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- PDB-9c9i: Structure of the TSC1:WIPI3 complex -

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Basic information

Entry
Database: PDB / ID: 9c9i
TitleStructure of the TSC1:WIPI3 complex
Components
  • Hamartin
  • WD repeat domain phosphoinositide-interacting protein 3
KeywordsGENE REGULATION / TSC / Tuberous sclerosis complex / TSC1 / TSC2 / TBC1D7 / WIPI3 / end-some / MTOR / cell growth
Function / homology
Function and homology information


memory T cell differentiation / TSC1-TSC2 complex binding / TSC1-TSC2 complex / Inhibition of TSC complex formation by PKB / cellular response to decreased oxygen levels / glycophagy / nucleophagy / regulation of cell-matrix adhesion / protein localization to phagophore assembly site / phagophore assembly site membrane ...memory T cell differentiation / TSC1-TSC2 complex binding / TSC1-TSC2 complex / Inhibition of TSC complex formation by PKB / cellular response to decreased oxygen levels / glycophagy / nucleophagy / regulation of cell-matrix adhesion / protein localization to phagophore assembly site / phagophore assembly site membrane / cardiac muscle cell differentiation / cell projection organization / negative regulation of ATP-dependent activity / Energy dependent regulation of mTOR by LKB1-AMPK / ATPase inhibitor activity / autophagy of mitochondrion / pexophagy / negative regulation of cell size / phosphatidylinositol-3-phosphate binding / regulation of stress fiber assembly / activation of GTPase activity / phagophore assembly site / phosphatidylinositol-3,5-bisphosphate binding / negative regulation of TOR signaling / TBC/RABGAPs / protein folding chaperone complex / negative regulation of macroautophagy / Macroautophagy / D-glucose import / associative learning / autophagosome assembly / positive regulation of focal adhesion assembly / negative regulation of TORC1 signaling / lipid droplet / myelination / Hsp70 protein binding / protein folding chaperone / adult locomotory behavior / hippocampus development / cellular response to starvation / cell-matrix adhesion / kidney development / TP53 Regulates Metabolic Genes / response to insulin / neural tube closure / Hsp90 protein binding / synapse organization / potassium ion transport / cerebral cortex development / lamellipodium / protein-folding chaperone binding / cell cortex / protein-macromolecule adaptor activity / adaptive immune response / lysosome / cell population proliferation / regulation of cell cycle / postsynaptic density / protein stabilization / ciliary basal body / lysosomal membrane / negative regulation of cell population proliferation / perinuclear region of cytoplasm / protein-containing complex / nucleus / membrane / plasma membrane / cytosol / cytoplasm
Similarity search - Function
Hamartin / Hamartin protein / : / PROPPIN / WD40 repeats / WD40 repeat / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily
Similarity search - Domain/homology
WD repeat domain phosphoinositide-interacting protein 3 / Hamartin
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.18 Å
AuthorsD'Andrea, L. / Bayly-Jones, C. / Lupton, C.J. / Ellisdon, A.M.
Funding support United States, 1items
OrganizationGrant numberCountry
Department of Defense (DOD, United States)TS180061 United States
CitationJournal: Sci Adv / Year: 2024
Title: Structure of the human TSC:WIPI3 lysosomal recruitment complex.
Authors: Charles Bayly-Jones / Christopher J Lupton / Laura D'Andrea / Yong-Gang Chang / Gareth D Jones / Joel R Steele / Hari Venugopal / Ralf B Schittenhelm / Michelle L Halls / Andrew M Ellisdon /
Abstract: Tuberous sclerosis complex (TSC) is targeted to the lysosomal membrane, where it hydrolyzes RAS homolog-mTORC1 binding (RHEB) from its GTP-bound to GDP-bound state, inhibiting mechanistic target of ...Tuberous sclerosis complex (TSC) is targeted to the lysosomal membrane, where it hydrolyzes RAS homolog-mTORC1 binding (RHEB) from its GTP-bound to GDP-bound state, inhibiting mechanistic target of rapamycin complex 1 (mTORC1). Loss-of-function mutations in TSC cause TSC disease, marked by excessive tumor growth. Here, we overcome a high degree of continuous conformational heterogeneity to determine the 2.8-Å cryo-electron microscopy (cryo-EM) structure of the complete human TSC in complex with the lysosomal recruitment factor WD repeat domain phosphoinositide-interacting protein 3 (WIPI3). We discover a previously undetected amino-terminal TSC1 HEAT repeat dimer that clamps onto a single TSC wing and forms a phosphatidylinositol phosphate (PIP)-binding pocket, which specifically binds monophosphorylated PIPs. These structural advances provide a model by which WIPI3 and PIP-signaling networks coordinate to recruit TSC to the lysosomal membrane to inhibit mTORC1. The high-resolution TSC structure reveals previously unrecognized mutational hotspots and uncovers crucial insights into the mechanisms of TSC dysregulation in disease.
History
DepositionJun 14, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 4, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: WD repeat domain phosphoinositide-interacting protein 3
C: WD repeat domain phosphoinositide-interacting protein 3
E: WD repeat domain phosphoinositide-interacting protein 3
G: WD repeat domain phosphoinositide-interacting protein 3
X: Hamartin
B: Hamartin
D: Hamartin
F: Hamartin
H: Hamartin


Theoretical massNumber of molelcules
Total (without water)158,3409
Polymers158,3409
Non-polymers00
Water00
1
A: WD repeat domain phosphoinositide-interacting protein 3
B: Hamartin


Theoretical massNumber of molelcules
Total (without water)38,7122
Polymers38,7122
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1480 Å2
ΔGint-13 kcal/mol
Surface area14350 Å2
MethodPISA
2
C: WD repeat domain phosphoinositide-interacting protein 3
D: Hamartin


Theoretical massNumber of molelcules
Total (without water)38,7122
Polymers38,7122
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1450 Å2
ΔGint-13 kcal/mol
Surface area13490 Å2
MethodPISA
3
E: WD repeat domain phosphoinositide-interacting protein 3
X: Hamartin
F: Hamartin


Theoretical massNumber of molelcules
Total (without water)42,2023
Polymers42,2023
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area2100 Å2
ΔGint-15 kcal/mol
Surface area14940 Å2
MethodPISA
4
G: WD repeat domain phosphoinositide-interacting protein 3
H: Hamartin


Theoretical massNumber of molelcules
Total (without water)38,7122
Polymers38,7122
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1510 Å2
ΔGint-12 kcal/mol
Surface area13530 Å2
MethodPISA
Unit cell
Length a, b, c (Å)94.241, 94.241, 199.386
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number76
Space group name H-MP41

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Components

#1: Protein
WD repeat domain phosphoinositide-interacting protein 3 / WIPI-3 / WD repeat-containing protein 45-like / WDR45-like protein / WD repeat-containing protein ...WIPI-3 / WD repeat-containing protein 45-like / WDR45-like protein / WD repeat-containing protein 45B / WIPI49-like protein


Mass: 35222.359 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: WDR45B, WDR45L, WIPI3 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q5MNZ6
#2: Protein/peptide
Hamartin / Tuberous sclerosis 1 protein


Mass: 3490.041 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: TSC1, KIAA0243, TSC / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q92574
Has protein modificationN
Sequence detailsThe author states that the poly-UNK stretch could not be sequence assigned.

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.8 Å3/Da / Density % sol: 56.01 %
Crystal growTemperature: 293.15 K / Method: vapor diffusion, hanging drop
Details: 0.2 M ammonium tartrate, 20 % PEG3350, and 0.25 M sodium acetate.

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Australian Synchrotron / Beamline: MX2 / Wavelength: 0.95373 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Jun 24, 2021
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.95373 Å / Relative weight: 1
ReflectionResolution: 3.17→47.12 Å / Num. obs: 29039 / % possible obs: 98.9 % / Redundancy: 13.9 % / CC1/2: 0.997 / Net I/σ(I): 11.1
Reflection shellResolution: 3.17→3.37 Å / Num. unique obs: 4425 / CC1/2: 0.376 / % possible all: 93.4

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Processing

Software
NameVersionClassification
PHENIX1.20.1_4487refinement
XDSdata scaling
XDSdata reduction
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 3.18→47.12 Å / SU ML: 0.47 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 28.15 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2554 1484 5.16 %
Rwork0.1976 --
obs0.2005 28783 98.46 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 3.18→47.12 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms10118 0 0 0 10118
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00210358
X-RAY DIFFRACTIONf_angle_d0.42714014
X-RAY DIFFRACTIONf_dihedral_angle_d3.9371367
X-RAY DIFFRACTIONf_chiral_restr0.0471552
X-RAY DIFFRACTIONf_plane_restr0.0041808
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.18-3.280.40581010.3182140X-RAY DIFFRACTION85
3.28-3.40.28261420.27742504X-RAY DIFFRACTION100
3.4-3.530.32221160.25982527X-RAY DIFFRACTION100
3.53-3.690.32491430.24532525X-RAY DIFFRACTION100
3.69-3.890.27031330.21762496X-RAY DIFFRACTION100
3.89-4.130.31011510.20122520X-RAY DIFFRACTION100
4.13-4.450.22821170.17692520X-RAY DIFFRACTION100
4.45-4.90.1931470.13912527X-RAY DIFFRACTION100
4.9-5.610.22661720.16362472X-RAY DIFFRACTION100
5.61-7.060.25091440.21492527X-RAY DIFFRACTION100
7.06-47.120.251180.1892541X-RAY DIFFRACTION99
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
13.9026-1.5152-1.07522.1670.8942.69580.18380.26480.0142-0.487-0.2718-0.0479-0.0371-0.13570.04570.9621-0.03880.04410.5649-0.07661.0267-8.238813.261329.9322
24.76480.4484-0.77342.9784-0.05693.3811-0.27820.37-0.2242-0.16840.3619-0.1995-0.1760.0221-0.07390.5762-0.1095-0.0330.971-0.01430.944911.829626.378962.2485
31.9088-1.3543-0.94933.22680.78331.7835-0.323-0.3092-0.00060.2290.2041-0.04290.17930.01040.11930.5852-0.03910.05170.9317-0.0480.9669-33.893738.908770.339
43.55781.1221-0.05185.01580.99993.21360.3307-0.12020.12970.3007-0.29530.3671-0.05180.1521-0.04240.9623-0.10610.02710.55290.04040.92-20.822158.945837.8281
56.06993.7137-1.45583.9264-1.88935.6457-0.4779-0.7078-0.6012-0.9835-0.93810.51150.77930.37871.16620.96820.41480.05010.6997-0.08760.7202-21.748725.813349.9621
63.59550.04933.00763.2993-1.44753.188-0.88680.6294-1.5758-1.56370.7067-0.78481.1058-0.35510.13851.599-0.4880.31840.8536-0.05251.4307-10.4048-11.84233.6613
76.78270.7791.92023.96631.17428.8683-0.37380.4934-0.1731-0.33960.4934-0.45610.3662-1.7195-0.04180.6695-0.3526-0.01831.4906-0.2851.267332.295631.606361.8182
84.6546-0.90571.33634.54540.30213.5676-0.33190.1320.77890.8869-0.3890.95980.0244-0.29830.86651.0539-0.3479-0.03691.6005-0.06781.515-58.882436.659266.6357
94.470.4277-2.95250.62941.39166.97550.31080.25640.59320.55220.10720.26991.1723-0.2083-0.22591.2294-0.22610.31440.60970.05061.144-16.421380.104138.0805
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection details
1X-RAY DIFFRACTION1(chain 'A' and resid 11 through 315)
2X-RAY DIFFRACTION2(chain 'C' and resid 11 through 315)
3X-RAY DIFFRACTION3(chain 'E' and resid 11 through 315)
4X-RAY DIFFRACTION4(chain 'G' and resid 12 through 314)
5X-RAY DIFFRACTION5(chain 'X' and resid 8 through 18)
6X-RAY DIFFRACTION6(chain 'B' and resid 659 through 680)
7X-RAY DIFFRACTION7(chain 'D' and resid 667 through 681)
8X-RAY DIFFRACTION8(chain 'F' and resid 659 through 680)
9X-RAY DIFFRACTION9(chain 'H' and resid 666 through 681)

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