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- EMDB-37644: Cryo-EM structure of cyanobacterial nitrate/nitrite transporter N... -

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Basic information

Entry
Database: EMDB / ID: EMD-37644
TitleCryo-EM structure of cyanobacterial nitrate/nitrite transporter NrtBCD in complex with signalling protein PII
Map data
Sample
  • Complex: Complex of nitrate transporter NrtBCD with signling protein PII
    • Protein or peptide: Nitrate transport permease protein
    • Protein or peptide: Nitrate transport ATP-binding protein
    • Protein or peptide: Nitrate transport ATP-binding protein
    • Protein or peptide: Nitrogen regulatory protein P-II
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
KeywordsATP-dependent transporter / ABC transporter / nitrate/nitrite transporter / MEMBRANE PROTEIN
Function / homology
Function and homology information


nitrate transmembrane transporter activity / regulation of nitrogen utilization / enzyme regulator activity / monoatomic ion transport / nucleotide binding / ATP hydrolysis activity / ATP binding / membrane / plasma membrane
Similarity search - Function
Nitrate transport permease / Nitrate transport ATP-binding subunit C/D / Nitrate/bicarbonate ABC transporter substrate-binding domain / NMT1-like family / Nitrogen regulatory protein P-II, urydylation site / P-II protein uridylation site. / Nitrogen regulatory protein PII, conserved site / P-II protein C-terminal region signature. / Nitrogen regulatory protein P-II / Nitrogen regulatory protein P-II ...Nitrate transport permease / Nitrate transport ATP-binding subunit C/D / Nitrate/bicarbonate ABC transporter substrate-binding domain / NMT1-like family / Nitrogen regulatory protein P-II, urydylation site / P-II protein uridylation site. / Nitrogen regulatory protein PII, conserved site / P-II protein C-terminal region signature. / Nitrogen regulatory protein P-II / Nitrogen regulatory protein P-II / Nitrogen regulatory protein PII / P-II protein family profile. / ABC transporter type 1, transmembrane domain MetI-like / MetI-like superfamily / Binding-protein-dependent transport system inner membrane component / ABC transporter integral membrane type-1 domain profile. / Nitrogen regulatory PII-like, alpha/beta / Nitrogen regulatory protein PII/ATP phosphoribosyltransferase, C-terminal / ABC transporter-like, conserved site / ABC transporters family signature. / ABC transporter / ABC transporter-like, ATP-binding domain / ATP-binding cassette, ABC transporter-type domain profile. / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Nitrate transport ATP-binding protein / Nitrate transport ATP-binding protein / Nitrate transport permease protein / Nitrogen regulatory protein P-II
Similarity search - Component
Biological speciesNostoc sp. PCC 7120 = FACHB-418 (bacteria) / Nostoc sp. (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.53 Å
AuthorsLi B / Zhou CZ / Chen YX / Jiang YL
Funding support China, 1 items
OrganizationGrant numberCountry
Chinese Academy of SciencesXDB37020202 China
CitationJournal: Proc Natl Acad Sci U S A / Year: 2024
Title: Allosteric regulation of nitrate transporter NRT via the signaling protein PII.
Authors: Bo Li / Xiao-Qian Wang / Qin-Yao Li / Da Xu / Jing Li / Wen-Tao Hou / Yuxing Chen / Yong-Liang Jiang / Cong-Zhao Zhou /
Abstract: Coordinated carbon and nitrogen metabolism is crucial for bacteria living in the fluctuating environments. Intracellular carbon and nitrogen homeostasis is maintained by a sophisticated network, in ...Coordinated carbon and nitrogen metabolism is crucial for bacteria living in the fluctuating environments. Intracellular carbon and nitrogen homeostasis is maintained by a sophisticated network, in which the widespread signaling protein PII acts as a major regulatory hub. In cyanobacteria, PII was proposed to regulate the nitrate uptake by an ABC (ATP-binding cassette)-type nitrate transporter NrtABCD, in which the nucleotide-binding domain of NrtC is fused with a C-terminal regulatory domain (CRD). Here, we solved three cryoelectron microscopy structures of NrtBCD, bound to nitrate, ATP, and PII, respectively. Structural and biochemical analyses enable us to identify the key residues that form a hydrophobic and a hydrophilic cavity along the substrate translocation channel. The core structure of PII, but not the canonical T-loop, binds to NrtC and stabilizes the CRD, making it visible in the complex structure, narrows the substrate translocation channel in NrtB, and ultimately locks NrtBCD at an inhibited inward-facing conformation. Based on these results and previous reports, we propose a putative transport cycle driven by NrtABCD, which is allosterically inhibited by PII in response to the cellular level of 2-oxoglutarate. Our findings provide a distinct regulatory mechanism of ABC transporter via asymmetrically binding to a signaling protein.
History
DepositionOct 3, 2023-
Header (metadata) releaseFeb 28, 2024-
Map releaseFeb 28, 2024-
UpdateMar 20, 2024-
Current statusMar 20, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_37644.png

Downloads & links

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Map

FileDownload / File: emd_37644.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.07 Å
Density
Contour LevelBy AUTHOR: 0.243
Minimum - Maximum-1.7129498 - 2.9454226
Average (Standard dev.)0.0031051864 (±0.072175264)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 273.92 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_37644_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_37644_half_map_2.map
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 nitrate transporter NrtBCD with signling protein PII

EntireName: Complex of nitrate transporter NrtBCD with signling protein PII
Components
  • Complex: Complex of nitrate transporter NrtBCD with signling protein PII
    • Protein or peptide: Nitrate transport permease protein
    • Protein or peptide: Nitrate transport ATP-binding protein
    • Protein or peptide: Nitrate transport ATP-binding protein
    • Protein or peptide: Nitrogen regulatory protein P-II
  • Ligand: ADENOSINE-5'-DIPHOSPHATE

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Supramolecule #1: Complex of nitrate transporter NrtBCD with signling protein PII

SupramoleculeName: Complex of nitrate transporter NrtBCD with signling protein PII
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Nostoc sp. PCC 7120 = FACHB-418 (bacteria)

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Macromolecule #1: Nitrate transport permease protein

MacromoleculeName: Nitrate transport permease protein / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Nostoc sp. (bacteria) / Strain: PCC 7120 = FACHB-418
Molecular weightTheoretical: 30.483053 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MTAVLGNRAR VRKSQKAINN FLWKKVVPPL VALGIFLVIW QLLCLNPNFK LPGPIETFSE TWDPFIINPF FDNGESDKGL GWQILSSLG RVGLGFSLAA IAGIILGILI GVNPLVYNAV DPIFQVLRTV PPLAWLPISL AAFQQANPSA IFVIFITSIW P ILLNTTVG ...String:
MTAVLGNRAR VRKSQKAINN FLWKKVVPPL VALGIFLVIW QLLCLNPNFK LPGPIETFSE TWDPFIINPF FDNGESDKGL GWQILSSLG RVGLGFSLAA IAGIILGILI GVNPLVYNAV DPIFQVLRTV PPLAWLPISL AAFQQANPSA IFVIFITSIW P ILLNTTVG VQQIPQDYIN VAKVLRLKGV KYFFKIVFPA TVPYIFTGLR IGIGLSWLAI VAAEMLVGGV GIGSFIWDAY NT TTETNLS EIILALIYVG LVGLLLDRLV GFVASKVVAD QK

UniProtKB: Nitrate transport permease protein

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Macromolecule #2: Nitrate transport ATP-binding protein

MacromoleculeName: Nitrate transport ATP-binding protein / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: ec: 7.3.2.4
Source (natural)Organism: Nostoc sp. (bacteria) / Strain: PCC7120 = FACHB-418
Molecular weightTheoretical: 76.326141 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MPTFVEIDHV DRIFDLPNGG RYIALKNIEL KIKQGEFVSL IGHSGCGKST LLNIIAGLDR ASIGGVTLEG REIREPSPDR MVVFQNYSL LPWLTVRENV ALAVDEVYQG KSKGERRAII EEHIDMVGLR LAANKRPSEL SGGMKQRVAI ARALATRPKL L LLDEPFGA ...String:
MPTFVEIDHV DRIFDLPNGG RYIALKNIEL KIKQGEFVSL IGHSGCGKST LLNIIAGLDR ASIGGVTLEG REIREPSPDR MVVFQNYSL LPWLTVRENV ALAVDEVYQG KSKGERRAII EEHIDMVGLR LAANKRPSEL SGGMKQRVAI ARALATRPKL L LLDEPFGA LDALTRGSLQ EQLMKICNEH QITCVMVTHD VDEALLLSDR VVMLTNGPEA HIGQILEVPI SRPRQRLEVV KH PSYYNLR NEIIYFLNQQ KLAKKRQTQQ ASAPLGTAKA VIEIGFMPLT DSAPLIVAKE KGFFAKYGLD NVILNRANNW QAI ATGVVT GKLDAAQMVA GMPIALTLGA GSQTPTPVIN ALNLSRNANA ITFSKRLYNQ GVRSLADLKA VIDSSPDQIL TLGV VHSAS MQNLILRYWL AAGGIDPDRD VSLTVIPPTQ MVSQLKAGNI DGYCAGEPWN YQAVHDDLGF VAATALEIWS GQPKK VLGV REDWAQKYPE TYLNLVKALI EACKYCDDLR NREEILEILC RPEYLDVNPA YVRSGFIDPY DRGDGTPPQQ LTAYNQ FYL NKTNYPNRTE ILWMITQMAR WGLTPFPKNW VEITERVCRT DIFGAAARDL GLLDIGEDDP IHLFDGKLFN PSEPIEY LK SLEIRRQIRI EEVFISSGDY KDHDGDYKDH DIDYKDDDDK

UniProtKB: Nitrate transport ATP-binding protein

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Macromolecule #3: Nitrate transport ATP-binding protein

MacromoleculeName: Nitrate transport ATP-binding protein / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Nostoc sp. (bacteria) / Strain: PCC 7120 = FACHB-418
Molecular weightTheoretical: 31.399137 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MQIINRNNQT NLKPQKTDNF LVVEGVSKIY PTPEGPYTVL DGIDLKVREG EFVCLIGHSG CGKSTLLNMI SGFNTPSEGV VLLQDKPIT EPGPDRMMVF QNYCLLPWLN VFENVYLAVD AVFPNKPQAE KRAIVREHLA MVGLTEAAEK KPSQISGGMK Q RVAIARAL ...String:
MQIINRNNQT NLKPQKTDNF LVVEGVSKIY PTPEGPYTVL DGIDLKVREG EFVCLIGHSG CGKSTLLNMI SGFNTPSEGV VLLQDKPIT EPGPDRMMVF QNYCLLPWLN VFENVYLAVD AVFPNKPQAE KRAIVREHLA MVGLTEAAEK KPSQISGGMK Q RVAIARAL SIRPQVLILD EPFGALDAIT KEELQEELLQ IWSDHQVTVL MITHDIDEAL FLADRVVMMT NGPAAQIGEI LD IPFDRPR NRRRIMEDPK YYDLRNYALD FLFNRFAHNE

UniProtKB: Nitrate transport ATP-binding protein

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Macromolecule #4: Nitrogen regulatory protein P-II

MacromoleculeName: Nitrogen regulatory protein P-II / type: protein_or_peptide / ID: 4 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Nostoc sp. (bacteria) / Strain: PCC 7120 = FACHB-418
Molecular weightTheoretical: 13.339398 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MKKVEAIIRP FKLDEVKIAL VNAGIVGMTV SEVRGFGRQK GQTERYRGSE YTVEFLQKLK VEIVVEDNQV DMVVDKIIAA ARTGEIGDG KIFISPVEQV IRIRTGEKNT EAVHHHHHH

UniProtKB: Nitrogen regulatory protein P-II

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Macromolecule #5: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 5 / Number of copies: 5 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM / Adenosine diphosphate

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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

Concentration8 mg/mL
BufferpH: 7.5
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV

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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 / Nominal defocus max: 2.3000000000000003 µm / Nominal defocus min: 1.5 µm
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 55.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.53 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 304482

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