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- PDB-8wm7: Cryo-EM structure of cyanobacterial nitrate/nitrite transporter N... -

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Basic information

Entry
Database: PDB / ID: 8wm7
TitleCryo-EM structure of cyanobacterial nitrate/nitrite transporter NrtBCD in complex with signalling protein PII
Components
  • (Nitrate transport ATP-binding protein) x 2
  • Nitrate transport permease protein
  • Nitrogen regulatory protein P-II
KeywordsMEMBRANE PROTEIN / ATP-dependent transporter / ABC transporter / nitrate/nitrite transporter
Function / homology
Function and homology information


nitrate transmembrane transporter activity / regulation of nitrogen utilization / enzyme regulator activity / monoatomic ion transport / ATP hydrolysis activity / ATP binding / plasma membrane / cytosol
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 ...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 / P-II protein family profile. / Nitrogen regulatory protein PII / Nitrogen regulatory protein P-II / 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
ADENOSINE-5'-DIPHOSPHATE / 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. (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.53 Å
AuthorsLi, B. / Zhou, C.Z. / Chen, Y.X. / Jiang, Y.L.
Funding support China, 1items
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, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Feb 28, 2024Provider: repository / Type: Initial release
Revision 1.1Mar 20, 2024Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
B: Nitrate transport permease protein
A: Nitrate transport permease protein
C: Nitrate transport ATP-binding protein
D: Nitrate transport ATP-binding protein
E: Nitrogen regulatory protein P-II
F: Nitrogen regulatory protein P-II
G: Nitrogen regulatory protein P-II
hetero molecules


Theoretical massNumber of molelcules
Total (without water)210,84612
Polymers208,7107
Non-polymers2,1365
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Nitrate transport permease protein / ABC nitrate transport permease protein


Mass: 30483.053 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Nostoc sp. (bacteria) / Strain: PCC 7120 = FACHB-418 / Gene: nrtB / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): C43 / References: UniProt: Q8YZ77
#2: Protein Nitrate transport ATP-binding protein


Mass: 76326.141 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Nostoc sp. (bacteria) / Strain: PCC7120 = FACHB-418 / Gene: nrtC / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): C43 / References: UniProt: Q8YZ76, EC: 7.3.2.4
#3: Protein Nitrate transport ATP-binding protein


Mass: 31399.137 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Nostoc sp. (bacteria) / Strain: PCC 7120 = FACHB-418 / Gene: nrtD / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): C43 / References: UniProt: Q8YZ75
#4: Protein Nitrogen regulatory protein P-II


Mass: 13339.398 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Nostoc sp. (bacteria) / Strain: PCC 7120 = FACHB-418 / Gene: glnB / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): C43 / References: UniProt: Q9L422
#5: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
Has ligand of interestY

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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

ComponentName: Complex of nitrate transporter NrtBCD with signling protein PII
Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT
Source (natural)Organism: Nostoc sp. PCC 7120 = FACHB-418 (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: C43(DE3)
Buffer solutionpH: 7.5
SpecimenConc.: 8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2300 nm / Nominal defocus min: 1500 nm
Image recordingElectron dose: 55 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.19.2-4158 / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.53 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 304482 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 61.56 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.002413789
ELECTRON MICROSCOPYf_angle_d0.609718758
ELECTRON MICROSCOPYf_chiral_restr0.04382186
ELECTRON MICROSCOPYf_plane_restr0.00392368
ELECTRON MICROSCOPYf_dihedral_angle_d5.49721881

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