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- PDB-8qgy: Cryo-EM structure of C-terminally truncated Apoptosis signal-regu... -

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Basic information

Entry
Database: PDB / ID: 8qgy
TitleCryo-EM structure of C-terminally truncated Apoptosis signal-regulating kinase 1 (ASK1)
ComponentsMitogen-activated protein kinase kinase kinase 5
KeywordsAPOPTOSIS / ASK1 / MAP3K / MAPK signaling / thioredoxin
Function / homology
Function and homology information


cellular response to reactive nitrogen species / neuron intrinsic apoptotic signaling pathway in response to oxidative stress / IRE1-TRAF2-ASK1 complex / protein kinase complex / mitogen-activated protein kinase kinase kinase / programmed necrotic cell death / JUN kinase kinase kinase activity / endothelial cell apoptotic process / positive regulation of p38MAPK cascade / intrinsic apoptotic signaling pathway in response to oxidative stress ...cellular response to reactive nitrogen species / neuron intrinsic apoptotic signaling pathway in response to oxidative stress / IRE1-TRAF2-ASK1 complex / protein kinase complex / mitogen-activated protein kinase kinase kinase / programmed necrotic cell death / JUN kinase kinase kinase activity / endothelial cell apoptotic process / positive regulation of p38MAPK cascade / intrinsic apoptotic signaling pathway in response to oxidative stress / positive regulation of cardiac muscle cell apoptotic process / p38MAPK cascade / positive regulation of cysteine-type endopeptidase activity involved in apoptotic process / MAP kinase kinase kinase activity / intrinsic apoptotic signaling pathway in response to endoplasmic reticulum stress / positive regulation of protein kinase activity / positive regulation of myoblast differentiation / stress-activated MAPK cascade / positive regulation of JUN kinase activity / JNK cascade / positive regulation of vascular associated smooth muscle cell proliferation / cellular response to amino acid starvation / response to endoplasmic reticulum stress / response to ischemia / apoptotic signaling pathway / positive regulation of JNK cascade / cellular response to hydrogen peroxide / MAPK cascade / cellular senescence / cellular response to tumor necrosis factor / protein phosphatase binding / Oxidative Stress Induced Senescence / neuron apoptotic process / protein kinase activity / positive regulation of apoptotic process / protein domain specific binding / external side of plasma membrane / protein phosphorylation / innate immune response / protein serine kinase activity / protein serine/threonine kinase activity / protein kinase binding / positive regulation of DNA-templated transcription / magnesium ion binding / protein homodimerization activity / protein-containing complex / ATP binding / identical protein binding / cytoplasm / cytosol
Similarity search - Function
MAP3K, deoxyribohydrolase domain / MAP3K, HisK-N-like globin domain / HisK-N-like globin domain of the ASK signalosome / Deoxyribohydrolase (DRHyd) domain of the ASK signalosome / MAP3K, TRAFs-binding domain / MAP3K, PH domain / MAP3K TRAFs-binding domain / ASK kinase PH domain / Sterile alpha motif/pointed domain superfamily / Serine/threonine-protein kinase, active site ...MAP3K, deoxyribohydrolase domain / MAP3K, HisK-N-like globin domain / HisK-N-like globin domain of the ASK signalosome / Deoxyribohydrolase (DRHyd) domain of the ASK signalosome / MAP3K, TRAFs-binding domain / MAP3K, PH domain / MAP3K TRAFs-binding domain / ASK kinase PH domain / Sterile alpha motif/pointed domain superfamily / Serine/threonine-protein kinase, active site / Serine/Threonine protein kinases active-site signature. / Protein kinase domain / Serine/Threonine protein kinases, catalytic domain / Protein kinase, ATP binding site / Protein kinases ATP-binding region signature. / Protein kinase domain profile. / Protein kinase domain / Protein kinase-like domain superfamily
Similarity search - Domain/homology
Mitogen-activated protein kinase kinase kinase 5
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.71 Å
AuthorsKosek, D. / Honzejkova, K. / Obsilova, V. / Obsil, T.
Funding support Czech Republic, 2items
OrganizationGrant numberCountry
Czech Science Foundation19-00121S Czech Republic
Czech Academy of Sciences67985823 Czech Republic
CitationJournal: Elife / Year: 2024
Title: The cryo-EM structure of ASK1 reveals an asymmetric architecture allosterically modulated by TRX1.
Authors: Karolina Honzejkova / Dalibor Kosek / Veronika Obsilova / Tomas Obsil /
Abstract: Apoptosis signal-regulating kinase 1 (ASK1) is a crucial stress sensor, directing cells toward apoptosis, differentiation, and senescence via the p38 and JNK signaling pathways. ASK1 dysregulation ...Apoptosis signal-regulating kinase 1 (ASK1) is a crucial stress sensor, directing cells toward apoptosis, differentiation, and senescence via the p38 and JNK signaling pathways. ASK1 dysregulation has been associated with cancer and inflammatory, cardiovascular, and neurodegenerative diseases, among others. However, our limited knowledge of the underlying structural mechanism of ASK1 regulation hampers our ability to target this member of the MAP3K protein family towards developing therapeutic interventions for these disorders. Nevertheless, as a multidomain Ser/Thr protein kinase, ASK1 is regulated by a complex mechanism involving dimerization and interactions with several other proteins, including thioredoxin 1 (TRX1). Thus, the present study aims at structurally characterizing ASK1 and its complex with TRX1 using several biophysical techniques. As shown by cryo-EM analysis, in a state close to its active form, ASK1 is a compact and asymmetric dimer, which enables extensive interdomain and interchain interactions. These interactions stabilize the active conformation of the ASK1 kinase domain. In turn, TRX1 functions as a negative allosteric effector of ASK1, modifying the structure of the TRX1-binding domain and changing its interaction with the tetratricopeptide repeats domain. Consequently, TRX1 reduces access to the activation segment of the kinase domain. Overall, our findings not only clarify the role of ASK1 dimerization and inter-domain contacts but also provide key mechanistic insights into its regulation, thereby highlighting the potential of ASK1 protein-protein interactions as targets for anti-inflammatory therapy.
History
DepositionSep 6, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 3, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Mitogen-activated protein kinase kinase kinase 5
B: Mitogen-activated protein kinase kinase kinase 5


Theoretical massNumber of molelcules
Total (without water)203,8172
Polymers203,8172
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable, assay for oligomerization, Sedimentation velocity
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Mitogen-activated protein kinase kinase kinase 5


Mass: 101908.625 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MAP3K5 / Production host: Escherichia coli (E. coli) / References: UniProt: Q99683

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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: C-terminally truncated Apoptosis signal-regulating kinase 1
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.2035 MDa / Experimental value: YES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
Details: 20 mM Tris-HCl 7.5 150 mM NaCl 2 mM 2-mercaptoethanol 3.8 mM CHAPSO
SpecimenConc.: 0.7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Gatan Solarus II 955 / Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 298 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: OTHER
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 2800 nm / Nominal defocus min: 700 nm
Image recordingElectron dose: 40 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 2 / Num. of real images: 5781
Details: 40 frames 2704 micrographs with 0 degree tilt, 8691 micrographs with 40 degree tilt, 5781 selected for analysis

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARC4.1.2particle selection
2SerialEMimage acquisition
7Coot0.9.8.4model fitting
9cryoSPARC4.1.2initial Euler assignment
10cryoSPARC4.1.2final Euler assignment
11cryoSPARC4.1.2classification
12cryoSPARC4.1.23D reconstruction
13PHENIX1.19.1-4122model refinementreal-space refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1126189
3D reconstructionResolution: 3.71 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 131110
Details: Sharpening was done using phenix.autosharpen (1.19.1-4122)
Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model building

3D fitting-ID: 1

IDPDB-IDPdb chain-IDAccession codeChain-IDDetailsInitial refinement model-IDSource nameTypeChain residue range
12CLQ

2clq

A2CLQAkinase domain1PDBexperimental model
25ULM

5ulm

A5ULMACRR2PDBexperimental model
3AF-Q99683-F1TBD initial model3AlphaFoldin silico model94-271
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00313020
ELECTRON MICROSCOPYf_angle_d0.59617604
ELECTRON MICROSCOPYf_dihedral_angle_d11.9454810
ELECTRON MICROSCOPYf_chiral_restr0.0581964
ELECTRON MICROSCOPYf_plane_restr0.0042243

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