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- PDB-7bcs: ASCT2 in the presence of the inhibitor Lc-BPE (position "down") i... -

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Basic information

Entry
Database: PDB / ID: 7bcs
TitleASCT2 in the presence of the inhibitor Lc-BPE (position "down") in the outward-open conformation.
ComponentsNeutral amino acid transporter B(0)
KeywordsMEMBRANE PROTEIN / Solute carrier transporter / homology modeling / cancer metabolism / glutamine deprivation / cryo-EM
Function / homology
Function and homology information


glutamine secretion / L-glutamine import across plasma membrane / L-serine transmembrane transporter activity / glutamine transport / L-glutamine transmembrane transporter activity / ligand-gated channel activity / neutral amino acid transport / amino acid transmembrane transporter activity / Amino acid transport across the plasma membrane / neutral L-amino acid transmembrane transporter activity ...glutamine secretion / L-glutamine import across plasma membrane / L-serine transmembrane transporter activity / glutamine transport / L-glutamine transmembrane transporter activity / ligand-gated channel activity / neutral amino acid transport / amino acid transmembrane transporter activity / Amino acid transport across the plasma membrane / neutral L-amino acid transmembrane transporter activity / L-aspartate transmembrane transporter activity / L-aspartate import across plasma membrane / symporter activity / amino acid transport / antiporter activity / RHOJ GTPase cycle / RHOQ GTPase cycle / protein homotrimerization / RHOH GTPase cycle / transport across blood-brain barrier / RAC3 GTPase cycle / RAC1 GTPase cycle / basal plasma membrane / erythrocyte differentiation / melanosome / virus receptor activity / signaling receptor activity / extracellular exosome / membrane / metal ion binding / plasma membrane
Similarity search - Function
Sodium:dicarboxylate symporter family signature 2. / Sodium:dicarboxylate symporter / Sodium:dicarboxylate symporter, conserved site / Sodium:dicarboxylate symporter superfamily / Sodium:dicarboxylate symporter family / Sodium:dicarboxylate symporter family signature 1.
Similarity search - Domain/homology
Chem-TJ5 / Neutral amino acid transporter B(0)
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.43 Å
AuthorsGaribsingh, R.A. / Ndaru, E. / Garaeva, A.A. / Shi, Y. / Zielewicz, L. / Bonomi, M. / Slotboom, D.J. / Paulino, C. / Grewer, C. / Schlessinger, A.
Funding support Netherlands, United States, 7items
OrganizationGrant numberCountry
Netherlands Organisation for Scientific Research (NWO)722.017.001 Netherlands
Netherlands Organisation for Scientific Research (NWO)740.018.016 Netherlands
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM108911 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)T32 CA078207 United States
National Science Foundation (NSF, United States)1515028 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R15 GM135843-01 United States
Netherlands Organisation for Scientific Research (NWO)714.018.003 Netherlands
Citation
Journal: Proc Natl Acad Sci U S A / Year: 2021
Title: Rational design of ASCT2 inhibitors using an integrated experimental-computational approach.
Authors: Rachel-Ann A Garibsingh / Elias Ndaru / Alisa A Garaeva / Yueyue Shi / Laura Zielewicz / Paul Zakrepine / Massimiliano Bonomi / Dirk J Slotboom / Cristina Paulino / Christof Grewer / Avner Schlessinger /
Abstract: ASCT2 (SLC1A5) is a sodium-dependent neutral amino acid transporter that controls amino acid homeostasis in peripheral tissues. In cancer, ASCT2 is up-regulated where it modulates intracellular ...ASCT2 (SLC1A5) is a sodium-dependent neutral amino acid transporter that controls amino acid homeostasis in peripheral tissues. In cancer, ASCT2 is up-regulated where it modulates intracellular glutamine levels, fueling cell proliferation. Nutrient deprivation via ASCT2 inhibition provides a potential strategy for cancer therapy. Here, we rationally designed stereospecific inhibitors exploiting specific subpockets in the substrate binding site using computational modeling and cryo-electron microscopy (cryo-EM). The final structures combined with molecular dynamics simulations reveal multiple pharmacologically relevant conformations in the ASCT2 binding site as well as a previously unknown mechanism of stereospecific inhibition. Furthermore, this integrated analysis guided the design of a series of unique ASCT2 inhibitors. Our results provide a framework for future development of cancer therapeutics targeting nutrient transport via ASCT2, as well as demonstrate the utility of combining computational modeling and cryo-EM for solute carrier ligand discovery.
#1: Journal: Biorxiv
Title: Structural basis for stereospecific inhibition of ASCT2 from rational design
Authors: Garibsingh, R.A. / Ndaru, E. / Garaeva, A.A. / Shi, Y. / Zielewicz, L. / Bonomi, M. / Slotboom, D.J. / Paulino, C. / Grewer, C. / Schlessinger, A.
History
DepositionDec 21, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 22, 2021Provider: repository / Type: Initial release

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Structure visualization

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Assembly

Deposited unit
A: Neutral amino acid transporter B(0)
B: Neutral amino acid transporter B(0)
C: Neutral amino acid transporter B(0)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)170,8516
Polymers169,9173
Non-polymers9343
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area8120 Å2
ΔGint-70 kcal/mol
Surface area51640 Å2
MethodPISA

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Components

#1: Protein Neutral amino acid transporter B(0) / ATB(0) / Baboon M7 virus receptor / RD114/simian type D retrovirus receptor / Sodium-dependent ...ATB(0) / Baboon M7 virus receptor / RD114/simian type D retrovirus receptor / Sodium-dependent neutral amino acid transporter type 2 / Solute carrier family 1 member 5


Mass: 56638.902 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SLC1A5, ASCT2, M7V1, RDR, RDRC / Production host: Komagataella pastoris (fungus) / References: UniProt: Q15758
#2: Chemical ChemComp-TJ5 / (2~{S},4~{S})-4-(4-phenylphenyl)carbonyloxypyrrolidine-2-carboxylic acid


Mass: 311.332 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C18H17NO4 / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: ASCT2 in the presence of the inhibitor Lc-BPE / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Komagataella pastoris (fungus)
Buffer solutionpH: 7.4 / Details: 20 mM Tris-HCl, pH 7.4, 200 mM NaCl
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: at 5mA / Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE-PROPANE / Humidity: 100 % / Chamber temperature: 288 K

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 130000 X / Calibrated magnification: 49407 X / Nominal defocus max: 2000 nm / Nominal defocus min: 300 nm / Calibrated defocus min: 300 nm / Calibrated defocus max: 2000 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 105 K / Temperature (min): 90 K
Image recordingAverage exposure time: 9 sec. / Electron dose: 53 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 3 / Num. of real images: 6233
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansWidth: 3838 / Height: 3710 / Movie frames/image: 60 / Used frames/image: 1-60

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
EM software
IDNameVersionCategoryDetails
1cryoSPARC2.14.2particle selection
2EPU2.3image acquisition
4CTFFIND4.1.13CTF correction
7Coot0.9-premodel fitting
8UCSF Chimera0.93model fittingChimeraX
10cryoSPARC2.14.2initial Euler assignment
11RELION3.0.8final Euler assignment
13RELION3.0.83D reconstruction
14PHENIX1.18.2-3874-000model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3666842
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 3.43 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 300899 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingSpace: REAL
Atomic model buildingPDB-ID: 6MPB

6mpb

Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0110044
ELECTRON MICROSCOPYf_angle_d1.0713692
ELECTRON MICROSCOPYf_dihedral_angle_d24.031431
ELECTRON MICROSCOPYf_chiral_restr0.1541704
ELECTRON MICROSCOPYf_plane_restr0.0061707

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