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

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Basic information

Entry
Database: PDB / ID: 7upi
TitleCryo-EM structure of SHOC2-PP1c-MRAS holophosphatase complex
Components
  • Leucine-rich repeat protein SHOC-2
  • Ras-related protein M-Ras
  • Serine/threonine-protein phosphatase PP1-alpha catalytic subunit
KeywordsCELL CYCLE / shoc2 / leucine-rich repeat / MRAs / protein phosphatase / RAS signaling / MAPK
Function / homology
Function and homology information


cellular response to growth hormone stimulus / regulation of glycogen catabolic process / positive regulation of termination of RNA polymerase II transcription, poly(A)-coupled / PTW/PP1 phosphatase complex / negative regulation of neural precursor cell proliferation / protein phosphatase type 1 complex / volume-sensitive anion channel activity / glycogen granule / RNA polymerase II promoter clearance / RNA polymerase II CTD heptapeptide repeat S5 phosphatase activity ...cellular response to growth hormone stimulus / regulation of glycogen catabolic process / positive regulation of termination of RNA polymerase II transcription, poly(A)-coupled / PTW/PP1 phosphatase complex / negative regulation of neural precursor cell proliferation / protein phosphatase type 1 complex / volume-sensitive anion channel activity / glycogen granule / RNA polymerase II promoter clearance / RNA polymerase II CTD heptapeptide repeat S5 phosphatase activity / nerve growth factor signaling pathway / cyclic-GMP-AMP transmembrane import across plasma membrane / cadherin binding involved in cell-cell adhesion / protein phosphatase 1 binding / regulation of translational initiation in response to stress / protein phosphatase regulator activity / GTP-dependent protein binding / 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 / dephosphorylation / positive regulation of Ras protein signal transduction / regulation of canonical Wnt signaling pathway / glycogen metabolic process / protein-serine/threonine phosphatase / negative regulation of neuron differentiation / branching morphogenesis of an epithelial tube / entrainment of circadian clock by photoperiod / Triglyceride catabolism / Maturation of hRSV A proteins / protein serine/threonine phosphatase activity / phosphatase activity / telomere maintenance in response to DNA damage / regulation of MAPK cascade / phosphoprotein phosphatase activity / negative regulation of transcription elongation by RNA polymerase II / transition metal ion binding / DARPP-32 events / fibroblast growth factor receptor signaling pathway / positive regulation of glycogen biosynthetic process / ribonucleoprotein complex binding / protein dephosphorylation / positive regulation of neuron differentiation / lung development / cellular response to leukemia inhibitory factor / Downregulation of TGF-beta receptor signaling / small monomeric GTPase / adherens junction / circadian regulation of gene expression / positive regulation of transcription elongation by RNA polymerase II / RAF activation / positive regulation of neuron projection development / regulation of circadian rhythm / response to lead ion / GDP binding / : / presynapse / G protein activity / actin cytoskeleton organization / protein phosphatase binding / perikaryon / dendritic spine / Ras protein signal transduction / intracellular signal transduction / protein stabilization / iron ion binding / cell division / GTPase activity / GTP binding / nucleolus / glutamatergic synapse / extracellular exosome / nucleoplasm / 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 domain profile. / Leucine rich repeat / Leucine-rich repeat, typical subtype / Leucine-rich repeats, typical (most populated) subfamily / Ran (Ras-related nuclear proteins) /TC4 subfamily of small GTPases / 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
GUANOSINE-5'-TRIPHOSPHATE / : / 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.89 Å
AuthorsFuller, J.R. / Hajian, B. / Lemke, C. / Kwon, J. / Bian, Y. / Aguirre, A.
Funding support United States, 1items
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, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 4, 2022Provider: repository / Type: Initial release
Revision 1.1Aug 17, 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 1.2Sep 21, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.3Jun 12, 2024Group: 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: Ras-related protein M-Ras
B: Serine/threonine-protein phosphatase PP1-alpha catalytic subunit
C: Leucine-rich repeat protein SHOC-2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)124,2338
Polymers123,5403
Non-polymers6935
Water00
1


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

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Components

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Protein , 3 types, 3 molecules ABC

#1: Protein Ras-related protein M-Ras / Ras-related protein R-Ras3


Mass: 20894.898 Da / Num. of mol.: 1 / Mutation: Q71L
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MRAS, RRAS3 / Production host: Escherichia coli (E. coli) / References: UniProt: O14807, small monomeric GTPase
#2: Protein Serine/threonine-protein phosphatase PP1-alpha catalytic subunit / PP-1A


Mass: 37615.102 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PPP1CA, PPP1A / Production host: Escherichia coli (E. coli)
References: UniProt: P62136, protein-serine/threonine phosphatase
#3: Protein Leucine-rich repeat protein SHOC-2 / Protein soc-2 homolog / Protein sur-8 homolog


Mass: 65029.926 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

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Non-polymers , 4 types, 5 molecules

#4: Chemical ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE


Mass: 523.180 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O14P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: GTP, energy-carrying molecule*YM
#5: Chemical ChemComp-MG / MAGNESIUM ION


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


Mass: 54.938 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mn / Feature type: SUBJECT OF INVESTIGATION
#7: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Cl / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY

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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: SHOC2-PP1C-MRAS holophosphatase / Type: COMPLEX / Entity ID: #1-#3 / Source: MULTIPLE SOURCES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
21Spodoptera frugiperda (fall armyworm)7108
31Escherichia coli (E. coli)562
Buffer solutionpH: 7.4
Details: Fluorinated octyl maltoside added immediately prior to vitrification
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMsodium chlorideNaCl1
250 mMHEPESC8H18N2O4S1
30.5 mMTCEPC9H15O6P1
40.025 %Fluorinated octyl maltosideC20H25F13O111
SpecimenConc.: 2.75 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Gatan Solarus using ambient air with power set to 20 watts
Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 291 K

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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: 81000 X / Nominal defocus max: 2300 nm / Nominal defocus min: 500 nm / Calibrated defocus min: 290 nm / Calibrated defocus max: 2840 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 4721
Details: Movies were recorded in CDS + super-resolution mode, fractionating 60 e-/A2 over 52 movie frames
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refine1.20_4459refinement
PHENIX1.20_4459refinement
EM software
IDNameVersionCategory
1RELION4.0beta2particle selection
2Topaz0.2.5particle selection
3EPU2.9image acquisition
5CTFFIND4.1.14CTF correction
6RELION4.0beta2CTF correction
9UCSF Chimera1.15model fitting
11RELION4.0beta2initial Euler assignment
12RELION4.0beta2final Euler assignment
13RELION4.0beta2classification
14RELION4.0beta23D reconstruction
15PHENIX1.19model refinement
16Coot0.9.6model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.89 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 449384 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model building

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

IDPDB-IDAccession codeInitial refinement model-ID
13E7A

3e7a

3E7A1
23KKO

3kko

3KKO2
RefinementCross valid method: NONE

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