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- PDB-9nbo: Closed conformation of ArsA from L. ferriphilum in complex with M... -

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Basic information

Entry
Database: PDB / ID: 9nbo
TitleClosed conformation of ArsA from L. ferriphilum in complex with MgATP and arsenite
ComponentsArsenite transporter ATPase-like protein,arsA
KeywordsHYDROLASE / ATPase / closed conformation / closed state / arsenite / arsenic / ATP / Intradimeric Walker A
Function / homology
Function and homology information


arsenite-transporting ATPase / ATPase-coupled arsenite transmembrane transporter activity / ATP hydrolysis activity / ATP binding
Similarity search - Function
Arsenical pump-driving ATPase / Arsenical pump ATPase, ArsA/GET3 / Anion-transporting ATPase-like domain / Anion-transporting ATPase / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ARSENIC / ADENOSINE-5'-TRIPHOSPHATE / arsenite-transporting ATPase
Similarity search - Component
Biological speciesLeptospirillum ferriphilum (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å
AuthorsMahajan, S. / Li, Y.E. / Rees, D.C. / Clemons, W.M.
Funding support United States, 1items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2025
Title: Nucleotide- and metalloid-driven conformational changes in the arsenite efflux ATPase ArsA.
Authors: Shivansh Mahajan / Ashley E Pall / Yancheng E Li / Timothy L Stemmler / Douglas C Rees / William M Clemons /
Abstract: Arsenite (As) is toxic to all organisms due to its ability to tightly bind exposed thiols within cells. An important As resistance mechanism in prokaryotes involves proteins encoded by the operon. A ...Arsenite (As) is toxic to all organisms due to its ability to tightly bind exposed thiols within cells. An important As resistance mechanism in prokaryotes involves proteins encoded by the operon. A central component of the operon in many bacteria is the cytoplasmic ATPase, ArsA, which orchestrates a series of nucleotide-dependent handoffs, starting with the capture of As by the ArsD metallochaperone and culminating in its removal from the cell by the ArsB efflux pump. Although the mechanism of ArsA has been widely studied, the molecular details of how nucleotide hydrolysis modulates these events remain unclear. ArsA is an archetypal member of the intradimeric Walker A (IWA) family of ATPases, implicated in a diversity of complex biological functions. Conformational changes typical of IWA ATPases have been postulated to drive these molecular events but have not been demonstrated. We report cryogenic electron microscopy (cryo-EM) structures of ArsA in MgADP-bound and MgATP-bound states, as well as a distinct MgATP-bound state liganded to As. X-ray absorption spectroscopy (XAS) confirmed three-coordinate binding of As to the conserved cysteines at the metalloid-binding site of the closed state. Coupled with biochemical characterization, our cryo-EM structures reveal key conformational changes in the ArsA catalytic cycle consistent with other IWA ATPases and provide the structural basis for allosteric activation of nucleotide hydrolysis by As. This work establishes how the nucleotide state of ArsA transiently creates a high-affinity binding site that can sequester metalloid within the cell, followed by a nucleotide-driven handoff to ArsB for efflux.
History
DepositionFeb 14, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 20, 2025Provider: repository / Type: Initial release
Revision 1.1Sep 10, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Arsenite transporter ATPase-like protein,arsA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)65,1086
Polymers63,9701
Non-polymers1,1385
Water905
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 Arsenite transporter ATPase-like protein,arsA


Mass: 63970.035 Da / Num. of mol.: 1 / Mutation: D46N,D364N
Source method: isolated from a genetically manipulated source
Details: non-hydrolyzing mutant / Source: (gene. exp.) Leptospirillum ferriphilum (bacteria) / Strain: ML-04 / Gene: LFML04_2459 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: J9ZFA3
#2: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
#3: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-ARS / ARSENIC


Mass: 74.922 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: As / Feature type: SUBJECT OF INVESTIGATION
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 5 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY
Has protein modificationN

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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: ArsA closed state in complex with MgATP and arsenite / Type: COMPLEX
Details: ArsA incubated with 2 mM each of MgCl2, ATP and sodium arsenite for 30 min
Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Leptospirillum ferriphilum (bacteria) / Strain: ML-04
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5
SpecimenConc.: 10 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: ArsA incubated with 2 mM each of MgCl2, ATP and sodium arsenite for 30 min
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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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 magnification: 130000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 500 nm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 70 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM softwareName: SerialEM / Category: image acquisition
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 91645 / Symmetry type: POINT

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