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- EMDB-44602: Cryo-EM structure of the mammalian peptide transporter PepT2 boun... -

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Basic information

Entry
Database: EMDB / ID: EMD-44602
TitleCryo-EM structure of the mammalian peptide transporter PepT2 bound to cloxacillin, pose 2
Map dataunsharpened map
Sample
  • Complex: Complex of the mammalian peptide transporter PepT2 with nanobody and bound cloxacillin, pose 2
    • Complex: PepT2
      • Protein or peptide: Solute carrier family 15 member 2
    • Complex: nanobody
      • Protein or peptide: nanobody
  • Ligand: CLOXACILLIN
Keywordsprotein-coupled peptide transporter / peptide transport / antibiotics / MEMBRANE PROTEIN
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 / antibacterial innate immune response / regulation of nucleotide-binding domain, leucine rich repeat containing receptor signaling pathway / peptide transport / 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)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsParker JL / Deme JC / Lea SM / Newstead S
Funding support United Kingdom, United States, 4 items
OrganizationGrant numberCountry
Wellcome Trust215519 United Kingdom
Wellcome Trust219531 United Kingdom
Wellcome Trust218514 United Kingdom
National Institutes of Health/National Cancer Institute (NIH/NCI)Intramural Research Program United States
CitationJournal: Nat Commun / Year: 2024
Title: Structural basis for antibiotic transport and inhibition in PepT2.
Authors: Joanne L Parker / Justin C Deme / Simon M Lichtinger / Gabriel Kuteyi / Philip C Biggin / Susan M Lea / Simon Newstead /
Abstract: The uptake and elimination of beta-lactam antibiotics in the human body are facilitated by the proton-coupled peptide transporters PepT1 (SLC15A1) and PepT2 (SLC15A2). The mechanism by which SLC15 ...The uptake and elimination of beta-lactam antibiotics in the human body are facilitated by the proton-coupled peptide transporters PepT1 (SLC15A1) and PepT2 (SLC15A2). The mechanism by which SLC15 family transporters recognize and discriminate between different drug classes and dietary peptides remains unclear, hampering efforts to improve antibiotic pharmacokinetics through targeted drug design and delivery. Here, we present cryo-EM structures of the proton-coupled peptide transporter, PepT2 from Rattus norvegicus, in complex with the widely used beta-lactam antibiotics cefadroxil, amoxicillin and cloxacillin. Our structures, combined with pharmacophore mapping, molecular dynamics simulations and biochemical assays, establish the mechanism of beta-lactam antibiotic recognition and the important role of protonation in drug binding and transport.
History
DepositionApr 24, 2024-
Header (metadata) releaseJul 24, 2024-
Map releaseJul 24, 2024-
UpdateOct 30, 2024-
Current statusOct 30, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_44602.png

Downloads & links

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Map

FileDownload / File: emd_44602.map.gz / Format: CCP4 / Size: 325 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationunsharpened map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.73 Å/pix.
x 440 pix.
= 322.08 Å		0.73 Å/pix.
x 440 pix.
= 322.08 Å		0.73 Å/pix.
x 440 pix.
= 322.08 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.732 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.25
Minimum - Maximum-1.9767834 - 3.4046814
Average (Standard dev.)-0.00003836795 (±0.03568692)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions440440440
Spacing440440440
CellA=B=C: 322.08 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_44602_msk_1.map
Projections & Slices
AxesZYX

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Histogram

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Additional map: unsharpened map

Fileemd_44602_additional_2.map
Annotationunsharpened map
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Half map: unsharpened map

Fileemd_44602_half_map_1.map
Annotationunsharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: unsharpened map

Fileemd_44602_half_map_2.map
Annotationunsharpened map
Projections & Slices
AxesZYX

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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 and bound cloxacillin, pose 2
Components
  • Complex: Complex of the mammalian peptide transporter PepT2 with nanobody and bound cloxacillin, pose 2
    • Complex: PepT2
      • Protein or peptide: Solute carrier family 15 member 2
    • Complex: nanobody
      • Protein or peptide: nanobody
  • Ligand: CLOXACILLIN

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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 and bound cloxacillin, pose 2
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Rattus norvegicus (Norway rat)

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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)

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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

UniProtKB: Solute carrier family 15 member 2

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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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Macromolecule #3: CLOXACILLIN

MacromoleculeName: CLOXACILLIN / type: ligand / ID: 3 / Number of copies: 1 / Formula: CXN
Molecular weightTheoretical: 435.881 Da
Chemical component information

ChemComp-CXN:
CLOXACILLIN / antibiotic*YM

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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

MicroscopeTFS KRIOS
Image recordingFilm or detector model: TFS FALCON 4i (4k x 4k) / Average electron dose: 57.6 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 201206
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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