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- PDB-7p6k: Structure of homomeric LRRC8A Volume-Regulated Anion Channel in c... -

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Basic information

Entry
Database: PDB / ID: 7p6k
TitleStructure of homomeric LRRC8A Volume-Regulated Anion Channel in complex with synthetic nanobody Sb5
Components
  • Volume-regulated anion channel subunit LRRC8A
  • synthetic nanobody Sb5
KeywordsMEMBRANE PROTEIN / LRRC8 family / Volume-Regulated Anion Channel / leucine-rich repeat / sybody / cryo-EM
Function / homology
Function and homology information


Miscellaneous transport and binding events / pre-B cell differentiation / volume-sensitive anion channel activity / taurine transmembrane transport / monoatomic anion transmembrane transport / aspartate transmembrane transport / cyclic-GMP-AMP transmembrane transporter activity / cyclic-GMP-AMP transmembrane import across plasma membrane / monoatomic anion transport / cell volume homeostasis ...Miscellaneous transport and binding events / pre-B cell differentiation / volume-sensitive anion channel activity / taurine transmembrane transport / monoatomic anion transmembrane transport / aspartate transmembrane transport / cyclic-GMP-AMP transmembrane transporter activity / cyclic-GMP-AMP transmembrane import across plasma membrane / monoatomic anion transport / cell volume homeostasis / protein hexamerization / response to osmotic stress / monoatomic ion channel complex / intracellular glucose homeostasis / positive regulation of myoblast differentiation / chloride transmembrane transport / positive regulation of insulin secretion / spermatogenesis / lysosomal membrane / cell surface / signal transduction / membrane / identical protein binding / plasma membrane
Similarity search - Function
LRRC8, pannexin-like TM region / Pannexin-like TM region of LRRC8 / Leucine-rich repeat, SDS22-like subfamily / Leucine rich repeat / Leucine-rich repeat, typical subtype / Leucine-rich repeats, typical (most populated) subfamily / Leucine-rich repeat profile. / Leucine-rich repeat / Leucine-rich repeat domain superfamily
Similarity search - Domain/homology
Volume-regulated anion channel subunit LRRC8A
Similarity search - Component
Biological speciesMus musculus (house mouse)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsDeneka, D. / Rutz, S. / Sawicka, M.
Funding support1items
OrganizationGrant numberCountry
Swiss National Science Foundation
CitationJournal: Nat Commun / Year: 2021
Title: Allosteric modulation of LRRC8 channels by targeting their cytoplasmic domains.
Authors: Dawid Deneka / Sonja Rutz / Cedric A J Hutter / Markus A Seeger / Marta Sawicka / Raimund Dutzler /
Abstract: Members of the LRRC8 family form heteromeric assemblies, which function as volume-regulated anion channels. These modular proteins consist of a transmembrane pore and cytoplasmic leucine-rich repeat ...Members of the LRRC8 family form heteromeric assemblies, which function as volume-regulated anion channels. These modular proteins consist of a transmembrane pore and cytoplasmic leucine-rich repeat (LRR) domains. Despite their known molecular architecture, the mechanism of activation and the role of the LRR domains in this process has remained elusive. Here we address this question by generating synthetic nanobodies, termed sybodies, which target the LRR domain of the obligatory subunit LRRC8A. We use these binders to investigate their interaction with homomeric LRRC8A channels by cryo-electron microscopy and the consequent effect on channel activation by electrophysiology. The five identified sybodies either inhibit or enhance activity by binding to distinct epitopes of the LRR domain, thereby altering channel conformations. In combination, our work provides a set of specific modulators of LRRC8 proteins and reveals the role of their cytoplasmic domains as regulators of channel activity by allosteric mechanisms.
History
DepositionJul 16, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 15, 2021Provider: repository / Type: Initial release
Revision 1.1Oct 13, 2021Group: Data collection / Database references
Category: citation / citation_author ...citation / citation_author / database_PDB_rev / database_PDB_rev_record / em_admin / pdbx_database_proc
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _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

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Assembly

Deposited unit
A: Volume-regulated anion channel subunit LRRC8A
B: Volume-regulated anion channel subunit LRRC8A
C: Volume-regulated anion channel subunit LRRC8A
D: Volume-regulated anion channel subunit LRRC8A
E: Volume-regulated anion channel subunit LRRC8A
F: Volume-regulated anion channel subunit LRRC8A
I: synthetic nanobody Sb5
G: synthetic nanobody Sb5
H: synthetic nanobody Sb5


Theoretical massNumber of molelcules
Total (without water)611,8259
Polymers611,8259
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Volume-regulated anion channel subunit LRRC8A / / Leucine-rich repeat-containing protein 8A


Mass: 94239.383 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Lrrc8a, Lrrc8 / Production host: Homo sapiens (human) / References: UniProt: Q80WG5
#2: Antibody synthetic nanobody Sb5


Mass: 15463.057 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli)

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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: Homomeric LRRC8A Volume-Regulated Anion Channel in complex with synthetic nanobody Sb5
Type: COMPLEX / Entity ID: all / Source: MULTIPLE SOURCES
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE-PROPANE

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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 FIELDBright-field microscopy
Image recordingElectron dose: 67 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.1_4122: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 48917 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00238952
ELECTRON MICROSCOPYf_angle_d0.4252800
ELECTRON MICROSCOPYf_dihedral_angle_d11.36514538
ELECTRON MICROSCOPYf_chiral_restr0.0386081
ELECTRON MICROSCOPYf_plane_restr0.0036558

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