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- PDB-7nqk: Cryo-EM structure of the mammalian peptide transporter PepT2 -

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Basic information

Entry
Database: PDB / ID: 7nqk
TitleCryo-EM structure of the mammalian peptide transporter PepT2
Components
  • Solute carrier family 15 member 2
  • nanobody
KeywordsMEMBRANE PROTEIN / proton-coupled peptide transporter
Function / homology
Function and homology information


high-affinity oligopeptide transmembrane transporter activity / Proton/oligopeptide cotransporters / tripeptide import across plasma membrane / peptidoglycan transport / tripeptide transmembrane transporter activity / dipeptide transport / metanephric proximal tubule development / dipeptide import across plasma membrane / oligopeptide transport / peptide:proton symporter activity ...high-affinity oligopeptide transmembrane transporter activity / Proton/oligopeptide cotransporters / tripeptide import across plasma membrane / peptidoglycan transport / tripeptide transmembrane transporter activity / dipeptide transport / metanephric proximal tubule development / dipeptide import across plasma membrane / oligopeptide transport / peptide:proton symporter activity / dipeptide transmembrane transporter activity / antibacterial innate immune response / regulation of nucleotide-binding domain, leucine rich repeat containing receptor signaling pathway / xenobiotic transport / renal absorption / phagocytic vesicle membrane / protein transport / apical plasma membrane / membrane / plasma membrane
Similarity search - Function
Oligopeptide transporter / PTR2 family proton/oligopeptide symporters signature 1. / PTR2 family proton/oligopeptide symporters signature 2. / PTR2 family proton/oligopeptide symporter, conserved site / Proton-dependent oligopeptide transporter family / POT family / Alpha/beta knot methyltransferases / MFS transporter superfamily
Similarity search - Domain/homology
Solute carrier family 15 member 2
Similarity search - Component
Biological speciesRattus norvegicus (Norway rat)
Lama glama (llama)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsParker, J.L. / Deme, J.C. / Lea, S.M. / Newstead, S.
Funding support United Kingdom, 11items
OrganizationGrant numberCountry
Wellcome Trust201536 United Kingdom
Wolfson FoundationWL16005 United Kingdom
Engineering and Physical Sciences Research CouncilEP/L000253/1 United Kingdom
Wellcome Trust209194 United Kingdom
Wellcome Trust100298 United Kingdom
Wellcome Trust219531 United Kingdom
Wellcome Trust215519 United Kingdom
Wellcome Trust219531 United Kingdom
Medical Research Council (MRC, United Kingdom)MR/M011984/1 United Kingdom
Medical Research Council (MRC, United Kingdom)MR/S021043/1 United Kingdom
Wellcome Trust203741 United Kingdom
CitationJournal: Sci Adv / Year: 2021
Title: Cryo-EM structure of PepT2 reveals structural basis for proton-coupled peptide and prodrug transport in mammals.
Authors: Joanne L Parker / Justin C Deme / Zhiyi Wu / Gabriel Kuteyi / Jiandong Huo / Raymond J Owens / Philip C Biggin / Susan M Lea / Simon Newstead /
Abstract: The SLC15 family of proton-coupled solute carriers PepT1 and PepT2 play a central role in human physiology as the principal route for acquiring and retaining dietary nitrogen. A remarkable feature of ...The SLC15 family of proton-coupled solute carriers PepT1 and PepT2 play a central role in human physiology as the principal route for acquiring and retaining dietary nitrogen. A remarkable feature of the SLC15 family is their extreme substrate promiscuity, which has enabled the targeting of these transporters for the improvement of oral bioavailability for several prodrug molecules. Although recent structural and biochemical studies on bacterial homologs have identified conserved sites of proton and peptide binding, the mechanism of peptide capture and ligand promiscuity remains unclear for mammalian family members. Here, we present the cryo-electron microscopy structure of the outward open conformation of the rat peptide transporter PepT2 in complex with an inhibitory nanobody. Our structure, combined with molecular dynamics simulations and biochemical and cell-based assays, establishes a framework for understanding peptide and prodrug recognition within this pharmaceutically important transporter family.
History
DepositionMar 1, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 7, 2021Provider: repository / Type: Initial release
Revision 1.1Oct 13, 2021Group: Data collection / Database references
Category: citation / citation_author ...citation / citation_author / database_2 / em_admin / pdbx_database_proc
Item: _citation.journal_volume / _citation.pdbx_database_id_DOI ..._citation.journal_volume / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name / _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_admin.last_update

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Assembly

Deposited unit
A: Solute carrier family 15 member 2
B: nanobody


Theoretical massNumber of molelcules
Total (without water)96,9722
Polymers96,9722
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area1840 Å2
ΔGint-5 kcal/mol
Surface area37210 Å2
MethodPISA

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Components

#1: Protein Solute carrier family 15 member 2 / Kidney H(+)/peptide cotransporter / Oligopeptide transporter / kidney isoform / Peptide transporter 2


Mass: 82477.766 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Slc15a2, Pept2 / Production host: Homo sapiens (human) / References: UniProt: Q63424
#2: Antibody nanobody


Mass: 14494.183 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Lama glama (llama)

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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

Component
IDNameTypeEntity IDParent-IDSource
1Complex of the mammalian peptide transporter PepT2 with nanobodyCOMPLEXall0MULTIPLE SOURCES
2PepT2COMPLEX#11RECOMBINANT
3nanobodyCOMPLEX#21NATURAL
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Rattus norvegicus (Norway rat)10116
33Lama glama (llama)9844
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
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: 58 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 298562 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0056282
ELECTRON MICROSCOPYf_angle_d0.7438531
ELECTRON MICROSCOPYf_dihedral_angle_d8.723840
ELECTRON MICROSCOPYf_chiral_restr0.046966
ELECTRON MICROSCOPYf_plane_restr0.0051067

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