National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
R35GM118099
United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
1S10OD026881
United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
1S10OD020054
United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
1S10OD021741
United States
Citation
Journal: Nat Commun / Year: 2023 Title: Hsp90 provides a platform for kinase dephosphorylation by PP5. Authors: Maru Jaime-Garza / Carlos A Nowotny / Daniel Coutandin / Feng Wang / Mariano Tabios / David A Agard / Abstract: The Hsp90 molecular chaperone collaborates with the phosphorylated Cdc37 cochaperone for the folding and activation of its many client kinases. As with many kinases, the Hsp90 client kinase CRaf is ...The Hsp90 molecular chaperone collaborates with the phosphorylated Cdc37 cochaperone for the folding and activation of its many client kinases. As with many kinases, the Hsp90 client kinase CRaf is activated by phosphorylation at specific regulatory sites. The cochaperone phosphatase PP5 dephosphorylates CRaf and Cdc37 in an Hsp90-dependent manner. Although dephosphorylating Cdc37 has been proposed as a mechanism for releasing Hsp90-bound kinases, here we show that Hsp90 bound kinases sterically inhibit Cdc37 dephosphorylation indicating kinase release must occur before Cdc37 dephosphorylation. Our cryo-EM structure of PP5 in complex with Hsp90:Cdc37:CRaf reveals how Hsp90 both activates PP5 and scaffolds its association with the bound CRaf to dephosphorylate phosphorylation sites neighboring the kinase domain. Thus, we directly show how Hsp90's role in maintaining protein homeostasis goes beyond folding and activation to include post translationally modifying its client kinases.
Protein or peptide: RAF proto-oncogene serine/threonine-protein kinase
Protein or peptide: Heat shock protein HSP 90-beta
Protein or peptide: Hsp90 co-chaperone Cdc37, N-terminally processed
Protein or peptide: Serine/threonine-protein phosphatase 5
Ligand: ADENOSINE-5'-DIPHOSPHATE
Ligand: MAGNESIUM ION
Ligand: POTASSIUM ION
Ligand: ADENOSINE-5'-TRIPHOSPHATE
Ligand: MANGANESE (II) ION
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Supramolecule #1: Hsp90:Cdc37:CRaf:PP5 complex
Supramolecule
Name: Hsp90:Cdc37:CRaf:PP5 complex / type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: #1-#4 Details: Yeast purified Hsp90:Cdc37:CRaf complex incubated with e. Coli purified PP5 (mutant H304A). Sample then cross-linked with 0.05% glutaraldehyde for 15m at room temperature, and ran over S200 sizing column.
Source (natural)
Organism: Homo sapiens (human)
Molecular weight
Theoretical: 57 KDa
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Supramolecule #2: Protein Phosphatase 5 (H304A)
Supramolecule
Name: Protein Phosphatase 5 (H304A) / type: complex / ID: 2 / Chimera: Yes / Parent: 1 / Macromolecule list: #3 Details: E coli purified Protein Phosphatase 5, with inactivating H304A mutation.
Source (natural)
Organism: Homo sapiens (human)
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Macromolecule #1: Heat shock protein HSP 90-beta
Macromolecule
Name: Heat shock protein HSP 90-beta / type: protein_or_peptide / ID: 1 Details: Hsp90 sequence with HRV 3C cleavage site and one residue glycine linker. Number of copies: 2 / Enantiomer: LEVO
Name: Hsp90 co-chaperone Cdc37, N-terminally processed / type: protein_or_peptide / ID: 2 Details: Cdc37 followed by HRV 3C cleavage site at C-terminal of construct. Number of copies: 1 / Enantiomer: LEVO
Name: RAF proto-oncogene serine/threonine-protein kinase / type: protein_or_peptide / ID: 3 Details: CRaf/Raf1 kinase domain followed by a LPESG linker, Strep Tag II sequence (WSHPQFEK) and a HRV 3C cleavage site (LEVLFQ). Number of copies: 1 / Enantiomer: LEVO / EC number: non-specific serine/threonine protein kinase
Name: MANGANESE (II) ION / type: ligand / ID: 9 / Number of copies: 2 / Formula: MN
Molecular weight
Theoretical: 54.938 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
0.09 mg/mL
Buffer
pH: 7.5 Component:
Concentration
Formula
Name
20.0 mM
C8H18N2O4S
HEPES
50.0 mM
KCl
KCl
10.0 mM
MgCl2
Magnesium Chloride
0.5 mM
C9H15O6P
TCEP
1.0 mM
C10H16N2O8
EDTA
Grid
Model: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GRAPHENE OXIDE / Support film - topology: CONTINUOUS / Support film - Film thickness: 1.0 nm
Vitrification
Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 283.15 K Details: 3uL OF SAMPLE, 10C, 100% HUMIDITY, 30S WAIT TIME, 3S BLOT TIME, -2 BLOT FORCE.
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Electron microscopy
Microscope
FEI TITAN KRIOS
Software
Name: SerialEM (ver. 3.8-beta)
Image recording
Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number real images: 4160 / Average electron dose: 69.0 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Details: 5FWL was used to fit the Hsp90 complex. 1WAO and 1S95 were used to fit the PP5 components.
Final reconstruction
Number classes used: 3 / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF Software: (Name: RELION (ver. 3.1.3), UCSF ChimeraX (ver. 1.2.5)) Details: Three different maps were used for final composite map: Hsp90:Cdc37: 288k particles, 3.2A PP5 TPR: 215k particles, 3.3A PP5 catalytic domain: 43k particles, 3.8A Composite half maps were ...Details: Three different maps were used for final composite map: Hsp90:Cdc37: 288k particles, 3.2A PP5 TPR: 215k particles, 3.3A PP5 catalytic domain: 43k particles, 3.8A Composite half maps were used to get the final resolution in Relion PostProcessing. All original maps provided in the "Related entries" tab. Number images used: 545237
Initial angle assignment
Type: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1.3)
Final angle assignment
Type: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1.3)
Final 3D classification
Software - Name: RELION (ver. 3.1.3) Details: Initial refinement on the Hsp90 component of the complex was followed up with focused classification with subtraction to obtain the separate maps used in this composite entry.
This model was built using rigid body docking in Chimera and ChimeraX for all main chains, and RosettaCM to add remaining fragments not included in previous models. Refinement was done using iterative Phenix and RosettaRelax, and finalized with ISOLDE.
Refinement
Protocol: RIGID BODY FIT
Output model
PDB-8gft: Hsp90 provides platform for CRaf dephosphorylation by PP5
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