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- PDB-6jkd: Crystal structure of tetrameric PepTSo2 in I4 space group -

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Basic information

Entry
Database: PDB / ID: 6jkd
TitleCrystal structure of tetrameric PepTSo2 in I4 space group
ComponentsProton:oligopeptide symporter POT family
KeywordsMEMBRANE PROTEIN / Peptide transporter / LCP crystallization
Function / homology
Function and homology information


dipeptide transmembrane transport / tripeptide transmembrane transporter activity / peptide:proton symporter activity / dipeptide transmembrane transporter activity / peptide transport / identical protein binding / plasma membrane
Similarity search - Function
: / Dipeptide/tripeptide permease / MFS general substrate transporter like domains / Proton-dependent oligopeptide transporter family / POT family / Growth Hormone; Chain: A; / MFS transporter superfamily / Up-down Bundle / Mainly Alpha
Similarity search - Domain/homology
Proton:oligopeptide symporter POT family
Similarity search - Component
Biological speciesShewanella oneidensis MR-1 (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.9 Å
AuthorsNagamura, R. / Fukuda, M. / Ishitani, R. / Nureki, O.
Funding support Japan, 2items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science24227004 Japan
Japan Society for the Promotion of Science25291011 Japan
CitationJournal: Acta Crystallogr F Struct Biol Commun / Year: 2019
Title: Structural basis for oligomerization of the prokaryotic peptide transporter PepT.
Authors: Reina Nagamura / Masahiro Fukuda / Akihiro Kawamoto / Kyoko Matoba / Naoshi Dohmae / Ryuichiro Ishitani / Junichi Takagi / Osamu Nureki /
Abstract: Proton-dependent oligopeptide transporters (POTs) belong to the major facilitator superfamily (MFS) and transport dipeptides and tripeptides from the extracellular environment into the target cell. ...Proton-dependent oligopeptide transporters (POTs) belong to the major facilitator superfamily (MFS) and transport dipeptides and tripeptides from the extracellular environment into the target cell. The human POTs PepT1 and PepT2 are also involved in the absorption of various orally ingested drugs. Previously reported structures revealed that the bacterial POTs possess 14 helices, of which H1-H6 and H7-H12 constitute the typical MFS fold and the residual two helices are involved in the cytoplasmic linker. PepT from Shewanella oneidensis is a unique POT which reportedly assembles as a 200 kDa tetramer. Although the previously reported structures suggested the importance of H12 for tetramer formation, the structural basis for the PepT-specific oligomerization remains unclear owing to the lack of a high-resolution tetrameric structure. In this study, the expression and purification conditions for tetrameric PepT were optimized. A single-particle cryo-EM analysis revealed the tetrameric structure of PepT incorporated into Salipro nanoparticles at 4.1 Å resolution. Furthermore, a combination of lipidic cubic phase (LCP) crystallization and an automated data-processing system for multiple microcrystals enabled crystal structures of PepT to be determined at resolutions of 3.5 and 3.9 Å. The present structures in a lipid bilayer revealed the detailed mechanism for the tetrameric assembly of PepT, in which a characteristic extracellular loop (ECL) interacts with two asparagine residues on H12 which were reported to be important for tetramerization and plays an essential role in oligomeric assembly. This study provides valuable insights into the oligomerization mechanism of this MFS-type transporter, which will further pave the way for understanding other oligomeric membrane proteins.
History
DepositionFeb 28, 2019Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0May 15, 2019Provider: repository / Type: Initial release
Revision 1.1Nov 22, 2023Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Proton:oligopeptide symporter POT family


Theoretical massNumber of molelcules
Total (without water)57,6271
Polymers57,6271
Non-polymers00
Water00
1
A: Proton:oligopeptide symporter POT family

A: Proton:oligopeptide symporter POT family

A: Proton:oligopeptide symporter POT family

A: Proton:oligopeptide symporter POT family


Theoretical massNumber of molelcules
Total (without water)230,5064
Polymers230,5064
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_555-x,-y,z1
crystal symmetry operation3_555-y,x,z1
crystal symmetry operation4_555y,-x,z1
Buried area5880 Å2
ΔGint-56 kcal/mol
Surface area76010 Å2
MethodPISA
Unit cell
Length a, b, c (Å)115.150, 115.150, 110.100
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number79
Space group name H-MI4
Space group name HallI4

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Components

#1: Protein Proton:oligopeptide symporter POT family


Mass: 57626.559 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Shewanella oneidensis MR-1 (bacteria) / Strain: MR-1 / Gene: SO_1277 / Production host: Escherichia coli (E. coli) / References: UniProt: Q8EHE6

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 3.1 Å3/Da / Density % sol: 66.16 %
Crystal growTemperature: 293 K / Method: lipidic cubic phase / pH: 4
Details: 40% PEG 200, 100 mM Na malonate, pH 7.0, 100 mM Na acetate, pH 4.0

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SPring-8 / Beamline: BL32XU / Wavelength: 1 Å
DetectorType: DECTRIS EIGER X 9M / Detector: PIXEL / Date: Dec 20, 2017
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 3.9→50 Å / Num. obs: 6448 / % possible obs: 97.4 % / Redundancy: 3.6 % / Biso Wilson estimate: 79.96 Å2 / CC1/2: 0.946 / Rmerge(I) obs: 0.394 / Rrim(I) all: 0.455 / Net I/σ(I): 4.38
Reflection shellResolution: 3.9→4.14 Å / Redundancy: 3.7 % / Rmerge(I) obs: 1.109 / Num. unique obs: 1048 / CC1/2: 0.459 / Rrim(I) all: 1.277 / % possible all: 97.8

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Processing

Software
NameVersionClassification
REFMACrefinement
PHENIX1.14_3260refinement
XDSdata reduction
XSCALEdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 6JKC

6jkc


Resolution: 3.9→46.65 Å / SU ML: 0.569 / Cross valid method: THROUGHOUT / σ(F): 1.34 / Phase error: 30.6447
RfactorNum. reflection% reflectionSelection details
Rfree0.2832 630 9.81 %RANDOM
Rwork0.2524 ---
obs0.2556 6420 97.01 %-
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso mean: 70.28 Å2
Refinement stepCycle: LAST / Resolution: 3.9→46.65 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3476 0 0 0 3476
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00213564
X-RAY DIFFRACTIONf_angle_d0.50664860
X-RAY DIFFRACTIONf_chiral_restr0.0379569
X-RAY DIFFRACTIONf_plane_restr0.0039594
X-RAY DIFFRACTIONf_dihedral_angle_d12.67532014
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.9-4.040.3389530.2958566X-RAY DIFFRACTION96.87
4.04-4.20.3008670.2859575X-RAY DIFFRACTION97.42
4.2-4.390.3241650.2811596X-RAY DIFFRACTION97.06
4.39-4.620.4379580.3262553X-RAY DIFFRACTION95.62
4.62-4.910.2406600.2499592X-RAY DIFFRACTION97.46
4.91-5.290.2637570.2608579X-RAY DIFFRACTION97.25
5.29-5.820.2348660.2772582X-RAY DIFFRACTION97.74
5.82-6.660.3157700.2671573X-RAY DIFFRACTION97.57
6.66-8.390.2735620.2179588X-RAY DIFFRACTION97.45
8.39-46.650.2231720.1771586X-RAY DIFFRACTION95.92

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