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- PDB-9bit: Cryo-EM structure of the mammalian peptide transporter PepT2 boun... -

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Basic information

Entry
Database: PDB / ID: 9bit
TitleCryo-EM structure of the mammalian peptide transporter PepT2 bound to cloxacillin, pose 1
Components
  • Solute carrier family 15 member 2
  • nanobody
KeywordsMEMBRANE PROTEIN / protein-coupled peptide transporter / peptide transport / antibiotics
Function / homology
Function and homology information


high-affinity oligopeptide transmembrane transporter activity / tripeptide import across plasma membrane / oligopeptide transport / dipeptide transport / peptidoglycan transport / dipeptide import across plasma membrane / tripeptide transmembrane transporter activity / metanephric proximal tubule development / peptide:proton symporter activity / antibacterial innate immune response ...high-affinity oligopeptide transmembrane transporter activity / tripeptide import across plasma membrane / oligopeptide transport / dipeptide transport / peptidoglycan transport / dipeptide import across plasma membrane / tripeptide transmembrane transporter activity / metanephric proximal tubule development / peptide:proton symporter activity / antibacterial innate immune response / 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 / 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
CLOXACILLIN / 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: 3.1 Å
AuthorsParker, J.L. / Deme, J.C. / Lea, S.M. / Newstead, S.
Funding support United Kingdom, United States, 4items
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, 2024Deposition site: RCSB / Processing site: RCSB
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Revision 1.1Oct 30, 2024Group: Data collection / Database references / Structure summary
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)97,4083
Polymers96,9722
Non-polymers4361
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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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)
#3: Chemical ChemComp-CXN / CLOXACILLIN / [3-(O-CHLOROPHENYL)-5-METHYL-4-ISOXAZOLYL]PENICILLIN


Mass: 435.881 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C19H18ClN3O5S / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
Has protein modificationY

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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 nanobody and bound cloxacillin, pose 1COMPLEX#1-#20MULTIPLE SOURCES
2PepT2COMPLEX#11RECOMBINANT
3nanobodyCOMPLEX#21NATURAL
Molecular weight
IDEntity assembly-IDExperimental value
11NO
21NO
31NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Rattus norvegicus (Norway rat)10116
32Rattus norvegicus (Norway rat)10116
43Lama glama (llama)9844
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
21Homo sapiens (human)9606
32Homo sapiens (human)9606
43Homo sapiens (human)9606
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: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 500 nm
Image recordingElectron dose: 57.6 e/Å2 / Film or detector model: TFS FALCON 4i (4k x 4k)

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Processing

EM softwareName: PHENIX / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 106684 / Symmetry type: POINT

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