Journal: 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 .
Full 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.
Name: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 1 / Formula: MG
Molecular weight
Theoretical: 24.305 Da
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Macromolecule #6: MANGANESE (II) ION
Macromolecule
Name: MANGANESE (II) ION / type: ligand / ID: 6 / Number of copies: 2 / Formula: MN
Molecular weight
Theoretical: 54.938 Da
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Macromolecule #7: CHLORIDE ION
Macromolecule
Name: CHLORIDE ION / type: ligand / ID: 7 / Number of copies: 1 / Formula: CL
Molecular weight
Theoretical: 35.453 Da
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Experimental details
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Structure determination
Method
cryo EM
Processing
single particle reconstruction
Aggregation state
particle
-
Sample preparation
Concentration
2.75 mg/mL
Buffer
pH: 7.4 Component:
Concentration
Formula
Name
150.0 mM
NaCl
sodium chloride
50.0 mM
C8H18N2O4S
HEPES
0.5 mM
C9H15O6P
TCEP
0.025 %
C20H25F13O11
Fluorinated octyl maltoside
Details: Fluorinated octyl maltoside added immediately prior to vitrification
Grid
Model: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 35 sec. / Pretreatment - Atmosphere: AIR Details: Gatan Solarus using ambient air with power set to 20 watts
Vitrification
Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 291 K / Instrument: FEI VITROBOT MARK IV
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Electron microscopy
Microscope
FEI TITAN KRIOS
Specialist optics
Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recording
Film 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
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
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