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- PDB-8fdu: Engineered human dynein motor domain in the microtubule-unbound s... -

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Entry
Database: PDB / ID: 8fdu
TitleEngineered human dynein motor domain in the microtubule-unbound state with LIS1 complex in the buffer containing ATP-Vi (local refined on AAA3-AAA5 and LIS1)
Components
  • Cytoplasmic dynein 1 heavy chain 1,Serine--tRNA ligase
  • Platelet-activating factor acetylhydrolase IB subunit beta,Platelet-activating factor acetylhydrolase IB subunit beta,human LIS1 protein with a SNAP tag
KeywordsMOTOR PROTEIN / Dynein / motor domain / microtubule-unbound / LIS1
Function / homology
Function and homology information


microtubule cytoskeleton organization involved in establishment of planar polarity / establishment of planar polarity of embryonic epithelium / 1-alkyl-2-acetylglycerophosphocholine esterase complex / L-selenocysteine biosynthetic process / corpus callosum morphogenesis / maintenance of centrosome location / ameboidal-type cell migration / platelet activating factor metabolic process / radial glia-guided pyramidal neuron migration / serine-tRNA ligase ...microtubule cytoskeleton organization involved in establishment of planar polarity / establishment of planar polarity of embryonic epithelium / 1-alkyl-2-acetylglycerophosphocholine esterase complex / L-selenocysteine biosynthetic process / corpus callosum morphogenesis / maintenance of centrosome location / ameboidal-type cell migration / platelet activating factor metabolic process / radial glia-guided pyramidal neuron migration / serine-tRNA ligase / serine-tRNA ligase activity / seryl-tRNA aminoacylation / establishment of centrosome localization / acrosome assembly / cerebral cortex neuron differentiation / central region of growth cone / nuclear membrane disassembly / microtubule sliding / positive regulation of cytokine-mediated signaling pathway / positive regulation of embryonic development / microtubule organizing center organization / layer formation in cerebral cortex / interneuron migration / astral microtubule / auditory receptor cell development / cortical microtubule organization / myeloid leukocyte migration / reelin-mediated signaling pathway / positive regulation of intracellular transport / regulation of metaphase plate congression / positive regulation of spindle assembly / positive regulation of dendritic spine morphogenesis / osteoclast development / establishment of spindle localization / stereocilium / microtubule plus-end binding / brain morphogenesis / motile cilium / vesicle transport along microtubule / retrograde axonal transport / COPI-independent Golgi-to-ER retrograde traffic / minus-end-directed microtubule motor activity / microtubule associated complex / negative regulation of JNK cascade / P-body assembly / dynein light intermediate chain binding / cytoplasmic dynein complex / kinesin complex / neuromuscular process controlling balance / stem cell division / nuclear migration / establishment of mitotic spindle orientation / dynein intermediate chain binding / cell leading edge / germ cell development / transmission of nerve impulse / dynein complex binding / neuroblast proliferation / dynactin binding / cochlea development / protein secretion / microtubule-based process / positive regulation of axon extension / lipid catabolic process / COPI-mediated anterograde transport / cytoplasmic microtubule / phospholipase binding / JNK cascade / cytoplasmic microtubule organization / Loss of Nlp from mitotic centrosomes / Loss of proteins required for interphase microtubule organization from the centrosome / axon cytoplasm / Amplification of signal from unattached kinetochores via a MAD2 inhibitory signal / Recruitment of mitotic centrosome proteins and complexes / MHC class II antigen presentation / positive regulation of mitotic cell cycle / Recruitment of NuMA to mitotic centrosomes / Anchoring of the basal body to the plasma membrane / HSP90 chaperone cycle for steroid hormone receptors (SHR) in the presence of ligand / Mitotic Prometaphase / EML4 and NUDC in mitotic spindle formation / AURKA Activation by TPX2 / Resolution of Sister Chromatid Cohesion / stress granule assembly / mitotic spindle organization / regulation of microtubule cytoskeleton organization / regulation of mitotic spindle organization / filopodium / adult locomotory behavior / hippocampus development / phosphoprotein binding / RHO GTPases Activate Formins / cerebral cortex development / kinetochore / modulation of chemical synaptic transmission / microtubule cytoskeleton organization / Schaffer collateral - CA1 synapse / neuron migration / HCMV Early Events / Aggrephagy
Similarity search - Function
: / : / PAC1 LisH domain / Serine-tRNA synthetase, type1, N-terminal / Seryl-tRNA synthetase N-terminal domain / Serine-tRNA ligase, type1 / Serine-tRNA ligase catalytic core domain / Serine-tRNA synthetase, type1, N-terminal domain superfamily / Dynein regulator LIS1 / LIS1, N-terminal ...: / : / PAC1 LisH domain / Serine-tRNA synthetase, type1, N-terminal / Seryl-tRNA synthetase N-terminal domain / Serine-tRNA ligase, type1 / Serine-tRNA ligase catalytic core domain / Serine-tRNA synthetase, type1, N-terminal domain superfamily / Dynein regulator LIS1 / LIS1, N-terminal / Class I and II aminoacyl-tRNA synthetase, tRNA-binding arm / Lissencephaly type-1-like homology motif / Dynein heavy chain, AAA 5 extension domain / Dynein heavy chain AAA lid domain / Dynein heavy chain, C-terminal domain / Dynein heavy chain, C-terminal domain, barrel region / Dynein heavy chain C-terminal domain / LIS1 homology (LisH) motif profile. / LIS1 homology motif / Aminoacyl-tRNA synthetase, class II (G/ P/ S/T) / tRNA synthetase class II core domain (G, H, P, S and T) / : / Dynein heavy chain, ATPase lid domain / Dynein heavy chain, tail / Dynein heavy chain, N-terminal region 1 / P-loop containing dynein motor region / Dynein heavy chain region D6 P-loop domain / Dynein heavy chain, linker / Dynein heavy chain, AAA module D4 / Dynein heavy chain, coiled coil stalk / Dynein heavy chain / Dynein heavy chain, hydrolytic ATP-binding dynein motor region / Dynein heavy chain, ATP-binding dynein motor region / Dynein heavy chain AAA lid domain / Dynein heavy chain AAA lid domain superfamily / Dynein heavy chain, domain 2, N-terminal / Dynein heavy chain, linker, subdomain 3 / Dynein heavy chain, AAA1 domain, small subdomain / Dynein heavy chain region D6 P-loop domain / Dynein heavy chain, N-terminal region 2 / Hydrolytic ATP binding site of dynein motor region / Microtubule-binding stalk of dynein motor / P-loop containing dynein motor region D4 / ATP-binding dynein motor region / Dynein heavy chain AAA lid domain / Aminoacyl-tRNA synthetase, class II / Aminoacyl-transfer RNA synthetases class-II family profile. / Class II Aminoacyl-tRNA synthetase/Biotinyl protein ligase (BPL) and lipoyl protein ligase (LPL) / WD domain, G-beta repeat / G-protein beta WD-40 repeat / WD40 repeat, conserved site / Trp-Asp (WD) repeats signature. / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD40 repeats / WD40 repeat / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / Platelet-activating factor acetylhydrolase IB subunit beta / Cytoplasmic dynein 1 heavy chain 1 / Serine--tRNA ligase
Similarity search - Component
Biological speciesHomo sapiens (human)
Thermus thermophilus (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsTon, W. / Wang, Y. / Chai, P.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM139483 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM142959 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)S10OD023603 United States
CitationJournal: Nat Struct Mol Biol / Year: 2023
Title: Microtubule-binding-induced allostery triggers LIS1 dissociation from dynein prior to cargo transport.
Authors: William D Ton / Yue Wang / Pengxin Chai / Cisloynny Beauchamp-Perez / Nicholas T Flint / Lindsay G Lammers / Hao Xiong / Kai Zhang / Steven M Markus /
Abstract: The lissencephaly-related protein LIS1 is a critical regulator of cytoplasmic dynein that governs motor function and intracellular localization (for example, to microtubule plus-ends). Although LIS1 ...The lissencephaly-related protein LIS1 is a critical regulator of cytoplasmic dynein that governs motor function and intracellular localization (for example, to microtubule plus-ends). Although LIS1 binding is required for dynein activity, its unbinding prior to initiation of cargo transport is equally important, since preventing dissociation leads to dynein dysfunction. To understand whether and how dynein-LIS1 binding is modulated, we engineered dynein mutants locked in a microtubule-bound (MT-B) or microtubule-unbound (MT-U) state. Whereas the MT-B mutant exhibits low LIS1 affinity, the MT-U mutant binds LIS1 with high affinity, and as a consequence remains almost irreversibly associated with microtubule plus-ends. We find that a monomeric motor domain is sufficient to exhibit these opposing LIS1 affinities, and that this is evolutionarily conserved between yeast and humans. Three cryo-EM structures of human dynein with and without LIS1 reveal microtubule-binding induced conformational changes responsible for this regulation. Our work reveals key biochemical and structural insight into LIS1-mediated dynein activation.
History
DepositionDec 4, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 21, 2023Provider: repository / Type: Initial release
Revision 1.1Jun 28, 2023Group: Database references / Category: citation / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Sep 20, 2023Group: Data collection / Database references
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.3Nov 15, 2023Group: Database references / Source and taxonomy / Structure summary
Category: entity / entity_name_com ...entity / entity_name_com / entity_src_gen / struct_ref / struct_ref_seq / struct_ref_seq_dif
Item: _entity.details / _entity.pdbx_description ..._entity.details / _entity.pdbx_description / _entity.pdbx_ec / _entity.pdbx_mutation / _entity_name_com.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Cytoplasmic dynein 1 heavy chain 1,Serine--tRNA ligase
B: Platelet-activating factor acetylhydrolase IB subunit beta,Platelet-activating factor acetylhydrolase IB subunit beta,human LIS1 protein with a SNAP tag
C: Platelet-activating factor acetylhydrolase IB subunit beta,Platelet-activating factor acetylhydrolase IB subunit beta,human LIS1 protein with a SNAP tag
hetero molecules


Theoretical massNumber of molelcules
Total (without water)491,1615
Polymers490,3063
Non-polymers8542
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Cytoplasmic dynein 1 heavy chain 1,Serine--tRNA ligase / Cytoplasmic dynein heavy chain 1 / Dynein heavy chain / cytosolic / Seryl-tRNA synthetase / SerRS / ...Cytoplasmic dynein heavy chain 1 / Dynein heavy chain / cytosolic / Seryl-tRNA synthetase / SerRS / Seryl-tRNA(Ser/Sec) synthetase


Mass: 357459.125 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: residues 1458-3277 of dynein followed by residues 30-96 of serine-tRNA ligase, followed by residues 3412-4646 of dynein
Source: (gene. exp.) Homo sapiens (human), (gene. exp.) Thermus thermophilus (bacteria)
Gene: DYNC1H1, DHC1, DNCH1, DNCL, DNECL, DYHC, KIAA0325, serS, TTHA0875
Strain: HB8 / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: Q14204, UniProt: Q5SJX7, serine-tRNA ligase
#2: Protein Platelet-activating factor acetylhydrolase IB subunit beta,Platelet-activating factor acetylhydrolase IB subunit beta,human LIS1 protein with a SNAP tag / Lissencephaly-1 protein / LIS-1 / PAF acetylhydrolase 45 kDa subunit / PAF-AH 45 kDa subunit / PAF- ...Lissencephaly-1 protein / LIS-1 / PAF acetylhydrolase 45 kDa subunit / PAF-AH 45 kDa subunit / PAF-AH alpha / PAFAH alpha


Mass: 66423.547 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: SNAP tag present on C-terminus,SNAP tag present on C-terminus
Source: (gene. exp.) Homo sapiens (human) / Gene: PAFAH1B1, LIS1, MDCR, MDS, PAFAHA / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P43034
#3: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
Has ligand of interestY

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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: Engineered human dynein motor domain in microtubule-unbound state with LIS1 complex in the buffer containing ATP-Vi
Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.49 MDa / Experimental value: YES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.4
Details: 50 mM Tris pH 7.4, 150 mM potassium acetate, 2 mM magnesium acetate, 1 mM EGTA, 1 mM DTT, and 0.1 mM Mg-ATP
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277 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: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Calibrated magnification: 105000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1200 nm / Calibrated defocus min: 1200 nm / Calibrated defocus max: 3000 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 40 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: UCSF ChimeraX / Version: 1.4/v9 / Classification: model building / URL: https://www.rbvi.ucsf.edu/chimerax/ / Os: macOS / Type: package
EM software
IDNameVersionCategory
1cryoSPARC3particle selection
2EPUimage acquisition
4cryoSPARC3CTF correction
7UCSF ChimeraX1.4model fitting
9PHENIX2.9model refinement
10cryoSPARC3initial Euler assignment
11cryoSPARC3final Euler assignment
13cryoSPARC33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 53572 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL

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