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- EMDB-12528: Cryo-EM structure of the mammalian peptide transporter PepT2 -

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Basic information

Entry
Database: EMDB / ID: EMD-12528
TitleCryo-EM structure of the mammalian peptide transporter PepT2
Map datacryoSPARC local refinement, sharpened
Sample
  • Complex: Complex of the mammalian peptide transporter PepT2 with nanobody
    • Complex: PepT2
      • Protein or peptide: Solute carrier family 15 member 2
    • Complex: nanobodySingle-domain antibody
      • Protein or peptide: nanobodySingle-domain antibody
Function / homology
Function and homology information


high-affinity oligopeptide transmembrane transporter activity / Proton/oligopeptide cotransporters / tripeptide import across plasma membrane / dipeptide transport / peptidoglycan transport / tripeptide transmembrane transporter activity / dipeptide import across plasma membrane / metanephric proximal tubule development / oligopeptide transport / peptide:proton symporter activity ...high-affinity oligopeptide transmembrane transporter activity / Proton/oligopeptide cotransporters / tripeptide import across plasma membrane / dipeptide transport / peptidoglycan transport / tripeptide transmembrane transporter activity / dipeptide import across plasma membrane / metanephric proximal tubule development / oligopeptide transport / peptide:proton symporter activity / dipeptide transmembrane transporter activity / regulation of nucleotide-binding domain, leucine rich repeat containing receptor signaling pathway / xenobiotic transport / renal absorption / phagocytic vesicle membrane / protein transport / apical plasma membrane / innate immune response / 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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsParker JL / Deme JC / Lea SM / Newstead S
Funding support United Kingdom, 11 items
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, 2021-
Header (metadata) releaseJul 7, 2021-
Map releaseJul 7, 2021-
UpdateOct 13, 2021-
Current statusOct 13, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.43
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.43
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7nqk
  • Surface level: 0.43
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7nqk
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_12528.png

Downloads & links

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Map

FileDownload / File: emd_12528.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationcryoSPARC local refinement, sharpened
Voxel sizeX=Y=Z: 0.832 Å
Density
Contour LevelBy AUTHOR: 0.43 / Movie #1: 0.43
Minimum - Maximum-0.8915138 - 2.8229928
Average (Standard dev.)0.0016997944 (±0.044236466)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 266.24 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.8320.8320.832
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z266.240266.240266.240
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-0.8922.8230.002

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Supplemental data

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Mask #1

Fileemd_12528_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: cryoSPARC local refinement, unsharpened

Fileemd_12528_additional_1.map
AnnotationcryoSPARC local refinement, unsharpened
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map A

Fileemd_12528_half_map_1.map
Annotationhalf map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map B

Fileemd_12528_half_map_2.map
Annotationhalf map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Complex of the mammalian peptide transporter PepT2 with nanobody

EntireName: Complex of the mammalian peptide transporter PepT2 with nanobody
Components
  • Complex: Complex of the mammalian peptide transporter PepT2 with nanobody
    • Complex: PepT2
      • Protein or peptide: Solute carrier family 15 member 2
    • Complex: nanobodySingle-domain antibody
      • Protein or peptide: nanobodySingle-domain antibody

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Supramolecule #1: Complex of the mammalian peptide transporter PepT2 with nanobody

SupramoleculeName: Complex of the mammalian peptide transporter PepT2 with nanobody
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all

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Supramolecule #2: PepT2

SupramoleculeName: PepT2 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Rattus norvegicus (Norway rat)
Recombinant expressionOrganism: Homo sapiens (human)

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Supramolecule #3: nanobody

SupramoleculeName: nanobody / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Lama glama (llama)

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Macromolecule #1: Solute carrier family 15 member 2

MacromoleculeName: Solute carrier family 15 member 2 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Rattus norvegicus (Norway rat)
Molecular weightTheoretical: 82.477766 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MNPFQKNESK ETLFSPVSTE EMLPRPPSPP KKSPPKIFGS SYPVSIAFIV VNEFCERFSY YGMKAVLTLY FLYFLHWNED TSTSVYHAF SSLCYFTPIL GAAIADSWLG KFKTIIYLSL VYVLGHVFKS LGAIPILGGK MLHTILSLVG LSLIALGTGG I KPCVAAFG ...String:
MNPFQKNESK ETLFSPVSTE EMLPRPPSPP KKSPPKIFGS SYPVSIAFIV VNEFCERFSY YGMKAVLTLY FLYFLHWNED TSTSVYHAF SSLCYFTPIL GAAIADSWLG KFKTIIYLSL VYVLGHVFKS LGAIPILGGK MLHTILSLVG LSLIALGTGG I KPCVAAFG GDQFEEEHAE ARTRYFSVFY LAINAGSLIS TFITPMLRGD VKCFGQDCYA LAFGVPGLLM VLALVVFAMG SK MYRKPPP EGNIVAQVIK CIWFALCNRF RNRSGDLPKR QHWLDWAAEK YPKHLIADVK ALTRVLFLYI PLPMFWALLD QQG SRWTLQ ANKMNGDLGF FVLQPDQMQV LNPFLVLIFI PLFDLVIYRL ISKCRINFSS LRKMAVGMIL ACLAFAVAAL VETK INGMI HPQPASQEIF LQVLNLADGD VKVTVLGSRN NSLLVESVSS FQNTTHYSKL HLEAKSQDLH FHLKYNSLSV HNDHS VEEK NCYQLLIHQD GESISSMLVK DTGIKPANGM AAIRFINTLH KDLNISLDTD APLSVGKDYG VSAYRTVLRG KYPAVH CET EDKVFSLDLG QLDFGTTYLF VITNITSQGL QAWKAEDIPV NKLSIAWQLP QYVLVTAAEV MFSVTGLEFS YSQAPSS MK SVLQAAWLLT VAVGNIIVLV VAQFSGLAQW AEFVLFSCLL LVVCLIFSVM AYYYVPLKSE DTREATDKQI PAVQGNMI N LETKNTRLVE GENLYFQ

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Macromolecule #2: nanobody

MacromoleculeName: nanobody / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Lama glama (llama)
Molecular weightTheoretical: 14.494183 KDa
SequenceString:
GPSQVQLVES GGGLVQPGGS LRLLCVASGR PFNDYDMGWF RQAPGKEREF VASISWSGRV TDYSDSMKGR CTVSRDNAKG TMFLQMSNL VPRDTAVYYC AAARRRWTFK ATNTEEFYET WGQGTQVTVS SA

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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

BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 58.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 298562
FSC plot (resolution estimation)

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