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- PDB-8k1l: Cryo-EM structure of Na+,K+-ATPase alpha2 from Artemia salina in ... -

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Entry
Database: PDB / ID: 8k1l
TitleCryo-EM structure of Na+,K+-ATPase alpha2 from Artemia salina in cation-free E2P form
Components
  • Na+,K+-ATPase alpha2KK
  • Na+,K+-ATPase beta2
KeywordsTRANSPORT PROTEIN / P-type ATPase / sodium pump / membrane protein / transporter
Function / homologyTETRAFLUOROALUMINATE ION
Function and homology information
Biological speciesArtemia salina (crustacean)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.44 Å
AuthorsAbe, K. / Artigas, P.
Funding support Japan, 1items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)21H02426 Japan
CitationJournal: Proc Natl Acad Sci U S A / Year: 2023
Title: A Na pump with reduced stoichiometry is up-regulated by brine shrimp in extreme salinities.
Authors: Pablo Artigas / Dylan J Meyer / Victoria C Young / Kerri Spontarelli / Jessica Eastman / Evan Strandquist / Huan Rui / Benoît Roux / Matthew A Birk / Hanayo Nakanishi / Kazuhiro Abe / Craig Gatto /
Abstract: Brine shrimp () are the only animals to thrive at sodium concentrations above 4 M. Salt excretion is powered by the Na,K-ATPase (NKA), a heterodimeric (αβ) pump that usually exports 3Na in exchange ...Brine shrimp () are the only animals to thrive at sodium concentrations above 4 M. Salt excretion is powered by the Na,K-ATPase (NKA), a heterodimeric (αβ) pump that usually exports 3Na in exchange for 2 K per hydrolyzed ATP. express several NKA catalytic α-subunit subtypes. High-salinity adaptation increases abundance of α2, an isoform that contains two lysines (Lys308 and Lys758 in transmembrane segments TM4 and TM5, respectively) at positions where canonical NKAs have asparagines ( α1's Asn333 and Asn785). Using de novo transcriptome assembly and qPCR, we found that express two salinity-independent canonical α subunits (α1 and α3), as well as two β variants, in addition to the salinity-controlled α2. These β subunits permitted heterologous expression of the α2 pump and determination of its CryoEM structure in a closed, ion-free conformation, showing Lys758 residing within the ion-binding cavity. We used electrophysiology to characterize the function of α2 pumps and compared it to that of α1 (and its α2-mimicking single- and double-lysine substitutions). The double substitution N333K/N785K confers α2-like characteristics to α1, and mutant cycle analysis reveals energetic coupling between these two residues, illustrating how α2's Lys308 helps to maintain high affinity for external K when Lys758 occupies an ion-binding site. By measuring uptake under voltage clamp of the K-congener Rb, we prove that double-lysine-substituted pumps transport 2Na and 1 K per catalytic cycle. Our results show how the two lysines contribute to generate a pump with reduced stoichiometry allowing to maintain steeper Na gradients in hypersaline environments.
History
DepositionJul 11, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Nov 29, 2023Provider: repository / Type: Initial release
Revision 1.1Jan 31, 2024Group: Database references / Category: citation / citation_author
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

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Structure visualization

Structure viewerMolecule:
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Assembly

Deposited unit
A: Na+,K+-ATPase alpha2KK
B: Na+,K+-ATPase beta2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)149,7443
Polymers149,6412
Non-polymers1031
Water0
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 Na+,K+-ATPase alpha2KK


Mass: 111144.211 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Artemia salina (crustacean) / Production host: Homo sapiens (human)
#2: Protein Na+,K+-ATPase beta2


Mass: 38496.422 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Artemia salina (crustacean) / Production host: Homo sapiens (human)
#3: Chemical ChemComp-ALF / TETRAFLUOROALUMINATE ION


Mass: 102.975 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: AlF4

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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: Na+,K+-ATPase alpha2/beta2 from Artemia Salina / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.13 MDa / Experimental value: YES
Source (natural)Organism: Artemia salina (crustacean)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7
SpecimenConc.: 8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

MicroscopyModel: JEOL CRYO ARM 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 1500 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

CTF correctionType: NONE
3D reconstructionResolution: 3.44 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 39483 / Symmetry type: POINT
RefinementCross valid method: NONE

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