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- EMDB-26667: Cryo-EM structure of SHOC2-PP1c-MRAS holophosphatase complex -

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Entry
Database: EMDB / ID: EMD-26667
TitleCryo-EM structure of SHOC2-PP1c-MRAS holophosphatase complex
Map dataRELION 3D auto-refine, full map
Sample
  • Complex: SHOC2-PP1C-MRAS holophosphatase
    • Protein or peptide: Ras-related protein M-Ras
    • Protein or peptide: Serine/threonine-protein phosphatase PP1-alpha catalytic subunit
    • Protein or peptide: Leucine-rich repeat protein SHOC-2
  • Ligand: GUANOSINE-5'-TRIPHOSPHATEGuanosine triphosphate
  • Ligand: MAGNESIUM ION
  • Ligand: MANGANESE (II) ION
  • Ligand: CHLORIDE IONChloride
Function / homology
Function and homology information


cellular response to growth hormone stimulus / protein phosphatase type 1 complex / regulation of glycogen catabolic process / negative regulation of neural precursor cell proliferation / PTW/PP1 phosphatase complex / glycogen granule / regulation of glycogen biosynthetic process / nerve growth factor signaling pathway / protein phosphatase regulator activity / protein phosphatase 1 binding ...cellular response to growth hormone stimulus / protein phosphatase type 1 complex / regulation of glycogen catabolic process / negative regulation of neural precursor cell proliferation / PTW/PP1 phosphatase complex / glycogen granule / regulation of glycogen biosynthetic process / nerve growth factor signaling pathway / protein phosphatase regulator activity / protein phosphatase 1 binding / GTP-dependent protein binding / cadherin binding involved in cell-cell adhesion / SHOC2 M1731 mutant abolishes MRAS complex function / Gain-of-function MRAS complexes activate RAF signaling / positive regulation of extrinsic apoptotic signaling pathway in absence of ligand / positive regulation of Ras protein signal transduction / regulation of canonical Wnt signaling pathway / regulation of translational initiation / myosin phosphatase activity / protein serine/threonine phosphatase activity / branching morphogenesis of an epithelial tube / glycogen metabolic process / protein-serine/threonine phosphatase / entrainment of circadian clock by photoperiod / Triglyceride catabolism / phosphatase activity / phosphoprotein phosphatase activity / DARPP-32 events / negative regulation of neuron differentiation / fibroblast growth factor receptor signaling pathway / ribonucleoprotein complex binding / dephosphorylation / positive regulation of neuron differentiation / cellular response to leukemia inhibitory factor / protein dephosphorylation / Downregulation of TGF-beta receptor signaling / small monomeric GTPase / G protein activity / adherens junction / response to lead ion / lung development / RAF activation / circadian regulation of gene expression / regulation of circadian rhythm / positive regulation of neuron projection development / GDP binding / Circadian Clock / presynapse / perikaryon / actin cytoskeleton organization / protein phosphatase binding / Ras protein signal transduction / dendritic spine / cell cycle / cell division / GTPase activity / glutamatergic synapse / nucleolus / GTP binding / signal transduction / extracellular exosome / nucleoplasm / metal ion binding / nucleus / plasma membrane / cytosol / cytoplasm
Similarity search - Function
Serine-threonine protein phosphatase, N-terminal / Serine-threonine protein phosphatase N-terminal domain / Serine/threonine specific protein phosphatases signature. / Protein phosphatase 2A homologues, catalytic domain. / Serine/threonine-specific protein phosphatase/bis(5-nucleosyl)-tetraphosphatase / Leucine-rich repeats, bacterial type / Leucine-rich repeat, SDS22-like subfamily / Calcineurin-like phosphoesterase domain, ApaH type / Calcineurin-like phosphoesterase / Metallo-dependent phosphatase-like ...Serine-threonine protein phosphatase, N-terminal / Serine-threonine protein phosphatase N-terminal domain / Serine/threonine specific protein phosphatases signature. / Protein phosphatase 2A homologues, catalytic domain. / Serine/threonine-specific protein phosphatase/bis(5-nucleosyl)-tetraphosphatase / Leucine-rich repeats, bacterial type / Leucine-rich repeat, SDS22-like subfamily / Calcineurin-like phosphoesterase domain, ApaH type / Calcineurin-like phosphoesterase / Metallo-dependent phosphatase-like / Small GTPase, Ras-type / small GTPase Ras family profile. / Ran (Ras-related nuclear proteins) /TC4 subfamily of small GTPases / Leucine rich repeat / Leucine-rich repeat, typical subtype / Leucine-rich repeats, typical (most populated) subfamily / Leucine-rich repeat profile. / Leucine-rich repeat / Rho (Ras homology) subfamily of Ras-like small GTPases / Ras subfamily of RAS small GTPases / Small GTPase / Ras family / Rab subfamily of small GTPases / Leucine-rich repeat domain superfamily / Small GTP-binding protein domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Ras-related protein M-Ras / Serine/threonine-protein phosphatase PP1-alpha catalytic subunit / Leucine-rich repeat protein SHOC-2
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.89 Å
AuthorsFuller JR / Hajian B / Lemke C / Kwon J / Bian Y / Aguirre A
Funding support United States, 1 items
OrganizationGrant numberCountry
Other private United States
CitationJournal: Nature / Year: 2022
Title: Structure-function analysis of the SHOC2-MRAS-PP1C holophosphatase complex.
Authors: Jason J Kwon / Behnoush Hajian / Yuemin Bian / Lucy C Young / Alvaro J Amor / James R Fuller / Cara V Fraley / Abbey M Sykes / Jonathan So / Joshua Pan / Laura Baker / Sun Joo Lee / Douglas ...Authors: Jason J Kwon / Behnoush Hajian / Yuemin Bian / Lucy C Young / Alvaro J Amor / James R Fuller / Cara V Fraley / Abbey M Sykes / Jonathan So / Joshua Pan / Laura Baker / Sun Joo Lee / Douglas B Wheeler / David L Mayhew / Nicole S Persky / Xiaoping Yang / David E Root / Anthony M Barsotti / Andrew W Stamford / Charles K Perry / Alex Burgin / Frank McCormick / Christopher T Lemke / William C Hahn / Andrew J Aguirre /
Abstract: Receptor tyrosine kinase (RTK)-RAS signalling through the downstream mitogen-activated protein kinase (MAPK) cascade regulates cell proliferation and survival. The SHOC2-MRAS-PP1C holophosphatase ...Receptor tyrosine kinase (RTK)-RAS signalling through the downstream mitogen-activated protein kinase (MAPK) cascade regulates cell proliferation and survival. The SHOC2-MRAS-PP1C holophosphatase complex functions as a key regulator of RTK-RAS signalling by removing an inhibitory phosphorylation event on the RAF family of proteins to potentiate MAPK signalling. SHOC2 forms a ternary complex with MRAS and PP1C, and human germline gain-of-function mutations in this complex result in congenital RASopathy syndromes. However, the structure and assembly of this complex are poorly understood. Here we use cryo-electron microscopy to resolve the structure of the SHOC2-MRAS-PP1C complex. We define the biophysical principles of holoenzyme interactions, elucidate the assembly order of the complex, and systematically interrogate the functional consequence of nearly all of the possible missense variants of SHOC2 through deep mutational scanning. We show that SHOC2 binds PP1C and MRAS through the concave surface of the leucine-rich repeat region and further engages PP1C through the N-terminal disordered region that contains a cryptic RVXF motif. Complex formation is initially mediated by interactions between SHOC2 and PP1C and is stabilized by the binding of GTP-loaded MRAS. These observations explain how mutant versions of SHOC2 in RASopathies and cancer stabilize the interactions of complex members to enhance holophosphatase activity. Together, this integrative structure-function model comprehensively defines key binding interactions within the SHOC2-MRAS-PP1C holophosphatase complex and will inform therapeutic development .
History
DepositionApr 15, 2022-
Header (metadata) releaseMay 4, 2022-
Map releaseMay 4, 2022-
UpdateSep 21, 2022-
Current statusSep 21, 2022Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26667.png

Downloads & links

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Map

FileDownload / File: emd_26667.map.gz / Format: CCP4 / Size: 67 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRELION 3D auto-refine, full map
Voxel sizeX=Y=Z: 1.068 Å
Density
Contour LevelBy AUTHOR: 0.012
Minimum - Maximum-0.009588601 - 0.040651537
Average (Standard dev.)2.1817114e-06 (±0.00089931133)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions260260260
Spacing260260260
CellA=B=C: 277.68 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_26667_msk_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Additional map: Full map, sharpened by an automatically determined B-factor...

Fileemd_26667_additional_1.map
AnnotationFull map, sharpened by an automatically determined B-factor (-90.7922) and filtered to local resolution. The model was refined primarily against this map, including model B-factors.
Projections & Slices
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Half map: RELION 3D auto-refine, half-map 1

Fileemd_26667_half_map_1.map
AnnotationRELION 3D auto-refine, half-map 1
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Half map: RELION 3D auto-refine, half-map 2

Fileemd_26667_half_map_2.map
AnnotationRELION 3D auto-refine, half-map 2
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Sample components

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Entire : SHOC2-PP1C-MRAS holophosphatase

EntireName: SHOC2-PP1C-MRAS holophosphatase
Components
  • Complex: SHOC2-PP1C-MRAS holophosphatase
    • Protein or peptide: Ras-related protein M-Ras
    • Protein or peptide: Serine/threonine-protein phosphatase PP1-alpha catalytic subunit
    • Protein or peptide: Leucine-rich repeat protein SHOC-2
  • Ligand: GUANOSINE-5'-TRIPHOSPHATEGuanosine triphosphate
  • Ligand: MAGNESIUM ION
  • Ligand: MANGANESE (II) ION
  • Ligand: CHLORIDE IONChloride

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Supramolecule #1: SHOC2-PP1C-MRAS holophosphatase

SupramoleculeName: SHOC2-PP1C-MRAS holophosphatase / type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Ras-related protein M-Ras

MacromoleculeName: Ras-related protein M-Ras / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: small monomeric GTPase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 20.894898 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
GMATSAVPSD NLPTYKLVVV GDGGVGKSAL TIQFFQKIFV PDYDPTIEDS YLKHTEIDNQ WAILDVLDTA GLEEFSAMRE QYMRTGDGF LIVYSVTDKA SFEHVDRFHQ LILRVKDRES FPMILVANKV DLMHLRKITR EQGKEMATKH NIPYIETSAK D PPLNVDKA FHDLVRVIRQ QIPEK

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Macromolecule #2: Serine/threonine-protein phosphatase PP1-alpha catalytic subunit

MacromoleculeName: Serine/threonine-protein phosphatase PP1-alpha catalytic subunit
type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: protein-serine/threonine phosphatase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 37.615102 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: GMSDSEKLNL DSIIGRLLEV QGSRPGKNVQ LTENEIRGLC LKSREIFLSQ PILLELEAPL KICGDIHGQY YDLLRLFEYG GFPPESNYL FLGDYVDRGK QSLETICLLL AYKIKYPENF FLLRGNHECA SINRIYGFYD ECKRRYNIKL WKTFTDCFNC L PIAAIVDE ...String:
GMSDSEKLNL DSIIGRLLEV QGSRPGKNVQ LTENEIRGLC LKSREIFLSQ PILLELEAPL KICGDIHGQY YDLLRLFEYG GFPPESNYL FLGDYVDRGK QSLETICLLL AYKIKYPENF FLLRGNHECA SINRIYGFYD ECKRRYNIKL WKTFTDCFNC L PIAAIVDE KIFCCHGGLS PDLQSMEQIR RIMRPTDVPD QGLLCDLLWS DPDKDVQGWG ENDRGVSFTF GAEVVAKFLH KH DLDLICR AHQVVEDGYE FFAKRQLVTL FSAPNYCGEF DNAGAMMSVD ETLMCSFQIL KPADKNKGKY GQFSGLNPGG RPI TPPRNS AKAKK

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Macromolecule #3: Leucine-rich repeat protein SHOC-2

MacromoleculeName: Leucine-rich repeat protein SHOC-2 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 65.029926 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: GMSSSLGKEK DSKEKDPKVP SAKEREKEAK ASGGFGKESK EKEPKTKGKD AKDGKKDSSA AQPGVAFSVD NTIKRPNPAP GTRKKSSNA EVIKELNKCR EENSMRLDLS KRSIHILPSS IKELTQLTEL YLYSNKLQSL PAEVGCLVNL MTLALSENSL T SLPDSLDN ...String:
GMSSSLGKEK DSKEKDPKVP SAKEREKEAK ASGGFGKESK EKEPKTKGKD AKDGKKDSSA AQPGVAFSVD NTIKRPNPAP GTRKKSSNA EVIKELNKCR EENSMRLDLS KRSIHILPSS IKELTQLTEL YLYSNKLQSL PAEVGCLVNL MTLALSENSL T SLPDSLDN LKKLRMLDLR HNKLREIPSV VYRLDSLTTL YLRFNRITTV EKDIKNLSKL SMLSIRENKI KQLPAEIGEL CN LITLDVA HNQLEHLPKE IGNCTQITNL DLQHNELLDL PDTIGNLSSL SRLGLRYNRL SAIPRSLAKC SALEELNLEN NNI STLPES LLSSLVKLNS LTLARNCFQL YPVGGPSQFS TIYSLNMEHN RINKIPFGIF SRAKVLSKLN MKDNQLTSLP LDFG TWTSM VELNLATNQL TKIPEDVSGL VSLEVLILSN NLLKKLPHGL GNLRKLRELD LEENKLESLP NEIAYLKDLQ KLVLT NNQL TTLPRGIGHL TNLTHLGLGE NLLTHLPEEI GTLENLEELY LNDNPNLHSL PFELALCSKL SIMSIENCPL SHLPPQ IVA GGPSFIIQFL KMQGPYRAMV

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Macromolecule #4: GUANOSINE-5'-TRIPHOSPHATE

MacromoleculeName: GUANOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 4 / Number of copies: 1 / Formula: GTP
Molecular weightTheoretical: 523.18 Da
Chemical component information

ChemComp-GTP:
GUANOSINE-5'-TRIPHOSPHATE / GTP, energy-carrying molecule*YM / Guanosine triphosphate

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #6: MANGANESE (II) ION

MacromoleculeName: MANGANESE (II) ION / type: ligand / ID: 6 / Number of copies: 2 / Formula: MN
Molecular weightTheoretical: 54.938 Da

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Macromolecule #7: CHLORIDE ION

MacromoleculeName: CHLORIDE ION / type: ligand / ID: 7 / Number of copies: 1 / Formula: CL
Molecular weightTheoretical: 35.453 Da

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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

Concentration2.75 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
150.0 mMNaClSodium chloridesodium chloride
50.0 mMC8H18N2O4SHEPES
0.5 mMC9H15O6PTCEP
0.025 %C20H25F13O11Fluorinated octyl maltoside

Details: Fluorinated octyl maltoside added immediately prior to vitrification
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Atmosphere: AIR
Details: Gatan Solarus using ambient air with power set to 20 watts
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 291 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated defocus max: 2.84 µm / Calibrated defocus min: 0.29 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.3000000000000003 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 81000
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 4721 / Average electron dose: 60.0 e/Å2
Details: Movies were recorded in CDS + super-resolution mode, fractionating 60 e-/A2 over 52 movie frames
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware: (Name: CTFFIND (ver. 4.1.14), RELION (ver. 4.0beta2))
Startup modelType of model: OTHER / Details: RELION ab initio volume
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 4.0beta2)
Final 3D classificationNumber classes: 9 / Software - Name: RELION (ver. 4.0beta2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 4.0beta2)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.89 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 4.0beta2) / Number images used: 449384
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

3e7a

chain_id: A

3kko

chain_id: A
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-7upi:
Cryo-EM structure of SHOC2-PP1c-MRAS holophosphatase complex

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