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- EMDB-12143: ASCT2 in the presence of the inhibitor ERA-21 in the outward-open... -

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Basic information

Entry
Database: EMDB / ID: EMD-12143
TitleASCT2 in the presence of the inhibitor ERA-21 in the outward-open conformation.
Map dataCryo-EM map of ASCT2 in the presence of the inhibitor ERA-21 at 3.37 A resolution in the outward-open conformation. Map was sharpened with a b factor of 173 A2
Sample
  • Complex: ASCT2 in the presence of the inhibitor ERA-21 in the outward-open conformation.
    • Protein or peptide: Neutral amino acid transporter B(0)
Function / homology
Function and homology information


glutamine secretion / L-glutamine import across plasma membrane / glutamine transport / L-glutamine transmembrane transporter activity / L-serine 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 / glutamine transport / L-glutamine transmembrane transporter activity / L-serine 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
Neutral amino acid transporter B(0)
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.37 Å
AuthorsGaribsingh RA / Ndaru E / Garaeva AA / Shi Y / Zielewicz L / Bonomi M / Slotboom DJ / Paulino C / Grewer C / Schlessinger A
Funding support Netherlands, United States, 7 items
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
CitationJournal: 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.
History
DepositionDec 21, 2020-
Header (metadata) releaseSep 22, 2021-
Map releaseSep 22, 2021-
UpdateSep 29, 2021-
Current statusSep 29, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0523
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.0523
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7bct
  • Surface level: 0.0523
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_12143.png

Downloads & links

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Map

FileDownload / File: emd_12143.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM map of ASCT2 in the presence of the inhibitor ERA-21 at 3.37 A resolution in the outward-open conformation. Map was sharpened with a b factor of 173 A2
Voxel sizeX=Y=Z: 1.012 Å
Density
Contour LevelBy AUTHOR: 0.0523 / Movie #1: 0.0523
Minimum - Maximum-0.27546647 - 0.31602994
Average (Standard dev.)0.00045099133 (±0.009487945)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 202.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0121.0121.012
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z202.400202.400202.400
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-0.2750.3160.000

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Supplemental data

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Mask #1

Fileemd_12143_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half-map 1 used for post processing step and...

Fileemd_12143_half_map_1.map
Annotationhalf-map 1 used for post processing step and FSC resolution calculation.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half-map 2 used for post processing step and...

Fileemd_12143_half_map_2.map
Annotationhalf-map 2 used for post processing step and FSC resolution calculation.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : ASCT2 in the presence of the inhibitor ERA-21 in the outward-open...

EntireName: ASCT2 in the presence of the inhibitor ERA-21 in the outward-open conformation.
Components
  • Complex: ASCT2 in the presence of the inhibitor ERA-21 in the outward-open conformation.
    • Protein or peptide: Neutral amino acid transporter B(0)

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Supramolecule #1: ASCT2 in the presence of the inhibitor ERA-21 in the outward-open...

SupramoleculeName: ASCT2 in the presence of the inhibitor ERA-21 in the outward-open conformation.
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Komagataella pastoris (fungus)

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Macromolecule #1: Neutral amino acid transporter B(0)

MacromoleculeName: Neutral amino acid transporter B(0) / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 56.638902 KDa
Recombinant expressionOrganism: Komagataella pastoris (fungus)
SequenceString: MVADPPRDSK GLAAAEPTAN GGLALASIED QGAAAGGYCG SRDQVRRCLR ANLLVLLTVV AVVAGVALGL GVSGAGGALA LGPERLSAF VFPGELLLRL LRMIILPLVV CSLIGGAASL DPGALGRLGA WALLFFLVTT LLASALGVGL ALALQPGAAS A AINASVGA ...String:
MVADPPRDSK GLAAAEPTAN GGLALASIED QGAAAGGYCG SRDQVRRCLR ANLLVLLTVV AVVAGVALGL GVSGAGGALA LGPERLSAF VFPGELLLRL LRMIILPLVV CSLIGGAASL DPGALGRLGA WALLFFLVTT LLASALGVGL ALALQPGAAS A AINASVGA AGSAENAPSK EVLDSFLDLA RNIFPSNLVS AAFRSYSTTY EERNITGTRV KVPVGQEVEG MNILGLVVFA IV FGVALRK LGPEGELLIR FFNSFNEATM VLVSWIMWYA PVGIMFLVAG KIVEMEDVGL LFARLGKYIL CCLLGHAIHG LLV LPLIYF LFTRKNPYRF LWGIVTPLAT AFGTSSSSAT LPLMMKCVEE NNGVAKHISR FILPIGATVN MDGAALFQCV AAVF IAQLS QQSLDFVKII TILVTATASS VGAAGIPAGG VLTLAIILEA VNLPVDHISL ILAVDWLVDR SCTVLNVEGD ALGAG LLQN YVDRTESRST EPELIQVKSE LPLDPLPVPT EEGNPLLKHY RGPAGDATVA SEKESVM

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1 mg/mL
BufferpH: 7.4 / Details: 20 mM Tris-HCl, pH 7.4, 200 mM NaCl
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Details: at 5mA
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 288 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Calibrated defocus max: 2.0 µm / Calibrated defocus min: 0.3 µm / Calibrated magnification: 49407 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.3 µm / Nominal magnification: 130000
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
TemperatureMin: 90.0 K / Max: 105.0 K
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Frames/image: 1-60 / Number grids imaged: 3 / Number real images: 9788 / Average exposure time: 9.0 sec. / Average electron dose: 53.0 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 4438395
CTF correctionSoftware - Name: CTFFIND (ver. 4.1.13)
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 3.1.0)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 3.1.0)
Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.37 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1.0) / Number images used: 224884

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Atomic model buiding 1

Initial modelPDB ID:

6mpb

RefinementSpace: REAL
Output model

PDB-7bct:
ASCT2 in the presence of the inhibitor ERA-21 in the outward-open conformation.

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