

molecular structures were created using PyMOL

(58).

PIP Strip assay - GST-tagged TSC2-N as well

as GST alone was expressed and purified like

described above, but eluted with 10 mL lysis

buffer including 20 mM GSH. PIP MicroStrips P-

M600 (Echelon, USA) were first blocked with

PBS (phosphate buffered saline) including 3 %

BSA for 1 h and then incubated with the target

proteins (1 µg mL-1

) in PBS with 3 % BSA.

Subsequently, the MicroStrips were washed

extensively in PBS containing 0.1 % Tween-20

and then incubated for 1 h with a mouse anti-GST

antibody (1:1000; G1160 Sigma Aldrich, USA) in

blocking solution. After washing, the MicroStrips

were incubated with a goat anti-mouse 680

antibody (1:15000; Invitrogen) for 1 h and washed

again. Bound protein was detected with an

Odyssey infrared imager (LI-COR).

Liposome sedimentation assay - Liposomes

were generated from neutral (dioleoyl-

phosphatidyl-choline, DOPC 81.2 mol%; dioleoyl-

phosphatidyl-ethanolamine, DOPE 18 mol%, N-

(7-nitro-2-1,3-benzoxadiazol-4-yl)-PE, NBD-PE

0.8 mol%) or vacuolar (59) (DOPC 43.2 mol%;

DOPE 18 mol%; dioleoyl-phosphatidyl-serine,

DOPS 4.4 mol%; dioleoyl-phosphatidic acid,

DOPA 2 mol%; soy phosphatidyl-inositol, PI 18

mol%; cardiolipin 1.6 mol%; ergosterol 8 mol%;

diacylglycerol, DAG 1 mol%; NBD-PE 0.8 mol%;

PI(3)P 2 mol%; PI(3,5)P2 1 mol%) lipid mixtures.

Lipids were dried overnight with a desiccator and

dissolved in 1 mL working buffer (20 mM PIPES

pH 8, 120 mM KCl and 5% sucrose (w/v)), so that

the final lipid concentration was 2 mM. The

liposome suspension was freeze/thawed for 10

times in liquid nitrogen and at 50°C, respectivly.

To co-pellet protein with liposomes, 0.75 mM

vacuolar liposomes and 1.5 µM of either TSC2-N

or Vam7 were added in 150 µL working buffer

without sucrose. After 10 minutes incubation at

room temperature the liposomes were spun down

at 20,000 g for 20 minutes at 4°C. Subsequently,

samples were kept on ice and the supernatant

(soluble fraction) was carefully separated from the

pellet (membrane fraction). Samples were TCA

precipitated and analyzed via SDS-PAGE and

subsequent coomassie staining.

Circular dichroism spectroscopy - Purified

TSC2-N and respective mutants were run on a

Superdex 200 10/300 column (GE Healthcare,

USA) equilibrated with PBS. Peak fractions were

collected and used for measurements at

concentrations of 0.1-0.35 mg mL-1

. Spectra were

recorded with a JASCO J-810 spectrophotometer

in a 1 mm path length cuvette at room temperature.


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Acknowledgements: We are grateful to Saskia Schuback and Nadine Lottmann for excellent technical

support and the Ungermann lab for help and suggestions. We thank the staff at beamline ID23-1 at the

ESRF and BL 14.1 at BESSY II for assistance during data collection, the HZB for the allocation of

synchrotron radiation beamtime and acknowledge the financial support by HZB. This work was supported

by grants from the DFG to DK (KU 2531/2-1, SFB 944-P17) and AO (OE 531/2-1).

Competing interests: The authors declare that no competing interests exist.

Author contributions: RZ performed protein purification, biochemistry, crystallization and structure

determination with help from DK and SK; UM collected and processed data; AO performed cell culture

experiments; DK designed experiments, analyzed data and wrote the paper.

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FOOTNOTES

We acknowledge the HZB for the allocation and financial support of synchrotron radiation beamtime.

This work was supported by grants from the DFG to D.K. (KU 2531/2-1).

Abbreviations used are: TSC, tuberous sclerosis complex; mTOR, mammalian target of rapamycin; GSH,

glutathione; GST, Glutathione-S-Transferase; DTT, dithiothreitol; HEK293, human embryonic kidney

293 cell; IP, immunoprecipitation; WB, western blotting; PD, pull-down; PBS, phosphate-buffered saline;

SeMet, Selenomethionine; SUMO, small ubiquitin-like modifier;


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FIGURE LEGENDS

FIGURE 1. Interaction mapping of the TSC complex. (a) Schematic representation of the domain

architecture of TSC1 and TSC2 from Chaetomium thermophilum and human hamartin and tuberin. (b)

Reconstitution of the complex between TSC1 and TSC2-N with recombinant proteins. His6-SUMO (HS)-

TSC1 and GST-TSC2 were co-expressed in E. coli and purified via GSH (glutathione) affinity

chromatography. TSC2-N binds TSC1-fl, but not TSC1-N. Note that TSC1 is partially degraded when

expressed alone (marked by an asterisk), and yield of TSC2-N is reduced when purified in a complex. (c)

The N-terminus of TSC2 is sufficient for binding to TSC1. Full-length Flag-tagged TSC1 was co-

transfected with HA-TSC2 constructs and their interaction probed by co-immunoprecipitation and

Western blot analysis. (d) TSC2 binds to the C-terminus of TSC1. Flag-TSC1 full-length and truncations

were co-transfected with HA-TSC2 and binding was probed by co-immunoprecipitation and Western blot

analysis. (e) Tuberin (tub) N-terminus binds the hamartin (ham) C-terminus. Flag-hamartin C-terminal

half was co-transfected with HA-tuberin full-length and N-terminus and binding was probed by co-

immunoprecipitation and Western blot analysis.

FIGURE 2. Structure of TSC2-N. (a) Limited proteolysis of TSC2 70-800 with trypsin led to the

identification of a stable fragment, marked by an asterisk. (b) TSC2-N (70-530) was loaded onto a

Superdex 200 HiLoad 16/600 column equilibrated with 25 mM HEPES, 500 mM NaCl, 1 mM TCEP, pH

7.0 and 5 % glycerol. The protein elutes as a monomer at 77 ml, corresponding to an apparent molecular

weight of 67 kDa. (c) Top’ (upper panel) and ‘side’ view (lower panel) of the TSC2 N-terminal domain.

Nine HEAT repeats (HR) and an additional C-terminal helix α19 are labeled. The structure can be divided

into two subdomains, colored in light and dark cyan, respectively. (d) Representative 2FO-FC electron

density map at 2σ contour level of chain A HEAT repeat 2 (residues 124-159). (e) Structurally related

proteins identified by a Dali search (33) in rainbow color code from N- (blue) to C-terminus (red):

importin α3 (PDBID: 4UAE), TSC1-N (PDBID: 4KK1) and HspBP1 (PDBID: 1XQR).

FIGURE 3. Modelling of the tuberin N-terminus. (a) A multiple sequence alignment of the N-terminal

region of TSC2 from C. thermophilum, S. pombe, D. melanogaster and humans was generated with

ClustalW (57) and visualized with ESPript (60). Similar and conserved positions are marked by yellow

and red boxes, respectively. Secondary structure elements of TSC2-N are labeled and shown above the

alignment. Below the sequence of human tuberin-N, secondary structure is indicated as predicted by the

PSIPRED server (32). Residues mutated in this study are marked by triangles (surface residues), asterisks

(structural mutations) or numbers (for the corresponding set mutations). (b) Based on the X-ray structure

of TSC2-N from Chaetomium thermophilum, (c) a model for the N-terminal domain of human tuberin was

created. The structures are rainbow color-coded from from N- (blue) to C-terminus (red).

FIGURE 4. Membrane binding properties of TSC2-N. (a) Surface potential map of TSC2-N generated

with the APBS tool in Pymol (39, 58) and contoured at -10 kT/e to 10 kT/e level (red to blue) and (b) of

the tuberin-N model in the same orientations for comparison. The circles mark basic patches at equivalent

positions on both proteins. (c) For a protein lipid overlay assay, GST-TSC2-N fusion protein and GST

alone as control were incubated with PIP MicroStrips. Binding of protein to specific lipids was probed by

decorating with an α-GST antibody. (32, 36). LPS: Lysophosphatidic Acid, LPC: Lysophosphocholine,

PI: Phosphatidylinositol, PI(3)P: Phosphatidylinositol-3-phosphate, PI(4)P: Phosphatidylinositol-4-

phosphate, PI(5)P: Phosphatidylinositol-5-phosphate, PE: Phosphatidylethanolamine, PC:

Phosphatidylcholine, S1P: Sphingosine-1-phosphate, PI(3,4)P2: Phosphatidylinositol-3,4-bisphosphate,

PI(3,5)P2: Phosphatidylinositol-3,5-bisphosphate, PI(4,5)P2: Phosphatidylinositol-4,5-bisphosphate,

PI(3,4,5)P3: Phosphatidylinositol-3,4,5-triphosphate, PA: Phosphatidic Acid, PS: Phosphatidylserine. (d)

Representative vesicle sedimentation assay of the vacuolar SNARE Vam7 and TSC2-N with liposomes

from a vacuolar or neutral lipid mix, and without liposomes as control. (e) Quantification of the liposome

sedimentation assay from three biological repeats.


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FIGURE 5. Analysis of conserved patches on the TSC2 surface. (a) Mapping of conserved residues on

TSC2-N. Conserved and similar residues are highlighted in red and yellow, respectively, as in figure 3a.

Mutated residues are labeled set 1-3. (b) Protein folding of the conserved patch mutants compared to wild-

type TSC2-N is not affected as monitored by circular dichroism spectroscopy. (c) Stability of the

conserved patch mutations in comparison to wild-type TSC2-N was tested by determining the melting

temperature with differential scanning fluorimetry. (d) Representative SDS-PAGE of complex formation

analysis between TSC1-fl and TSC2-N by purification of the recombinant complex. TSC2-N wild-type

and indicated mutants were co-expressed with TSC1-fl and purified via GSH affinity chromatography. (e)

Quantification of the complex purification assay from three biological repeats.

FIGURE 6. Role of pathogenic mutations on TSC2-N functionality. (a) Pathogenic missense mutations

mapped onto the model of tuberin-N. Mutated residues are shown with spheres and are colored according

to their structural classification: red mutations are helix breaking, yellow mutations affect intramolecular

packing and green mutations are surface exposed residues. (b) Stability of selected pathogenic mutations

in comparison to wild-type TSC2-N was tested by determining the melting temperature with differential

scanning fluorimetry. Surface mutants are of solvent exposed residues, whereas structural mutants are of

residues involved in intramolecular interactions. (c) Protein folding of the pathogenic surface mutants

compared to wild-type TSC2-N is not affected as monitored by circular dichroism spectroscopy. (d)

Representative complex formation assay between TSC1-fl and TSC2-N by purification of the recombinant

complex. TSC2-N wild-type and indicated mutants were co-expressed with TSC1-fl and purified via GSH

affinity pull-down. (e) Quantification of the complex purification assay from three biological repeats.

FIGURE 7. Conserved interaction interface on TSC2-N. (a) Co-immunoprecipitation of tuberin-N point

mutations with the C-terminus of hamartin. The pathogenic surface mutation E75G (equivalent L122G in

CtTSC2) and the structural mutation R261P (equivalent to S341P) show impaired interaction, whereas

R261W (equivalent to S341W) binding is comparable to wild-type. (b) Quantification of the co-

immunoprecipitation experiment from three biological repeats (c) Residues that affect TSC1 binding

(colored magenta) map to the base of HR 2 and 3 in TSC2-N. The locations of mutations that showed no

effect in our assay are shown in lime green.


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TABLE 1. Data processing and refinement statistics for the structure of TSC2-N.

TSC2-N

(5HIU)

SeMet TSC2-N

(peak)

Space group P1 P1

Wavelength [Å] 0.97626 0.97977

Unit cell a [Å] 42.7 43.7

b [Å] 78.1 155.0

c [Å] 153.8 78.8

α [°] 89.990 90.0

β [°] 89.9 101.4

γ [°] 78.9 90.1

Resolution [Å] * 41.9 -2.3 (2.42-2.3) 43.0-2.5 (2.64-2.5)

Unique reflections *# 83,768 (12,118) 69,157 (10,004)

Multiplicity * 3.6 (3.5) 3.9 (3.8)

I/σ * 21.8 (2.7) 12.7 (2.9)

Completeness [%] * 96.6 (95.8) 98.5 (97.2)

Rmeas [%] * 3.5 (61.1) 5.6 (42.0)

Wilson B factor [Å²] 58.4 51.9

Refinement [Å] 41.9 - 2.5

Reflections 62038

Rwork / Rfree [%] 22.3/26.2

rmsd bond distances [Å] 0.008

rmsd bond angles [°] 1.224

mean B value [Å²] 88.5

Ramachandran diagram [%]

most favored 92.4

additionally allowed 7.3

* values in parenthesis refer to outer shell of reflections


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TABLE 2. Pathogenic point mutations in the tuberin N-terminus.

Mutation

tuberinN

Position

CtTSC2N

Structural

classification

Biophysical

characterization

Arg 57 His Ser 104 Surface exposed residue

Glu 75 Gly Leu 122 Surface exposed residue soluble, stable

Val 126 Phe Thr 172 Intramolecular packing

Leu 146 Arg Leu 192 Intramolecular packing soluble, instable

Ala 161 Val Phe 207 Intramolecular packing

Leu 183 Trp Val 261 Intramolecular packing

Asn 187 Ile Asn 265 Intramolecular packing soluble, instable

Met 198 Thr Leu 277 Intramolecular packing

Leu 219 Pro Ile 298 Helix breaking


Cys 244 Tyr Ser 232 Intramolecular packing

Thr 246 Ala Ile 325 Intramolecular packing

Arg 261 Trp Ser 341 Surface exposed residue soluble, stable

Arg 261 Pro Ser 341 Helix breaking aggregated

Leu 264 Pro Cys 344 Helix breaking

His 278 Pro Asp 358 Helix breaking

Glu 281 Asp Arg361 Surface exposed residue soluble, stable

Leu 292 Pro Val 379 Intramolecular packing

Gly 294 Glu Gly 381 Intramolecular packing degraded

Val 299 Gly Leu 386 Intramolecular packing

Ala 328 Pro Ala 411 Helix breaking insoluble

Val 334 Gly Ser 416 Intramolecular packing

Glu 337 Lys Arg 420 Surface exposed residue soluble, stable

Leu 340 Pro Thr 423 Helix breaking

Leu 345 Arg Leu 428 Intramolecular packing insoluble


Leu 362 Pro Phe 453 Helix breaking

Arg 367 Pro Gln 458 Helix breaking

Gln 373 Pro Val 464 Surface exposed residue soluble, stable

Val 384 Pro Ala 480 Helix breaking

Leu 387 Pro Leu 497 Helix breaking insoluble

Tyr407 Asp Cys 518 Intramolecular packing

Leu 410 Arg Phe 521 Intramolecular packing


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Figure 1


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Figure 2


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Figure 3


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Figure 4


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Figure 5


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Figure 6


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Figure 7


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Reinhard Zech, Stephan Kiontke, Uwe Mueller, Andrea Oeckinghaus and Daniel Kümmelinto Complex Assembly and Tuberous Sclerosis Pathogenesis

Structure of the Tuberous Sclerosis Complex 2 (TSC2) N-terminus Provides Insight

published online August 4, 2016J. Biol. Chem.

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