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- PDB-7ccs: Consensus mutated xCT-CD98hc complex -

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Entry
Database: PDB / ID: 7ccs
TitleConsensus mutated xCT-CD98hc complex
Components
  • 4F2 cell-surface antigen heavy chain
  • Consensus mutated Anionic Amino Acid Transporter Light Chain, Xc- System
KeywordsTRANSPORT PROTEIN / Transporter / membrane protein / complex
Function / homology
Function and homology information


cystine:glutamate antiporter activity / regulation of neutrophil apoptotic process / L-kynurenine transmembrane transport / L-kynurenine transmembrane transporter activity / regulation of cysteine metabolic process / regulation of glutathione biosynthetic process / regulation of glutamate metabolic process / regulation of melanin biosynthetic process / regulation of AMPA glutamate receptor clustering / L-cystine transport ...cystine:glutamate antiporter activity / regulation of neutrophil apoptotic process / L-kynurenine transmembrane transport / L-kynurenine transmembrane transporter activity / regulation of cysteine metabolic process / regulation of glutathione biosynthetic process / regulation of glutamate metabolic process / regulation of melanin biosynthetic process / regulation of AMPA glutamate receptor clustering / L-cystine transport / Defective SLC7A7 causes lysinuric protein intolerance (LPI) / neutral L-amino acid secondary active transmembrane transporter activity / apical pole of neuron / aromatic amino acid transmembrane transporter activity / tyrosine transport / L-histidine transport / amino acid transport complex / dipeptide import across plasma membrane / L-leucine import across plasma membrane / L-alanine transmembrane transporter activity / L-alanine import across plasma membrane / regulation of protein transport / isoleucine transport / phenylalanine transport / methionine transport / L-amino acid transmembrane transporter activity / valine transport / L-leucine transmembrane transporter activity / calcium:sodium antiporter activity / L-leucine transport / thyroid hormone transport / proline transport / L-glutamate transmembrane transport / glutathione transmembrane transport / amino acid transmembrane transport / regulation of cellular response to oxidative stress / ventricular system development / lens fiber cell differentiation / intracellular glutamate homeostasis / Amino acid transport across the plasma membrane / neutral L-amino acid transmembrane transporter activity / L-glutamate import across plasma membrane / Tryptophan catabolism / striatum development / astrocyte projection / exogenous protein binding / anchoring junction / limb development / Basigin interactions / negative regulation of ferroptosis / response to redox state / microvillus membrane / NFE2L2 regulating anti-oxidant/detoxification enzymes / lung alveolus development / amino acid transport / regulation of synapse organization / adult behavior / response to exogenous dsRNA / tryptophan transport / glutathione metabolic process / basal plasma membrane / response to nicotine / brush border membrane / modulation of chemical synaptic transmission / visual learning / response to organic cyclic compound / platelet aggregation / response to toxic substance / calcium ion transport / double-stranded RNA binding / melanosome / apical part of cell / virus receptor activity / regulation of cell population proliferation / cellular response to oxidative stress / basolateral plasma membrane / carbohydrate metabolic process / cadherin binding / symbiont entry into host cell / protein heterodimerization activity / apical plasma membrane / lysosomal membrane / synapse / cell surface / protein homodimerization activity / RNA binding / extracellular exosome / nucleoplasm / membrane / plasma membrane
Similarity search - Function
L-type amino acid transporter / Solute carrier family 3 member 2, N-terminal domain / 4F2 cell-surface antigen heavy chain / Solute carrier family 3 member 2 N-terminus / : / Amino acid/polyamine transporter I / Amino acid permease / Alpha amylase, catalytic domain / Glycosyl hydrolase, family 13, catalytic domain / Alpha-amylase domain ...L-type amino acid transporter / Solute carrier family 3 member 2, N-terminal domain / 4F2 cell-surface antigen heavy chain / Solute carrier family 3 member 2 N-terminus / : / Amino acid/polyamine transporter I / Amino acid permease / Alpha amylase, catalytic domain / Glycosyl hydrolase, family 13, catalytic domain / Alpha-amylase domain / Glycosyl hydrolase, all-beta / Glycoside hydrolase superfamily
Similarity search - Domain/homology
Amino acid transporter heavy chain SLC3A2 / Cystine/glutamate transporter
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 6.2 Å
AuthorsOda, K. / Lee, Y. / Takemoto, M. / Yamashita, K. / Nishizawa, T. / Nureki, O.
CitationJournal: Protein Sci / Year: 2020
Title: Consensus mutagenesis approach improves the thermal stability of system x transporter, xCT, and enables cryo-EM analyses.
Authors: Kazumasa Oda / Yongchan Lee / Pattama Wiriyasermkul / Yoko Tanaka / Mizuki Takemoto / Keitaro Yamashita / Shushi Nagamori / Tomohiro Nishizawa / Osamu Nureki /
Abstract: System x is an amino acid antiporter that imports L-cystine into cells and exports intracellular L-glutamate, at a 1:1 ratio. As L-cystine is an essential precursor for glutathione synthesis, system ...System x is an amino acid antiporter that imports L-cystine into cells and exports intracellular L-glutamate, at a 1:1 ratio. As L-cystine is an essential precursor for glutathione synthesis, system x supports tumor cell growth through glutathione-based oxidative stress resistance and is considered as a potential therapeutic target for cancer treatment. System x consists of two subunits, the light chain subunit SLC7A11 (xCT) and the heavy chain subunit SLC3A2 (also known as CD98hc or 4F2hc), which are linked by a conserved disulfide bridge. Although the recent structures of another SLC7 member, L-type amino acid transporter 1 (LAT1) in complex with CD98hc, have provided the structural basis toward understanding the amino acid transport mechanism, the detailed molecular mechanism of xCT remains unknown. To revealthe molecular mechanism, we performed single-particle analyses of the xCT-CD98hc complex. As wild-type xCT-CD98hc displayed poor stability and could not be purified to homogeneity, we applied a consensus mutagenesis approach to xCT. The consensus mutated construct exhibited increased stability as compared to the wild-type, and enabled the cryoelectron microscopy (cryo-EM) map to be obtained at 6.2 Å resolution by single-particle analysis. The cryo-EM map revealed sufficient electron density to assign secondary structures. In the xCT structure, the hash and arm domains are well resolved, whereas the bundle domain shows some flexibility. CD98hc is positioned next to the xCT transmembrane domain. This study provides the structural basis of xCT, and our consensus-based strategy could represent a good choice toward solving unstable protein structures.
History
DepositionJun 17, 2020Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Dec 9, 2020Provider: repository / Type: Initial release
Revision 1.1Dec 16, 2020Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.title / _citation_author.identifier_ORCID

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Structure visualization

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Assembly

Deposited unit
A: 4F2 cell-surface antigen heavy chain
B: Consensus mutated Anionic Amino Acid Transporter Light Chain, Xc- System


Theoretical massNumber of molelcules
Total (without water)124,0032
Polymers124,0032
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein 4F2 cell-surface antigen heavy chain / Glycoprotein / 4F2hc / 4F2 heavy chain antigen / Lymphocyte activation antigen 4F2 large subunit / ...Glycoprotein / 4F2hc / 4F2 heavy chain antigen / Lymphocyte activation antigen 4F2 large subunit / Solute carrier family 3 member 2


Mass: 68069.625 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SLC3A2, MDU1 / Cell line (production host): HEK293SGnTI- / Production host: Homo sapiens (human) / References: UniProt: P08195
#2: Protein Consensus mutated Anionic Amino Acid Transporter Light Chain, Xc- System


Mass: 55933.852 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SLC7A11 / Cell line (production host): HEK293SGnTI- / Production host: Homo sapiens (human) / References: UniProt: Q9UPY5*PLUS

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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: Consensus mutated xCT-CD98hc complex / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.12 MDa / Experimental value: YES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER/RHODIUM / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Image recordingElectron dose: 49.76 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 6.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 86395
Details: This pdb entry contains an issue about peptide linkage (bond outlier). Residues GLU A 213 and LEU A 214 that are next to each other are not properly linked: distance between C and N is 3.35. ...Details: This pdb entry contains an issue about peptide linkage (bond outlier). Residues GLU A 213 and LEU A 214 that are next to each other are not properly linked: distance between C and N is 3.35. This was introduced upon the fitting of the homology model into the low resolution cryo-EM map by using Rosetta. The model has not undergone any further structure refinement.
Symmetry type: POINT

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