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- EMDB-18877: Cryo EM structure of a stable LGL/aPKC Iota/Par-6 complex -

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Basic information

Entry
Database: EMDB / ID: EMD-18877
TitleCryo EM structure of a stable LGL/aPKC Iota/Par-6 complex
Map dataUnfiltered map
Sample
  • Complex: aPKCiota-Par6-Llgl1 complex
    • Protein or peptide: Protein kinase C iota type
    • Protein or peptide: Lethal(2) giant larvae protein homolog 1
    • Protein or peptide: Partitioning defective 6 homolog alpha
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER
KeywordsKinase / Polarity / Kinase substrate complex. / CYTOSOLIC PROTEIN
Function / homology
Function and homology information


establishment of spindle orientation / regulation of cellular localization / regulation of establishment or maintenance of cell polarity / Golgi vesicle budding / Golgi cis cisterna / PAR polarity complex / cell-cell junction maintenance / Tight junction interactions / diacylglycerol-dependent, calcium-independent serine/threonine kinase activity / calcium,diacylglycerol-dependent serine/threonine kinase activity ...establishment of spindle orientation / regulation of cellular localization / regulation of establishment or maintenance of cell polarity / Golgi vesicle budding / Golgi cis cisterna / PAR polarity complex / cell-cell junction maintenance / Tight junction interactions / diacylglycerol-dependent, calcium-independent serine/threonine kinase activity / calcium,diacylglycerol-dependent serine/threonine kinase activity / protein kinase C / diacylglycerol-dependent serine/threonine kinase activity / establishment of apical/basal cell polarity / negative regulation of glial cell apoptotic process / regulation of Notch signaling pathway / eye photoreceptor cell development / myosin II binding / GTP-dependent protein binding / positive regulation of protein localization to centrosome / Schmidt-Lanterman incisure / Golgi to plasma membrane transport / membrane organization / cellular response to chemical stress / establishment or maintenance of epithelial cell apical/basal polarity / cell-cell junction organization / tight junction / centrosome cycle / protein targeting to membrane / cortical actin cytoskeleton organization / cortical actin cytoskeleton / RHOV GTPase cycle / positive regulation of Notch signaling pathway / establishment or maintenance of cell polarity / establishment of cell polarity / intercellular bridge / cell leading edge / exocytosis / brush border / RHOU GTPase cycle / CDC42 GTPase cycle / centriolar satellite / positive regulation of endothelial cell apoptotic process / regulation of postsynaptic membrane neurotransmitter receptor levels / viral process / bicellular tight junction / positive regulation of glial cell proliferation / vesicle-mediated transport / cytoskeleton organization / ruffle / RAC1 GTPase cycle / p75NTR recruits signalling complexes / response to interleukin-1 / axonogenesis / secretion / GTPase activator activity / actin filament organization / trans-Golgi network membrane / TGF-beta receptor signaling in EMT (epithelial to mesenchymal transition) / protein localization to plasma membrane / Asymmetric localization of PCP proteins / positive regulation of D-glucose import / positive regulation of protein secretion / adherens junction / positive regulation of protein localization to plasma membrane / Schaffer collateral - CA1 synapse / phospholipid binding / positive regulation of neuron projection development / small GTPase binding / Pre-NOTCH Transcription and Translation / cellular response to insulin stimulus / microtubule cytoskeleton / cell migration / KEAP1-NFE2L2 pathway / positive regulation of NF-kappaB transcription factor activity / early endosome membrane / cell cortex / protein-containing complex assembly / negative regulation of neuron apoptotic process / cytoskeleton / protein kinase activity / endosome / intracellular signal transduction / apical plasma membrane / protein phosphorylation / axon / Golgi membrane / cell division / protein serine kinase activity / protein serine/threonine kinase activity / centrosome / negative regulation of apoptotic process / protein kinase binding / glutamatergic synapse / structural molecule activity / extracellular exosome / nucleoplasm / ATP binding / nucleus / metal ion binding / plasma membrane
Similarity search - Function
Lethal(2) giant larvae protein / Lethal giant larvae homologue 2 / LLGL2 / Partitioning defective protein 6, PB1 domain / : / Atypical protein kinase C iota type, catalytic domain / Protein kinase C / Protein kinase C, PB1 domain / PB1 domain / PB1 domain ...Lethal(2) giant larvae protein / Lethal giant larvae homologue 2 / LLGL2 / Partitioning defective protein 6, PB1 domain / : / 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. / WD40 repeats / WD40 repeat / Serine/threonine-protein kinase, active site / Serine/Threonine protein kinases active-site signature. / WD40-repeat-containing domain superfamily / Protein kinase domain / 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
Protein kinase C iota type / Lethal(2) giant larvae protein homolog 1 / Partitioning defective 6 homolog alpha
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.68 Å
AuthorsEarl CP / Briggs DC / McDonald NQ
Funding support United Kingdom, 3 items
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, 2023-
Header (metadata) releaseNov 20, 2024-
Map releaseNov 20, 2024-
UpdateJan 15, 2025-
Current statusJan 15, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_18877.png

Downloads & links

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Map

FileDownload / File: emd_18877.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationUnfiltered map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)X (Row.)Y (Col.)
0.82 Å/pix.
x 280 pix.
= 229.6 Å		0.82 Å/pix.
x 280 pix.
= 229.6 Å		0.82 Å/pix.
x 280 pix.
= 229.6 Å

Surface

Projections

Slices (1/3)

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Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.82 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.2
Minimum - Maximum-1.2979945 - 2.359292
Average (Standard dev.)0.0027597866 (±0.048550677)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderYXZ
Origin000
Dimensions280280280
Spacing280280280
CellA=B=C: 229.59999 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Phenix Sharpened Map used for Model building

Fileemd_18877_additional_1.map
AnnotationPhenix Sharpened Map used for Model building
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Half map: Half Map A

Fileemd_18877_half_map_1.map
AnnotationHalf Map A
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map B

Fileemd_18877_half_map_2.map
AnnotationHalf Map B
Projections & Slices
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Histogram

Histogram (log scale)

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Sample components

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Entire : aPKCiota-Par6-Llgl1 complex

EntireName: aPKCiota-Par6-Llgl1 complex
Components
  • Complex: aPKCiota-Par6-Llgl1 complex
    • Protein or peptide: Protein kinase C iota type
    • Protein or peptide: Lethal(2) giant larvae protein homolog 1
    • Protein or peptide: Partitioning defective 6 homolog alpha
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER

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Supramolecule #1: aPKCiota-Par6-Llgl1 complex

SupramoleculeName: aPKCiota-Par6-Llgl1 complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 220 KDa

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Macromolecule #1: Protein kinase C iota type

MacromoleculeName: Protein kinase C iota type / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: protein kinase C
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 39.146004 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: SLGLQDFDLL RVIGRGSYAK VLLVRLKKTD RIYAMKVVKK ELVNDDEDID WVQTEKHVFE QASNHPFLVG LHSCFQTESR LFFVIEYVN GGDLMFHMQR QRKLPEEHAR FYSAEISLAL NYLHERGIIY RDLKLDNVLL DSEGHIKLTD YGMCKEGLRP G DTTS(TPO)FCG ...String:
SLGLQDFDLL RVIGRGSYAK VLLVRLKKTD RIYAMKVVKK ELVNDDEDID WVQTEKHVFE QASNHPFLVG LHSCFQTESR LFFVIEYVN GGDLMFHMQR QRKLPEEHAR FYSAEISLAL NYLHERGIIY RDLKLDNVLL DSEGHIKLTD YGMCKEGLRP G DTTS(TPO)FCG TPNYIAPEIL RGEDYGFSVD WWALGVLMFE MMAGRSPFDI VGSSDNPDQN TEDYLFQVIL EKQIRIPR S LSVKAASVLK SFLNKDPKER LGCHPQTGFA DIQGHPFFRN VDWDMMEQKQ VVPPFKPNIS GEFGLDNFDS QFTNEPVQL (TPO)PDDDDIVRK IDQSEFEGFE YI

UniProtKB: Protein kinase C iota type

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Macromolecule #2: Lethal(2) giant larvae protein homolog 1

MacromoleculeName: Lethal(2) giant larvae protein homolog 1 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 101.889805 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: REKLKQELFA FNKTVEHGFP NQPSALAFDP ELRIMAIGTR SGAVKIYGAP GVEFTGLHRD AATVTQMHFL TGQGRLLSLL DDSSLHLWE IVHHNGCAHL EEALSFQLPS RPGFDGASAP LSLTRVTVVL LVAAGDIAAL GTEGSSVFFL DVTTLTLLEG Q TLAPGEVL ...String:
REKLKQELFA FNKTVEHGFP NQPSALAFDP ELRIMAIGTR SGAVKIYGAP GVEFTGLHRD AATVTQMHFL TGQGRLLSLL DDSSLHLWE IVHHNGCAHL EEALSFQLPS RPGFDGASAP LSLTRVTVVL LVAAGDIAAL GTEGSSVFFL DVTTLTLLEG Q TLAPGEVL RSVPDDYRCG KALGPVESLQ GHLRDPTKIL IGYSRGLLVI WNQASQCVDH IFLGNQQLES LCWGRDSSTV VS SHSDGSY AVWSVDAGSF PTLQPTVATT PYGPFPCKAI NKILWRNCES GGHFIIFSGG MPRASYGDRH CVSVLRAETL VTL DFTSRI IDFFTVHSTR PEDEFDDPQA LAVLLEEELV VLDLQTPGWP AVPAPYLAPL HSSAITCSAH VASVPAKLWA RIVS AGEQQ SPQPVSSALS WPITGGRNLA QEPSQRGLLL TGHEDGTVRF WDASGVALRP LYKLSTAGLF QTDCEHADSL AQAAE DDWP PFRKVGCFDP YSDDPRLGVQ KVALCKYTAQ MVVAGTAGQV LVLELSDVPV EQAVSVAIID LLQDREGFTW KGHERL SPR TGPLPWPAGF QPRVLVQCLP PAAVTAVTLH TEWSLVAFGT SHGFGLFDYQ RKSPVLARCT LHPNDSLAME GPLSRVK SL KKSLRQ(SEP)FRR IRKSRVSGKK RAANASSKLQ EANAQLAEQA CPHDVEMTPV QRRIEPRSAD DSLSGVVRCL YFAD TFLRD GAHHGPTMWA GTNSGSVFAY ALEVPAAAVG GEKRPEQAVE AVLGKEVQLM HRAPVVAIAV LDGRGRPLPE PYEAS RDLA QAPDMQGGHA VLIASEEQFK VFTLPKVSAK TKFKLTAHEG CRVRKVALAT FASVACEDYA ETCLACLTNL GDVHVF SVP GLRPQVHYSC IRKEDISGIA SCVFTRHGQG FYLISPSEFE RFSLSARNIT EPLCSLDI

UniProtKB: Lethal(2) giant larvae protein homolog 1

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Macromolecule #3: Partitioning defective 6 homolog alpha

MacromoleculeName: Partitioning defective 6 homolog alpha / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 10.856517 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
THRRVRLHKH GSDRPLGFYI RDGMSVRVAP QGLERVPGIF ISRLVRGGLA ESTGLLAVSD EILEVNGIEV AGKTLDQVTD MMVANSHNL IVTVKPANQR

UniProtKB: Partitioning defective 6 homolog alpha

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Macromolecule #4: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER

MacromoleculeName: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / type: ligand / ID: 4 / Number of copies: 1 / Formula: ANP
Molecular weightTheoretical: 506.196 Da
Chemical component information

ChemComp-ANP:
PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / AMP-PNP, energy-carrying molecule analogue*YM

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.4 mg/mL
BufferpH: 7.5
Component:
ConcentrationName
20.0 mMHEPES pH 7.5
150.0 mMSodium Chloride
0.5 mMTCEP
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec. / Details: 45mA
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV
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..
DetailsSample was double affinity purified and gel filtered. Sample was monodisperse.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Quantum ER
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number real images: 4002 / Average exposure time: 8.0 sec. / Average electron dose: 48.1 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.2 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber 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.
Startup modelType of model: INSILICO MODEL
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.68 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2) / Number images used: 121194
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChainDetails

8r3x

chain_id: I, source_name: PDB, initial_model_type: experimental model

1nf3

chain_id: P, source_name: PDB, initial_model_type: experimental model

6n8q

chain_id: L, source_name: PDB, initial_model_type: experimental modelCrystal structure of LGL2 was used to generate a model of LGL1
DetailsInitial fitting was done in Chimera. Interactive Model building was done in COOT & Isolde Refinement was done using Phenix.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

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

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