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- PDB-7b00: Human LAT2-4F2hc complex in the apo-state -

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Basic information

Entry
Database: PDB / ID: 7b00
TitleHuman LAT2-4F2hc complex in the apo-state
Components
  • Isoform 2 of 4F2 cell-surface antigen heavy chain
  • Large neutral amino acids transporter small subunit 2
KeywordsMEMBRANE PROTEIN / HATs / amino-acid / transporter / membrane / protein / heterodimer
Function / homology
Function and homology information


proline transmembrane transport / glycine transmembrane transporter activity / organic cation transmembrane transporter activity / glycine transport / thyroid hormone transmembrane transporter activity / neutral L-amino acid secondary active transmembrane transporter activity / amino acid import across plasma membrane / Defective SLC7A7 causes lysinuric protein intolerance (LPI) / methionine transport / tyrosine transport ...proline transmembrane transport / glycine transmembrane transporter activity / organic cation transmembrane transporter activity / glycine transport / thyroid hormone transmembrane transporter activity / neutral L-amino acid secondary active transmembrane transporter activity / amino acid import across plasma membrane / Defective SLC7A7 causes lysinuric protein intolerance (LPI) / methionine transport / tyrosine transport / L-histidine transport / apical pole of neuron / aromatic amino acid transmembrane transporter activity / amino acid transport complex / L-amino acid transmembrane transporter activity / : / L-leucine import across plasma membrane / L-alanine transmembrane transporter activity / isoleucine transport / L-alanine import across plasma membrane / phenylalanine transport / valine transport / L-leucine transmembrane transporter activity / amino acid transmembrane transport / calcium:sodium antiporter activity / L-leucine transport / thyroid hormone transport / proline transport / toxin transmembrane transporter activity / neutral amino acid transport / amino acid transmembrane transporter activity / Amino acid transport across the plasma membrane / neutral L-amino acid transmembrane transporter activity / Tryptophan catabolism / exogenous protein binding / antiporter activity / anchoring junction / Basigin interactions / microvillus membrane / amino acid transport / response to exogenous dsRNA / tryptophan transport / transport across blood-brain barrier / basal plasma membrane / calcium ion transport / peptide antigen binding / double-stranded RNA binding / melanosome / virus receptor activity / basolateral plasma membrane / carbohydrate metabolic process / cadherin binding / symbiont entry into host cell / apical plasma membrane / protein heterodimerization activity / 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 / Glycosyl hydrolase, all-beta / Glycoside hydrolase superfamily
Similarity search - Domain/homology
Amino acid transporter heavy chain SLC3A2 / Large neutral amino acids transporter small subunit 2
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.98 Å
AuthorsRodriguez, C.F. / Escudero-Bravo, P. / Garcia-Martin, C. / Boskovic, J. / Errasti-Murugarren, E. / Palacin, M. / Llorca, O.
Funding support7items
OrganizationGrant numberCountry
La Caixa FoundationLCF/PR/HR20/52400017
Ministry of Economy and Competitiveness (MINECO)SAF2015-64869-R-FEDER
Ministry of Economy and Competitiveness (MINECO)RTI2018-094211-B-100-FEDER
Ministry of Economy and Competitiveness (MINECO)SAF2017-82632-P
Ministry of Economy and Competitiveness (MINECO)BES-2015-071348
Generalitat de Catalunya2017 SGR 961
Comunidad de MadridY2018/BIO4747
CitationJournal: Proc Natl Acad Sci U S A / Year: 2021
Title: Structural basis for substrate specificity of heteromeric transporters of neutral amino acids.
Authors: Carlos F Rodriguez / Paloma Escudero-Bravo / Lucía Díaz / Paola Bartoccioni / Carmen García-Martín / Joan G Gilabert / Jasminka Boskovic / Víctor Guallar / Ekaitz Errasti-Murugarren / ...Authors: Carlos F Rodriguez / Paloma Escudero-Bravo / Lucía Díaz / Paola Bartoccioni / Carmen García-Martín / Joan G Gilabert / Jasminka Boskovic / Víctor Guallar / Ekaitz Errasti-Murugarren / Oscar Llorca / Manuel Palacín /
Abstract: Despite having similar structures, each member of the heteromeric amino acid transporter (HAT) family shows exquisite preference for the exchange of certain amino acids. Substrate specificity ...Despite having similar structures, each member of the heteromeric amino acid transporter (HAT) family shows exquisite preference for the exchange of certain amino acids. Substrate specificity determines the physiological function of each HAT and their role in human diseases. However, HAT transport preference for some amino acids over others is not yet fully understood. Using cryo-electron microscopy of apo human LAT2/CD98hc and a multidisciplinary approach, we elucidate key molecular determinants governing neutral amino acid specificity in HATs. A few residues in the substrate-binding pocket determine substrate preference. Here, we describe mutations that interconvert the substrate profiles of LAT2/CD98hc, LAT1/CD98hc, and Asc1/CD98hc. In addition, a region far from the substrate-binding pocket critically influences the conformation of the substrate-binding site and substrate preference. This region accumulates mutations that alter substrate specificity and cause hearing loss and cataracts. Here, we uncover molecular mechanisms governing substrate specificity within the HAT family of neutral amino acid transporters and provide the structural bases for mutations in LAT2/CD98hc that alter substrate specificity and that are associated with several pathologies.
History
DepositionNov 17, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 27, 2021Provider: repository / Type: Initial release
Revision 2.0Feb 23, 2022Group: Advisory / Atomic model ...Advisory / Atomic model / Author supporting evidence / Data collection / Database references / Source and taxonomy / Structure summary
Category: atom_site / audit_author ...atom_site / audit_author / citation / citation_author / database_PDB_caveat / em_software / entity_src_gen / pdbx_audit_support / pdbx_contact_author / pdbx_nonpoly_scheme / pdbx_unobs_or_zero_occ_atoms / pdbx_validate_chiral / pdbx_validate_close_contact
Item: _audit_author.identifier_ORCID / _citation.country ..._audit_author.identifier_ORCID / _citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_software.category / _entity_src_gen.gene_src_common_name / _pdbx_audit_support.country / _pdbx_audit_support.funding_organization / _pdbx_nonpoly_scheme.auth_seq_num / _pdbx_nonpoly_scheme.pdb_seq_num
Description: Chirality error / Provider: author / Type: Coordinate replacement

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Structure visualization

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Assembly

Deposited unit
A: Large neutral amino acids transporter small subunit 2
B: Isoform 2 of 4F2 cell-surface antigen heavy chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)119,3517
Polymers116,4242
Non-polymers2,9275
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area4420 Å2
ΔGint-8 kcal/mol
Surface area44260 Å2
MethodPISA

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Components

#1: Protein Large neutral amino acids transporter small subunit 2 / L-type amino acid transporter 2 / hLAT2 / Solute carrier family 7 member 8


Mass: 58420.133 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SLC7A8, LAT2 / Cell line (production host): HEK293-6E / Production host: Homo sapiens (human) / References: UniProt: Q9UHI5
#2: Protein Isoform 2 of 4F2 cell-surface antigen heavy chain / 4F2hc / 4F2 heavy chain antigen / Lymphocyte activation antigen 4F2 large subunit / Solute carrier ...4F2hc / 4F2 heavy chain antigen / Lymphocyte activation antigen 4F2 large subunit / Solute carrier family 3 member 2


Mass: 58003.652 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): HEK293-6E / Production host: Homo sapiens (human) / References: UniProt: P08195
#3: Polysaccharide
2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}LINUCSPDB-CARE
#4: Chemical ChemComp-AJP / Digitonin


Mass: 1229.312 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C56H92O29 / Comment: detergent*YM
Has ligand of interestN

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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: Human LAT2 4F2hc complex in the apo state / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK293-6E
Buffer solutionpH: 7
SpecimenConc.: 1.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R0.6/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 297 K

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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: 1 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1

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Processing

SoftwareName: PHENIX / Version: 1.14_3260: / Classification: refinement
EM software
IDNameVersionCategory
2EPUimage acquisition
10RELION3.1final Euler assignment
12RELION3.13D reconstruction
CTF correctionType: NONE
3D reconstructionResolution: 3.98 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 176132 / Details: CTF refinement in cisTEM / Symmetry type: POINT
Atomic model buildingProtocol: BACKBONE TRACE / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0057498
ELECTRON MICROSCOPYf_angle_d0.93610229
ELECTRON MICROSCOPYf_dihedral_angle_d10.8884356
ELECTRON MICROSCOPYf_chiral_restr0.0541196
ELECTRON MICROSCOPYf_plane_restr0.0061270

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