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- PDB-8a8m: Structure of the MAPK p38alpha in complex with its activating MAP... -

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Basic information

Entry
Database: PDB / ID: 8a8m
TitleStructure of the MAPK p38alpha in complex with its activating MAP2K MKK6
Components
  • Dual specificity mitogen-activated protein kinase kinase 6
  • Mitogen-activated protein kinase 14
KeywordsIMMUNE SYSTEM / Kinase / Signalling / MAP kinase / phosphoryl transfer
Function / homology
Function and homology information


nucleotide-binding domain, leucine rich repeat containing receptor signaling pathway / mitogen-activated protein kinase kinase / Activation of PPARGC1A (PGC-1alpha) by phosphorylation / regulation of synaptic membrane adhesion / stress-induced premature senescence / negative regulation of cold-induced thermogenesis / positive regulation of myoblast fusion / cartilage condensation / stress-activated protein kinase signaling cascade / 3'-UTR-mediated mRNA stabilization ...nucleotide-binding domain, leucine rich repeat containing receptor signaling pathway / mitogen-activated protein kinase kinase / Activation of PPARGC1A (PGC-1alpha) by phosphorylation / regulation of synaptic membrane adhesion / stress-induced premature senescence / negative regulation of cold-induced thermogenesis / positive regulation of myoblast fusion / cartilage condensation / stress-activated protein kinase signaling cascade / 3'-UTR-mediated mRNA stabilization / CD163 mediating an anti-inflammatory response / positive regulation of cyclase activity / response to dietary excess / positive regulation of myotube differentiation / positive regulation of muscle cell differentiation / cellular response to UV-B / KSRP (KHSRP) binds and destabilizes mRNA / mitogen-activated protein kinase p38 binding / Myogenesis / Platelet sensitization by LDL / NFAT protein binding / cellular response to lipoteichoic acid / regulation of cytokine production involved in inflammatory response / Activation of the AP-1 family of transcription factors / p38MAPK cascade / ERK/MAPK targets / PI5P Regulates TP53 Acetylation / MAP kinase kinase activity / Regulation of MITF-M-dependent genes involved in pigmentation / MAP kinase activity / cellular response to vascular endothelial growth factor stimulus / Uptake and function of anthrax toxins / mitogen-activated protein kinase / vascular endothelial growth factor receptor signaling pathway / stress-activated MAPK cascade / RHO GTPases Activate NADPH Oxidases / positive regulation of myoblast differentiation / negative regulation of hippo signaling / osteoclast differentiation / positive regulation of interleukin-12 production / signal transduction in response to DNA damage / p38MAPK events / positive regulation of erythrocyte differentiation / regulation of signal transduction by p53 class mediator / tumor necrosis factor-mediated signaling pathway / response to insulin / cellular response to ionizing radiation / cellular response to virus / activated TAK1 mediates p38 MAPK activation / negative regulation of inflammatory response to antigenic stimulus / negative regulation of canonical Wnt signaling pathway / platelet activation / bone development / NOD1/2 Signaling Pathway / PKR-mediated signaling / VEGFA-VEGFR2 Pathway / cellular senescence / chemotaxis / positive regulation of reactive oxygen species metabolic process / osteoblast differentiation / Ubiquitin-Mediated Degradation of Phosphorylated Cdc25A / Interleukin-1 signaling / MAPK cascade / ADP signalling through P2Y purinoceptor 1 / cellular response to lipopolysaccharide / spindle pole / protein tyrosine kinase activity / secretory granule lumen / protein phosphatase binding / Oxidative Stress Induced Senescence / Regulation of TP53 Activity through Phosphorylation / ficolin-1-rich granule lumen / cytoskeleton / regulation of cell cycle / cell surface receptor signaling pathway / nuclear speck / intracellular signal transduction / apoptotic process / protein serine kinase activity / protein serine/threonine kinase activity / positive regulation of gene expression / regulation of transcription by RNA polymerase II / Neutrophil degranulation / protein kinase binding / glutamatergic synapse / enzyme binding / signal transduction / DNA-templated transcription / mitochondrion / nucleoplasm / extracellular region / ATP binding / identical protein binding / nucleus / cytosol / cytoplasm
Similarity search - Function
Mitogen-activated protein kinase 14 / Mitogen-activated protein (MAP) kinase p38-like / Mitogen-activated protein (MAP) kinase, conserved site / MAP kinase signature. / : / Transferase(Phosphotransferase) domain 1 / Transferase(Phosphotransferase); domain 1 / Serine/threonine-protein kinase, active site / Serine/Threonine protein kinases active-site signature. / Protein kinase domain ...Mitogen-activated protein kinase 14 / Mitogen-activated protein (MAP) kinase p38-like / Mitogen-activated protein (MAP) kinase, conserved site / MAP kinase signature. / : / Transferase(Phosphotransferase) domain 1 / Transferase(Phosphotransferase); domain 1 / 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 / Orthogonal Bundle / Mainly Alpha
Similarity search - Domain/homology
PHOSPHOMETHYLPHOSPHONIC ACID ADENOSYL ESTER / Dual specificity mitogen-activated protein kinase kinase 6 / Mitogen-activated protein kinase 14
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4 Å
AuthorsBowler, M.W. / Juyoux, P. / Pellegrini, E.
Funding support1items
OrganizationGrant numberCountry
Not funded
Citation
Journal: Science / Year: 2023
Title: Architecture of the MKK6-p38α complex defines the basis of MAPK specificity and activation.
Authors: Pauline Juyoux / Ioannis Galdadas / Dorothea Gobbo / Jill von Velsen / Martin Pelosse / Mark Tully / Oscar Vadas / Francesco Luigi Gervasio / Erika Pellegrini / Matthew W Bowler /
Abstract: The mitogen-activated protein kinase (MAPK) p38α is a central component of signaling in inflammation and the immune response and is, therefore, an important drug target. Little is known about the ...The mitogen-activated protein kinase (MAPK) p38α is a central component of signaling in inflammation and the immune response and is, therefore, an important drug target. Little is known about the molecular mechanism of its activation by double phosphorylation from MAPK kinases (MAP2Ks), because of the challenge of trapping a transient and dynamic heterokinase complex. We applied a multidisciplinary approach to generate a structural model of p38α in complex with its MAP2K, MKK6, and to understand the activation mechanism. Integrating cryo-electron microscopy with molecular dynamics simulations, hydrogen-deuterium exchange mass spectrometry, and experiments in cells, we demonstrate a dynamic, multistep phosphorylation mechanism, identify catalytically relevant interactions, and show that MAP2K-disordered amino termini determine pathway specificity. Our work captures a fundamental step of cell signaling: a kinase phosphorylating its downstream target kinase.
#1: Journal: Biorxiv / Year: 2022
Title: Architecture of the MKK6-p38 alpha complex defines the basis of MAPK specificity and activation
Authors: Juyoux, P. / Galdadas, I. / Gobbo, D. / Tully, M. / Gervasio, F.L. / Pellegrini, E. / Bowler, M.W.
History
DepositionJun 23, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 13, 2022Provider: repository / Type: Initial release
Revision 1.1Aug 3, 2022Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.pdbx_database_id_DOI / _citation.title / _citation.year
Revision 1.2Sep 20, 2023Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / citation_author / pdbx_initial_refinement_model
Revision 1.3Sep 27, 2023Group: Database references / Refinement description / Category: citation / citation_author / em_3d_fitting_list
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _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
Revision 1.4Nov 13, 2024Group: Data collection / Structure summary / Category: em_admin / pdbx_entry_details
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Mitogen-activated protein kinase 14
B: Dual specificity mitogen-activated protein kinase kinase 6
hetero molecules


Theoretical massNumber of molelcules
Total (without water)86,4188
Polymers85,4712
Non-polymers9486
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: SAXS, electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area4870 Å2
ΔGint-66 kcal/mol
Surface area31240 Å2
MethodPISA

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Components

#1: Protein Mitogen-activated protein kinase 14 / MAP kinase 14 / MAPK 14 / Cytokine suppressive anti-inflammatory drug-binding protein / CSAID- ...MAP kinase 14 / MAPK 14 / Cytokine suppressive anti-inflammatory drug-binding protein / CSAID-binding protein / CSBP / MAP kinase MXI2 / MAX-interacting protein 2 / Mitogen-activated protein kinase p38 alpha / MAP kinase p38 alpha / Stress-activated protein kinase 2a / SAPK2a


Mass: 43647.727 Da / Num. of mol.: 1 / Mutation: T180V
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MAPK14, CSBP, CSBP1, CSBP2, CSPB1, MXI2, SAPK2A / Plasmid: pET-28b+ / Production host: Escherichia coli (E. coli) / Strain (production host): DE3
References: UniProt: Q16539, mitogen-activated protein kinase
#2: Protein Dual specificity mitogen-activated protein kinase kinase 6 / MAP kinase kinase 6 / MAPKK 6 / MAPK/ERK kinase 6 / MEK 6 / Stress-activated protein kinase kinase ...MAP kinase kinase 6 / MAPKK 6 / MAPK/ERK kinase 6 / MEK 6 / Stress-activated protein kinase kinase 3 / SAPK kinase 3 / SAPKK-3 / SAPKK3


Mass: 41822.844 Da / Num. of mol.: 1 / Mutation: S207D, T211D
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MAP2K6, MEK6, MKK6, PRKMK6, SKK3 / Plasmid: pFastBac1 / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: P52564, mitogen-activated protein kinase kinase
#3: Chemical ChemComp-AP2 / PHOSPHOMETHYLPHOSPHONIC ACID ADENOSYL ESTER


Mass: 425.228 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C11H17N5O9P2 / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
Has protein modificationN

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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
1Complex between the MAP2K MKK6 and its substrate MAPK p38alphaCOMPLEX#1-#20MULTIPLE SOURCES
2Mitogen-activated protein kinase 14 (MAPK p38alpha)COMPLEX#11RECOMBINANT
3Dual specificity mitogen-activated protein kinase kinase 6 (MAP2K MKK6)COMPLEX#21RECOMBINANT
Molecular weightValue: 0.0802 MDa / Experimental value: YES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Homo sapiens (human)9606
33Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDStrainPlasmid
22Escherichia coli (E. coli)562DE3pET-28b+
33Spodoptera frugiperda (fall armyworm)7108
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
1200 mMsodium chlorideNaCl1
210 mMmagnesium chlorideMgCl21
350 mMHEPESHEPES1
SpecimenConc.: 0.48 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: 298 K / Details: blot for 3.5 seconds

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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: 215000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1500 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 70 K / Temperature (min): 70 K
Image recordingElectron dose: 62.77 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Num. of grids imaged: 3 / Num. of real images: 28633
Image scansMovie frames/image: 40

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
EM software
IDNameVersionCategoryDetails (eV)
1cryoSPARC3.3.2particle selectiontemplate + topaz
2SerialEMimage acquisition
4cryoSPARC3.3.2CTF correction
7PHENIXmodel fitting
9cryoSPARC3.3.2initial Euler assignment
10cryoSPARC3.2.2final Euler assignment
12cryoSPARC3.3.23D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 35123 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
15ETC

5etc

15ETC1PDBexperimental model
25ETA

5eta

15ETA2PDBexperimental model
36YG1

6yg1

16YG13PDBexperimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0045364
ELECTRON MICROSCOPYf_angle_d0.8947276
ELECTRON MICROSCOPYf_dihedral_angle_d6.732707
ELECTRON MICROSCOPYf_chiral_restr0.048814
ELECTRON MICROSCOPYf_plane_restr0.007921

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