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- PDB-7sd0: Cryo-EM structure of the SHOC2:PP1C:MRAS complex -

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Basic information

Entry
Database: PDB / ID: 7sd0
TitleCryo-EM structure of the SHOC2:PP1C:MRAS complex
Components
  • Leucine-rich repeat protein SHOC-2
  • Ras-related protein M-Ras
  • Serine/threonine-protein phosphatase PP1-gamma catalytic subunit
KeywordsSIGNALING PROTEIN / Phosphatase / leucine rich repeat / RAF / complex
Function / homology
Function and homology information


cellular response to growth hormone stimulus / negative regulation of neural precursor cell proliferation / PTW/PP1 phosphatase complex / protein phosphatase type 1 complex / regulation of nucleocytoplasmic transport / volume-sensitive anion channel activity / nerve growth factor signaling pathway / GTP-dependent protein binding / cyclic-GMP-AMP transmembrane import across plasma membrane / protein phosphatase 1 binding ...cellular response to growth hormone stimulus / negative regulation of neural precursor cell proliferation / PTW/PP1 phosphatase complex / protein phosphatase type 1 complex / regulation of nucleocytoplasmic transport / volume-sensitive anion channel activity / nerve growth factor signaling pathway / GTP-dependent protein binding / cyclic-GMP-AMP transmembrane import across plasma membrane / protein phosphatase 1 binding / lamin binding / protein phosphatase regulator activity / SHOC2 M1731 mutant abolishes MRAS complex function / Gain-of-function MRAS complexes activate RAF signaling / positive regulation of Ras protein signal transduction / protein serine/threonine phosphatase activity / glycogen metabolic process / histone H2AXS140 phosphatase activity / RNA polymerase II CTD heptapeptide repeat Y1 phosphatase activity / RNA polymerase II CTD heptapeptide repeat T4 phosphatase activity / RNA polymerase II CTD heptapeptide repeat S2 phosphatase activity / RNA polymerase II CTD heptapeptide repeat S5 phosphatase activity / RNA polymerase II CTD heptapeptide repeat S7 phosphatase activity / MAP kinase serine/threonine phosphatase activity / calmodulin-dependent protein phosphatase activity / myosin phosphatase activity / protein-serine/threonine phosphatase / Triglyceride catabolism / entrainment of circadian clock by photoperiod / Maturation of hRSV A proteins / phosphatase activity / microtubule organizing center / cleavage furrow / mitotic sister chromatid segregation / regulation of MAPK cascade / phosphoprotein phosphatase activity / blastocyst development / negative regulation of neuron differentiation / fibroblast growth factor receptor signaling pathway / protein dephosphorylation / Amplification of signal from unattached kinetochores via a MAD2 inhibitory signal / positive regulation of glial cell proliferation / Mitotic Prometaphase / EML4 and NUDC in mitotic spindle formation / positive regulation of neuron differentiation / Resolution of Sister Chromatid Cohesion / cellular response to leukemia inhibitory factor / Downregulation of TGF-beta receptor signaling / small monomeric GTPase / RHO GTPases Activate Formins / RAF activation / circadian regulation of gene expression / regulation of circadian rhythm / positive regulation of neuron projection development / kinetochore / neuron differentiation / GDP binding / Separation of Sister Chromatids / : / MAPK cascade / presynapse / G protein activity / actin cytoskeleton organization / midbody / protein phosphatase binding / spermatogenesis / dendritic spine / mitochondrial outer membrane / Ras protein signal transduction / intracellular signal transduction / nuclear speck / protein domain specific binding / cell division / focal adhesion / GTPase activity / protein-containing complex binding / protein kinase binding / GTP binding / nucleolus / glutamatergic synapse / protein-containing complex / mitochondrion / RNA binding / 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 repeat region / Metallo-dependent phosphatases ...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 repeat region / Metallo-dependent phosphatases / Purple Acid Phosphatase; chain A, domain 2 / 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. / Leucine rich repeat / Ran (Ras-related nuclear proteins) /TC4 subfamily of small GTPases / 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 / 4-Layer Sandwich / Small GTP-binding protein domain / P-loop containing nucleotide triphosphate hydrolases / Rossmann fold / P-loop containing nucleoside triphosphate hydrolase / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER / : / Ras-related protein M-Ras / Serine/threonine-protein phosphatase PP1-gamma catalytic subunit / Leucine-rich repeat protein SHOC-2
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.95 Å
AuthorsLiau, N.P.D. / Johnson, M.C. / Hymowitz, S.G. / Sudhamsu, J.
Funding support United States, 1items
OrganizationGrant numberCountry
Other private United States
CitationJournal: Nature / Year: 2022
Title: Structural basis for SHOC2 modulation of RAS signalling.
Authors: Nicholas P D Liau / Matthew C Johnson / Saeed Izadi / Luca Gerosa / Michal Hammel / John M Bruning / Timothy J Wendorff / Wilson Phung / Sarah G Hymowitz / Jawahar Sudhamsu /
Abstract: The RAS-RAF pathway is one of the most commonly dysregulated in human cancers. Despite decades of study, understanding of the molecular mechanisms underlying dimerization and activation of the kinase ...The RAS-RAF pathway is one of the most commonly dysregulated in human cancers. Despite decades of study, understanding of the molecular mechanisms underlying dimerization and activation of the kinase RAF remains limited. Recent structures of inactive RAF monomer and active RAF dimer bound to 14-3-3 have revealed the mechanisms by which 14-3-3 stabilizes both RAF conformations via specific phosphoserine residues. Prior to RAF dimerization, the protein phosphatase 1 catalytic subunit (PP1C) must dephosphorylate the N-terminal phosphoserine (NTpS) of RAF to relieve inhibition by 14-3-3, although PP1C in isolation lacks intrinsic substrate selectivity. SHOC2 is as an essential scaffolding protein that engages both PP1C and RAS to dephosphorylate RAF NTpS, but the structure of SHOC2 and the architecture of the presumptive SHOC2-PP1C-RAS complex remain unknown. Here we present a cryo-electron microscopy structure of the SHOC2-PP1C-MRAS complex to an overall resolution of 3 Å, revealing a tripartite molecular architecture in which a crescent-shaped SHOC2 acts as a cradle and brings together PP1C and MRAS. Our work demonstrates the GTP dependence of multiple RAS isoforms for complex formation, delineates the RAS-isoform preference for complex assembly, and uncovers how the SHOC2 scaffold and RAS collectively drive specificity of PP1C for RAF NTpS. Our data indicate that disease-relevant mutations affect complex assembly, reveal the simultaneous requirement of two RAS molecules for RAF activation, and establish rational avenues for discovery of new classes of inhibitors to target this pathway.
History
DepositionSep 29, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 20, 2022Provider: repository / Type: Initial release
Revision 1.1Jul 6, 2022Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Jul 20, 2022Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.3Jul 27, 2022Group: Database references / Category: citation / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.4Sep 21, 2022Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.5Nov 29, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Leucine-rich repeat protein SHOC-2
B: Ras-related protein M-Ras
C: Serine/threonine-protein phosphatase PP1-gamma catalytic subunit
hetero molecules


Theoretical massNumber of molelcules
Total (without water)127,0167
Polymers126,3603
Non-polymers6554
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: SAXS
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area6660 Å2
ΔGint-33 kcal/mol
Surface area38080 Å2

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Components

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Protein , 3 types, 3 molecules ABC

#1: Protein Leucine-rich repeat protein SHOC-2 / Protein soc-2 homolog / Protein sur-8 homolog


Mass: 65156.969 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SHOC2, KIAA0862 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q9UQ13
#2: Protein Ras-related protein M-Ras / Ras-related protein R-Ras3


Mass: 24028.621 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MRAS, RRAS3 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: O14807, small monomeric GTPase
#3: Protein Serine/threonine-protein phosphatase PP1-gamma catalytic subunit / PP-1G / Protein phosphatase 1C catalytic subunit


Mass: 37174.906 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PPP1CC / Production host: Escherichia coli (E. coli)
References: UniProt: P36873, protein-serine/threonine phosphatase

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Non-polymers , 3 types, 4 molecules

#4: Chemical ChemComp-GCP / PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER


Mass: 521.208 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C11H18N5O13P3 / Comment: GMP-PCP, energy-carrying molecule analogue*YM
#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#6: Chemical ChemComp-MN / MANGANESE (II) ION


Mass: 54.938 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mn

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Details

Has ligand of interestN

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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
1Ternary complex of SHOC2:PP1C:MRASCOMPLEX#1-#30MULTIPLE SOURCES
2SHOC2COMPLEX#11RECOMBINANT
3MRASCOMPLEX#21RECOMBINANT
4PP1CCOMPLEX#31RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.126 MDaYES
21.065 MDaYES
31.024 MDaYES
41.037 MDaYES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
11Homo sapiens (human)9606
22Homo sapiens (human)9606
33Homo sapiens (human)9606
44Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Spodoptera frugiperda (fall armyworm)7108
33Spodoptera frugiperda (fall armyworm)7108
44Escherichia coli (E. coli)562
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMTris1
2100 mMNaCl1
31 mMTCEP1
40.5 mMMgCl21
50.5 mMMnCl21
60.1 mMGMPPCP1
SpecimenConc.: 0.19 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K
Details: Used the "perpetually hydrated" method of applying graphene oxide. (Cheung et al., 2018)

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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 magnification: 105000 X / Nominal defocus max: 1500 nm / Nominal defocus min: 500 nm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 64 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1

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Processing

SoftwareName: PHENIX / Version: 1.19.1-4122_final: / Classification: refinement
EM software
IDNameCategory
1cryoSPARCparticle selection
2SerialEMimage acquisition
4cryoSPARCCTF correction
7UCSF ChimeraXmodel fitting
9PHENIXmodel refinement
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
12cryoSPARCclassification
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3996056
3D reconstructionResolution: 2.95 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 323910 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
11X1S

1x1s

11X1S1PDBexperimental model
24MOV

4mov

14MOV2PDBexperimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0087870
ELECTRON MICROSCOPYf_angle_d0.93910659
ELECTRON MICROSCOPYf_dihedral_angle_d13.2442990
ELECTRON MICROSCOPYf_chiral_restr0.0691228
ELECTRON MICROSCOPYf_plane_restr0.0051367

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