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- PDB-8dfl: Structure of human Kv1.3 with A0194009G09 nanobodies (alternate c... -

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Basic information

Entry
Database: PDB / ID: 8dfl
TitleStructure of human Kv1.3 with A0194009G09 nanobodies (alternate conformation)
Components
  • Nanobody A0194009G09
  • Potassium voltage-gated channel subfamily A member 3,Green fluorescent protein fusion
KeywordsIMMUNE SYSTEM / ion channel
Function / homology
Function and homology information


voltage-gated monoatomic ion channel activity / delayed rectifier potassium channel activity / Voltage gated Potassium channels / action potential / voltage-gated potassium channel activity / voltage-gated potassium channel complex / potassium ion transmembrane transport / bioluminescence / generation of precursor metabolites and energy / protein homooligomerization ...voltage-gated monoatomic ion channel activity / delayed rectifier potassium channel activity / Voltage gated Potassium channels / action potential / voltage-gated potassium channel activity / voltage-gated potassium channel complex / potassium ion transmembrane transport / bioluminescence / generation of precursor metabolites and energy / protein homooligomerization / potassium ion transport / axon / perinuclear region of cytoplasm / membrane / plasma membrane
Similarity search - Function
Potassium channel, voltage dependent, Kv1.3 / Potassium channel, voltage dependent, Kv1 / Potassium channel, voltage dependent, Kv / Potassium channel tetramerisation-type BTB domain / BTB/POZ domain / Voltage-gated potassium channel / Broad-Complex, Tramtrack and Bric a brac / BTB/POZ domain / Voltage-dependent channel domain superfamily / Green fluorescent protein, GFP ...Potassium channel, voltage dependent, Kv1.3 / Potassium channel, voltage dependent, Kv1 / Potassium channel, voltage dependent, Kv / Potassium channel tetramerisation-type BTB domain / BTB/POZ domain / Voltage-gated potassium channel / Broad-Complex, Tramtrack and Bric a brac / BTB/POZ domain / Voltage-dependent channel domain superfamily / Green fluorescent protein, GFP / Green fluorescent protein-related / Green fluorescent protein / Green fluorescent protein / SKP1/BTB/POZ domain superfamily / Ion transport domain / Ion transport protein / Immunoglobulins / Immunoglobulin-like / Sandwich / Mainly Beta
Similarity search - Domain/homology
: / Potassium voltage-gated channel subfamily A member 3 / Green fluorescent protein
Similarity search - Component
Biological speciesHomo sapiens (human)
Aequorea victoria (jellyfish)
Lama glama (llama)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.25 Å
AuthorsMeyerson, J.R. / Selvakumar, P.
Funding support United States, 1items
OrganizationGrant numberCountry
Not funded United States
CitationJournal: Nat Commun / Year: 2022
Title: Structures of the T cell potassium channel Kv1.3 with immunoglobulin modulators.
Authors: Purushotham Selvakumar / Ana I Fernández-Mariño / Nandish Khanra / Changhao He / Alice J Paquette / Bing Wang / Ruiqi Huang / Vaughn V Smider / William J Rice / Kenton J Swartz / Joel R Meyerson /
Abstract: The Kv1.3 potassium channel is expressed abundantly on activated T cells and mediates the cellular immune response. This role has made the channel a target for therapeutic immunomodulation to block ...The Kv1.3 potassium channel is expressed abundantly on activated T cells and mediates the cellular immune response. This role has made the channel a target for therapeutic immunomodulation to block its activity and suppress T cell activation. Here, we report structures of human Kv1.3 alone, with a nanobody inhibitor, and with an antibody-toxin fusion blocker. Rather than block the channel directly, four copies of the nanobody bind the tetramer's voltage sensing domains and the pore domain to induce an inactive pore conformation. In contrast, the antibody-toxin fusion docks its toxin domain at the extracellular mouth of the channel to insert a critical lysine into the pore. The lysine stabilizes an active conformation of the pore yet blocks ion permeation. This study visualizes Kv1.3 pore dynamics, defines two distinct mechanisms to suppress Kv1.3 channel activity with exogenous inhibitors, and provides a framework to aid development of emerging T cell immunotherapies.
History
DepositionJun 22, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 13, 2022Provider: repository / Type: Initial release
Revision 1.1May 1, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / citation
Item: _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Oct 16, 2024Group: Structure summary / Category: pdbx_entry_details / pdbx_modification_feature / Item: _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Potassium voltage-gated channel subfamily A member 3,Green fluorescent protein fusion
E: Nanobody A0194009G09
F: Nanobody A0194009G09
G: Nanobody A0194009G09
H: Nanobody A0194009G09
B: Potassium voltage-gated channel subfamily A member 3,Green fluorescent protein fusion
C: Potassium voltage-gated channel subfamily A member 3,Green fluorescent protein fusion
D: Potassium voltage-gated channel subfamily A member 3,Green fluorescent protein fusion
hetero molecules


Theoretical massNumber of molelcules
Total (without water)435,58911
Polymers435,4728
Non-polymers1173
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Potassium voltage-gated channel subfamily A member 3,Green fluorescent protein fusion / HGK5 / HLK3 / HPCN3 / Voltage-gated K(+) channel HuKIII / Voltage-gated potassium channel subunit Kv1.3


Mass: 95018.500 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human), (gene. exp.) Aequorea victoria (jellyfish)
Gene: KCNA3, HGK5, GFP / Production host: Homo sapiens (human) / References: UniProt: P22001, UniProt: P42212
#2: Antibody
Nanobody A0194009G09


Mass: 13849.449 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lama glama (llama) / Production host: Escherichia coli (E. coli)
#3: Chemical ChemComp-K / POTASSIUM ION


Mass: 39.098 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: K / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
Has protein modificationY

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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

Component
IDNameTypeEntity IDParent-IDSource
1Kv1.3 with nanobody A0194009G09COMPLEX#1-#20RECOMBINANT
2Potassium voltage-gated channel subfamily A member 3COMPLEX#11RECOMBINANT
3Nanobody A0194009G09COMPLEX#21RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23Lama glama (llama)9844
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23Escherichia coli (E. coli)562
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen 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: OTHER
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 1300 nm
Image recordingElectron dose: 54 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.25 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 123722 / Symmetry type: POINT

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