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- PDB-8r3y: Cryo EM structure of a stable LGL/aPKC Iota/Par-6 complex -

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Basic information

Entry
Database: PDB / ID: 8r3y
TitleCryo EM structure of a stable LGL/aPKC Iota/Par-6 complex
Components
  • Lethal(2) giant larvae protein homolog 1
  • Partitioning defective 6 homolog alpha
  • Protein kinase C iota type
KeywordsCYTOSOLIC PROTEIN / Kinase / Polarity / Kinase substrate complex.
Function / homology
Function and homology information


Golgi cis cisterna / regulation of cellular localization / establishment of spindle orientation / diacylglycerol-dependent, calcium-independent serine/threonine kinase activity / Golgi vesicle budding / PAR polarity complex / Tight junction interactions / regulation of establishment or maintenance of cell polarity / cell-cell junction maintenance / protein kinase C ...Golgi cis cisterna / regulation of cellular localization / establishment of spindle orientation / diacylglycerol-dependent, calcium-independent serine/threonine kinase activity / Golgi vesicle budding / PAR polarity complex / Tight junction interactions / regulation of establishment or maintenance of cell polarity / cell-cell junction maintenance / protein kinase C / establishment of apical/basal cell polarity / diacylglycerol-dependent serine/threonine kinase activity / myosin II binding / regulation of Notch signaling pathway / negative regulation of glial cell apoptotic process / eye photoreceptor cell development / positive regulation of protein localization to centrosome / Schmidt-Lanterman incisure / GTP-dependent protein binding / establishment or maintenance of epithelial cell apical/basal polarity / Golgi to plasma membrane transport / cellular response to chemical stress / membrane organization / cell-cell junction organization / centrosome cycle / cortical actin cytoskeleton organization / protein targeting to membrane / tight junction / RHOV GTPase cycle / cortical actin cytoskeleton / positive regulation of Notch signaling pathway / establishment of cell polarity / establishment or maintenance of cell polarity / cell leading edge / exocytosis / RHOU GTPase cycle / brush border / CDC42 GTPase cycle / positive regulation of glial cell proliferation / positive regulation of endothelial cell apoptotic process / viral process / bicellular tight junction / regulation of postsynaptic membrane neurotransmitter receptor levels / intercellular bridge / vesicle-mediated transport / ruffle / RAC1 GTPase cycle / cytoskeleton organization / secretion / p75NTR recruits signalling complexes / axonogenesis / response to interleukin-1 / GTPase activator activity / actin filament organization / trans-Golgi network membrane / TGF-beta receptor signaling in EMT (epithelial to mesenchymal transition) / positive regulation of protein secretion / positive regulation of D-glucose import / protein localization to plasma membrane / Asymmetric localization of PCP proteins / positive regulation of protein localization to plasma membrane / adherens junction / positive regulation of NF-kappaB transcription factor activity / positive regulation of neuron projection development / phospholipid binding / small GTPase binding / Pre-NOTCH Transcription and Translation / centriolar satellite / Schaffer collateral - CA1 synapse / cellular response to insulin stimulus / KEAP1-NFE2L2 pathway / cell migration / microtubule cytoskeleton / protein-containing complex assembly / early endosome membrane / cell cortex / negative regulation of neuron apoptotic process / cytoskeleton / protein phosphorylation / protein kinase activity / endosome / intracellular signal transduction / cilium / apical plasma membrane / Golgi membrane / axon / cell division / protein serine kinase activity / intracellular membrane-bounded organelle / protein serine/threonine kinase activity / centrosome / protein kinase binding / negative regulation of apoptotic process / glutamatergic synapse / structural molecule activity / extracellular exosome / zinc ion binding / nucleoplasm / ATP binding / nucleus
Similarity search - Function
Partitioning defective protein 6, PB1 domain / : / Lethal(2) giant larvae protein / Lethal giant larvae homologue 2 / LLGL2 / Atypical protein kinase C iota type, catalytic domain / Protein kinase C / Protein kinase C, PB1 domain / PB1 domain / PB1 domain ...Partitioning defective protein 6, PB1 domain / : / Lethal(2) giant larvae protein / Lethal giant larvae homologue 2 / LLGL2 / Atypical protein kinase C iota type, catalytic domain / Protein kinase C / Protein kinase C, PB1 domain / PB1 domain / PB1 domain / PB1 domain / : / PB1 domain profile. / Protein kinase, C-terminal / Protein kinase C terminal domain / Diacylglycerol/phorbol-ester binding / Phorbol esters/diacylglycerol binding domain (C1 domain) / Zinc finger phorbol-ester/DAG-type signature. / Zinc finger phorbol-ester/DAG-type profile. / Protein kinase C conserved region 1 (C1) domains (Cysteine-rich domains) / Protein kinase C-like, phorbol ester/diacylglycerol-binding domain / C1-like domain superfamily / Extension to Ser/Thr-type protein kinases / AGC-kinase, C-terminal / AGC-kinase C-terminal domain profile. / PDZ domain / PDZ domain profile. / Domain present in PSD-95, Dlg, and ZO-1/2. / PDZ domain / PDZ superfamily / Trp-Asp (WD) repeats signature. / Trp-Asp (WD) repeats profile. / Serine/threonine-protein kinase, active site / Serine/Threonine protein kinases active-site signature. / WD40 repeats / WD40 repeat / Protein kinase domain / WD40-repeat-containing domain superfamily / Serine/Threonine protein kinases, catalytic domain / WD40/YVTN repeat-like-containing domain superfamily / Protein kinase, ATP binding site / Protein kinases ATP-binding region signature. / Protein kinase domain profile. / Protein kinase domain / Protein kinase-like domain superfamily
Similarity search - Domain/homology
PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / Protein kinase C iota type / Lethal(2) giant larvae protein homolog 1 / Partitioning defective 6 homolog alpha
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.68 Å
AuthorsEarl, C.P. / Briggs, D.C. / McDonald, N.Q.
Funding support United Kingdom, 3items
OrganizationGrant numberCountry
Wellcome TrustCC2068 United Kingdom
Cancer Research UKCC2026 United Kingdom
Medical Research Council (MRC, United Kingdom)CC2068 United Kingdom
CitationJournal: Nat Struct Mol Biol / Year: 2025
Title: Capture, mutual inhibition and release mechanism for aPKC-Par6 and its multisite polarity substrate Lgl.
Authors: Christopher P Earl / Mathias Cobbaut / André Barros-Carvalho / Marina E Ivanova / David C Briggs / Eurico Morais-de-Sá / Peter J Parker / Neil Q McDonald /
Abstract: The mutually antagonistic relationship of atypical protein kinase C (aPKC) and partitioning-defective protein 6 (Par6) with the substrate lethal (2) giant larvae (Lgl) is essential for regulating ...The mutually antagonistic relationship of atypical protein kinase C (aPKC) and partitioning-defective protein 6 (Par6) with the substrate lethal (2) giant larvae (Lgl) is essential for regulating polarity across many cell types. Although aPKC-Par6 phosphorylates Lgl at three serine sites to exclude it from the apical domain, aPKC-Par6 and Lgl paradoxically form a stable kinase-substrate complex, with conflicting roles proposed for Par6. We report the structure of human aPKCι-Par6α bound to full-length Llgl1, captured through an aPKCι docking site and a Par6 contact. This complex traps a phospho-S663 Llgl1 intermediate bridging between aPKC and Par6, impeding phosphorylation progression. Thus, aPKCι is effectively inhibited by Llgl1 while Llgl1 is captured by aPKCι-Par6. Mutational disruption of the Lgl-aPKC interaction impedes complex assembly and Lgl phosphorylation, whereas disrupting the Lgl-Par6 contact promotes complex dissociation and Lgl phosphorylation. We demonstrate a Par6-regulated substrate capture-and-release model requiring binding by active Cdc42 and the apical partner Crumbs to drive complex disassembly. Our results suggest a mechanism for mutual regulation and spatial control of aPKC-Par6 and Lgl activities.
History
DepositionNov 10, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 20, 2024Provider: repository / Type: Initial release
Revision 1.1Jan 15, 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_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name / _em_admin.last_update
Revision 1.2Apr 23, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
I: Protein kinase C iota type
L: Lethal(2) giant larvae protein homolog 1
P: Partitioning defective 6 homolog alpha
hetero molecules


Theoretical massNumber of molelcules
Total (without water)152,3994
Polymers151,8923
Non-polymers5061
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable, gel filtration, Complex gel filters as a stable complex
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Protein kinase C iota type / Atypical protein kinase C-lambda/iota / PRKC-lambda/iota / aPKC-lambda/iota / nPKC-iota


Mass: 39146.004 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PRKCI, DXS1179E / Cell line (production host): HEK293 FreeStyle / Production host: Homo sapiens (human) / References: UniProt: P41743, protein kinase C
#2: Protein Lethal(2) giant larvae protein homolog 1 / LLGL / DLG4 / Hugl-1 / Human homolog to the D-lgl gene protein


Mass: 101889.805 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: LLGL1, DLG4, HUGL, HUGL1 / Cell line (production host): HEK293 Freestyle / Production host: Homo sapiens (human) / References: UniProt: Q15334
#3: Protein Partitioning defective 6 homolog alpha / PAR-6 / PAR-6 alpha / PAR-6A / PAR6C / Tax interaction protein 40 / TIP-40


Mass: 10856.517 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PARD6A, PAR6A / Cell line (production host): HEK293 Freestyle / Production host: Homo sapiens (human) / References: UniProt: Q9NPB6
#4: Chemical ChemComp-ANP / PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER


Mass: 506.196 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H17N6O12P3 / Comment: AMP-PNP, energy-carrying molecule analogue*YM
Has ligand of interestN
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

ComponentName: aPKCiota-Par6-Llgl1 complex / Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightValue: 0.22 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK293 Freestyle
Buffer solutionpH: 7.5
Buffer component
IDConc.NameBuffer-ID
120 mMHEPES pH 7.51
2150 mMSodium Chloride1
30.5 mMTCEP1
SpecimenConc.: 0.4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Sample was double affinity purified and gel filtered. Sample was monodisperse.
Specimen supportDetails: 45mA / Grid material: COPPER / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 298 K
Details: 4 ul of aPKCiota-Par6-Llgl1 complex at a concentration of 0.4 mg/ml was applied to R1.2/1.3 Quantifoil 300 mesh copper grids which had been glow-discharged for 45 s at 45 mA . Grids were ...Details: 4 ul of aPKCiota-Par6-Llgl1 complex at a concentration of 0.4 mg/ml was applied to R1.2/1.3 Quantifoil 300 mesh copper grids which had been glow-discharged for 45 s at 45 mA . Grids were blotted for 2.5 s at 100% humidity using an FEI Vitrobot MK IV.

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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: 3000 nm / Nominal defocus min: 1200 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 8 sec. / Electron dose: 48.1 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 4002
EM imaging opticsEnergyfilter name: GIF Quantum ER
Image scansMovie frames/image: 40

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Processing

EM software
IDNameVersionCategory
1Xmipp3particle selection
2Gautomatchparticle selection
3RELION3particle selection
6CTFFIND2CTF correction
9UCSF Chimeramodel fitting
10Coot0.9.8model fitting
11ISOLDEmodel fitting
16cryoSPARC23D reconstruction
23PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING ONLY
Particle selectionNum. of particles selected: 1069057
Details: Semi-automated picking with Xmipp3 and particle extraction in Relion-3 yielded 47,516 particles from 1000 micrographs 38. After reference-free 2D classification in Relion-3, eight 2D classes ...Details: Semi-automated picking with Xmipp3 and particle extraction in Relion-3 yielded 47,516 particles from 1000 micrographs 38. After reference-free 2D classification in Relion-3, eight 2D classes were selected and used as templates for reference-based particle picking in Gautomatch. A total of particles were extracted with 2-fold binning and submitted to 8 rounds of 2D classification in Relion-3.
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.68 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 121194 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Details: Initial fitting was done in Chimera. Interactive Model building was done in COOT & Isolde Refinement was done using Phenix.
Atomic model building

3D fitting-ID: 1 / Source name: PDB / Type: experimental model

IDPDB-IDPdb chain-IDAccession codeChain-IDInitial refinement model-IDDetails
18R3X

8r3x

I8R3XI
21NF3

1nf3

P1NF3P2
36N8Q

6n8q

L6N8QL3Crystal structure of LGL2 was used to generate a model of LGL1
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.03910367
ELECTRON MICROSCOPYf_angle_d3.57614086
ELECTRON MICROSCOPYf_dihedral_angle_d13.2791407
ELECTRON MICROSCOPYf_chiral_restr0.1581572
ELECTRON MICROSCOPYf_plane_restr0.031827

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