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- PDB-8vu5: Cryo-EM structure of MPL bound to TPO -

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Basic information

Entry
Database: PDB / ID: 8vu5
TitleCryo-EM structure of MPL bound to TPO
Components
  • Thrombopoietin
  • Thrombopoietin receptor
KeywordsSIGNALING PROTEIN / Receptor / cytokine / complex / platelet synthesis
Function / homology
Function and homology information


thrombopoietin receptor activity / regulation of chemokine production / positive regulation of hematopoietic stem cell proliferation / regulation of stem cell division / megakaryocyte differentiation / positive regulation of megakaryocyte differentiation / thrombopoietin-mediated signaling pathway / regulation of stem cell proliferation / myeloid cell differentiation / definitive hemopoiesis ...thrombopoietin receptor activity / regulation of chemokine production / positive regulation of hematopoietic stem cell proliferation / regulation of stem cell division / megakaryocyte differentiation / positive regulation of megakaryocyte differentiation / thrombopoietin-mediated signaling pathway / regulation of stem cell proliferation / myeloid cell differentiation / definitive hemopoiesis / platelet formation / homeostasis of number of cells / cytokine activity / hormone activity / positive regulation of protein phosphorylation / cell population proliferation / positive regulation of ERK1 and ERK2 cascade / positive regulation of phosphatidylinositol 3-kinase/protein kinase B signal transduction / positive regulation of gene expression / negative regulation of apoptotic process / cell surface / Golgi apparatus / extracellular space / plasma membrane
Similarity search - Function
Thrombopoietin / Erythropoietin/thrombopoietin / Erythropoietin/thrombopoeitin, conserved site / Erythropoietin/thrombopoietin / Erythropoietin / thrombopoeitin signature. / Long hematopoietin receptor, single chain, conserved site / Growth hormone/erythropoietin receptor, ligand binding / Erythropoietin receptor, ligand binding / Long hematopoietin receptor, single chain family signature. / Four-helical cytokine-like, core ...Thrombopoietin / Erythropoietin/thrombopoietin / Erythropoietin/thrombopoeitin, conserved site / Erythropoietin/thrombopoietin / Erythropoietin / thrombopoeitin signature. / Long hematopoietin receptor, single chain, conserved site / Growth hormone/erythropoietin receptor, ligand binding / Erythropoietin receptor, ligand binding / Long hematopoietin receptor, single chain family signature. / Four-helical cytokine-like, core / Fibronectin type 3 domain / Fibronectin type-III domain profile. / Fibronectin type III / Fibronectin type III superfamily / Immunoglobulin-like fold
Similarity search - Domain/homology
Thrombopoietin / Thrombopoietin receptor
Similarity search - Component
Biological speciesMus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.39 Å
AuthorsBratkowski, M. / Hao, Q. / Paddock, M.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Blood Vessel Thromb Hemost / Year: 2024
Title: Structural basis of MPL activation by thrombopoietin.
Authors: Amirhossein Mafi / Matthew Bratkowski / Jiefei Geng / Alyssa A Brito / Janani Sridar / Dongjian Hu / Anhdao T Darcy / Dhaval Nanavati / Nathan J Brown / Manoj K Rathinaswamy / Yuliya ...Authors: Amirhossein Mafi / Matthew Bratkowski / Jiefei Geng / Alyssa A Brito / Janani Sridar / Dongjian Hu / Anhdao T Darcy / Dhaval Nanavati / Nathan J Brown / Manoj K Rathinaswamy / Yuliya Kutskova / Dan Eaton / Qi Hao / Marcia Paddock
Abstract: Myeloproliferative leukemia protein (MPL), also known as thrombopoietin (TPO) receptor, is a class I cytokine receptor that is expressed on hematopoietic progenitors, promoting growth and ...Myeloproliferative leukemia protein (MPL), also known as thrombopoietin (TPO) receptor, is a class I cytokine receptor that is expressed on hematopoietic progenitors, promoting growth and differentiation toward the megakaryocyte lineage and is critical for normal platelet production. Mutations in MPL, TPO, or Janus kinase 2 (JAK2) have been implicated in multiple diseases from congenital thrombocytopenias to myeloproliferative neoplasms. The ligand for MPL, TPO, stimulates platelet production by inducing MPL dimerization and results in an active conformation that allows downstream JAK2/STAT5 signaling. Despite the biological importance of this pathway, the molecular signaling mechanism remained unclear. Here, we present a 3.39-Å cryo-electron microscopy structure of the ectodomain of mouse MPL bound to TPO. The structure revealed both low and high affinity sites between MPL and TPO that contain several pathologic mutations. To better understand TPO-driven MPL signaling, we expanded upon this structure by molecular dynamic (MD) simulations to model the full-length human MPL/TPO complex, and showed that MPL D4-D4 domain interactions are functionally relevant in activity assays. To build on our understanding of downstream activation, we added JAK2 to the MPL/TPO complex by MD simulations. This ternary complex illustrates JAK2 dimerization through the pseudokinase domain, illustrates residues important for MPL interactions, and reveals the constitutive activation mechanism of patient mutant V617F. The model also suggests the mechanism of JAK2 tyrosine kinase domain transphosphorylation. Overall, our studies illuminate TPO/MPL/JAK2 signaling mechanisms and provide additional insight into the nature of receptor signaling, which will further benefit human health.
History
DepositionJan 28, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 11, 2024Provider: repository / Type: Initial release
Revision 1.0Sep 11, 2024Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Sep 11, 2024Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
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Revision 1.0Sep 11, 2024Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
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Revision 1.1Nov 13, 2024Group: Data collection / Database references / Structure summary
Category: citation / em_admin ...citation / em_admin / pdbx_entry_details / pdbx_modification_feature
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Revision 1.2May 21, 2025Group: Data collection / Category: em_admin / em_software / Item: _em_admin.last_update / _em_software.name
Revision 1.1May 21, 2025Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Data processing / Experimental summary / Data content type: EM metadata / EM metadata / Category: em_admin / em_software / Data content type: EM metadata / EM metadata / Item: _em_admin.last_update / _em_software.name
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Thrombopoietin
B: Thrombopoietin receptor
C: Thrombopoietin receptor


Theoretical massNumber of molelcules
Total (without water)122,4263
Polymers122,4263
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable, gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Thrombopoietin / C-mpl ligand / ML / Megakaryocyte colony-stimulating factor / Megakaryocyte growth and development ...C-mpl ligand / ML / Megakaryocyte colony-stimulating factor / Megakaryocyte growth and development factor / MGDF / Myeloproliferative leukemia virus oncogene ligand


Mass: 18365.381 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Thpo / Cell line (production host): HEK239 / Production host: Homo sapiens (human) / References: UniProt: P40226
#2: Protein Thrombopoietin receptor / TPO-R / Myeloproliferative leukemia protein / Proto-oncogene c-Mpl


Mass: 52030.488 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Mpl, Tpor / Production host: Homo sapiens (human) / References: UniProt: Q08351
Has protein modificationY

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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: Thrombopoietin bound to thrombopoietin receptor / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.122 MDa / Experimental value: NO
Source (natural)Organism: Mus musculus (house mouse)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 8
SpecimenConc.: 0.8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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 defocus max: 3000 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 1.18 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM softwareName: PHENIX / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.39 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 207077 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0045765
ELECTRON MICROSCOPYf_angle_d0.6477853
ELECTRON MICROSCOPYf_dihedral_angle_d4.625760
ELECTRON MICROSCOPYf_chiral_restr0.042871
ELECTRON MICROSCOPYf_plane_restr0.0051011

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