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- PDB-8rgh: Structure of dynein-2 intermediate chain DYNC2I1 (WDR60) in compl... -

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Basic information

Entry
Database: PDB / ID: 8rgh
TitleStructure of dynein-2 intermediate chain DYNC2I1 (WDR60) in complex with the dynein-2 heavy chain DYNC2H1.
Components
  • Cytoplasmic dynein 2 intermediate chain 1
  • Cytoplasmic dynein 2 intermediate chain 2
  • Cytoplasmic dynein 2 light intermediate chain 1
  • Dynein light chain roadblock-type 1
  • Methylated-DNA--protein-cysteine methyltransferase,Cytoplasmic dynein 2 heavy chain 1
KeywordsTRANSPORT PROTEIN / dynein / cilia / intraflagellar transport / complex
Function / homology
Function and homology information


MGMT-mediated DNA damage reversal / intraciliary transport involved in cilium assembly / intraciliary retrograde transport / visual behavior / ciliary transition zone / dynein light chain binding / methylated-DNA-[protein]-cysteine S-methyltransferase / methylated-DNA-[protein]-cysteine S-methyltransferase activity / intraciliary transport / cilium movement involved in cell motility ...MGMT-mediated DNA damage reversal / intraciliary transport involved in cilium assembly / intraciliary retrograde transport / visual behavior / ciliary transition zone / dynein light chain binding / methylated-DNA-[protein]-cysteine S-methyltransferase / methylated-DNA-[protein]-cysteine S-methyltransferase activity / intraciliary transport / cilium movement involved in cell motility / 9+2 motile cilium / dynein heavy chain binding / regulation of cilium assembly / spinal cord motor neuron differentiation / DNA-methyltransferase activity / embryonic skeletal system morphogenesis / ciliary tip / Intraflagellar transport / DNA alkylation repair / DNA ligation / dynein complex / protein localization to cilium / minus-end-directed microtubule motor activity / cytoplasmic dynein complex / non-motile cilium assembly / coronary vasculature development / dynein light intermediate chain binding / positive regulation of smoothened signaling pathway / ciliary plasm / dorsal/ventral pattern formation / motile cilium / determination of left/right symmetry / embryonic limb morphogenesis / positive regulation of double-strand break repair / microtubule motor activity / ciliary base / dynein intermediate chain binding / microtubule-based movement / pericentriolar material / Golgi organization / axoneme / cytoskeletal motor activity / cilium assembly / Hedgehog 'off' state / forebrain development / centriole / ciliary basal body / filopodium / kidney development / methyltransferase activity / cilium / protein processing / apical part of cell / microtubule / DNA repair / centrosome / negative regulation of apoptotic process / Golgi apparatus / ATP hydrolysis activity / DNA binding / extracellular space / extracellular exosome / nucleoplasm / ATP binding / membrane / metal ion binding / nucleus / plasma membrane / cytosol / cytoplasm
Similarity search - Function
Cytoplasmic dynein 2 light intermediate chain 1 / Cytoplasmic dynein 2 intermediate chain 1 / : / Cytoplasmic dynein 2 heavy chain 1, AAA+ ATPase domain / Methylguanine DNA methyltransferase, ribonuclease-like domain / Dynein family light intermediate chain / 6-O-methylguanine DNA methyltransferase, ribonuclease-like domain / Dynein light intermediate chain (DLIC) / Dynein light chain roadblock-type 1/2 / Methylated DNA-protein cysteine methyltransferase domain superfamily ...Cytoplasmic dynein 2 light intermediate chain 1 / Cytoplasmic dynein 2 intermediate chain 1 / : / Cytoplasmic dynein 2 heavy chain 1, AAA+ ATPase domain / Methylguanine DNA methyltransferase, ribonuclease-like domain / Dynein family light intermediate chain / 6-O-methylguanine DNA methyltransferase, ribonuclease-like domain / Dynein light intermediate chain (DLIC) / Dynein light chain roadblock-type 1/2 / Methylated DNA-protein cysteine methyltransferase domain superfamily / Methylated-DNA-[protein]-cysteine S-methyltransferase, active site / Methylated-DNA--protein-cysteine methyltransferase active site. / Methylated-DNA-[protein]-cysteine S-methyltransferase, DNA binding / Methylated DNA-protein cysteine methyltransferase, DNA binding domain / 6-O-methylguanine DNA methyltransferase, DNA binding domain / Roadblock/LAMTOR2 domain / Roadblock/LC7 domain / Roadblock/LC7 domain / Dynein heavy chain, C-terminal domain / Dynein heavy chain, C-terminal domain, barrel region / Dynein heavy chain C-terminal domain / P-loop containing dynein motor region / Dynein heavy chain, tail / Dynein heavy chain, N-terminal region 1 / Dynein heavy chain / 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, 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 / WD domain, G-beta repeat / WD40 repeats / WD40 repeat / Trp-Asp (WD) repeats circular profile. / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily / Winged helix-like DNA-binding domain superfamily / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Methylated-DNA--protein-cysteine methyltransferase / Cytoplasmic dynein 2 heavy chain 1 / Cytoplasmic dynein 2 light intermediate chain 1 / Cytoplasmic dynein 2 intermediate chain 1 / Cytoplasmic dynein 2 intermediate chain 2 / Dynein light chain roadblock-type 1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsMukhopadhyay, A.G. / Toropova, K. / Daly, L. / Wells, J. / Vuolo, L. / Seda, M. / Jenkins, D. / Stephens, D.J. / Roberts, A.J.
Funding support United Kingdom, 9items
OrganizationGrant numberCountry
Wellcome Trust217186/Z/19/Z United Kingdom
Wellcome Trust202679/Z/16/Z United Kingdom
Wellcome Trust206166/Z/17/Z United Kingdom
Biotechnology and Biological Sciences Research Council (BBSRC)BB/P008348/1 United Kingdom
Biotechnology and Biological Sciences Research Council (BBSRC)BB/S007202/1 United Kingdom
Royal SocietyRG170260 United Kingdom
Biotechnology and Biological Sciences Research Council (BBSRC)BB/S013024/1 United Kingdom
Biotechnology and Biological Sciences Research Council (BBSRC)BB/S005390/1 United Kingdom
Wellcome Trust210585/Z/18/Z United Kingdom
CitationJournal: EMBO J / Year: 2024
Title: Structure and tethering mechanism of dynein-2 intermediate chains in intraflagellar transport.
Authors: Aakash G Mukhopadhyay / Katerina Toropova / Lydia Daly / Jennifer N Wells / Laura Vuolo / Miroslav Mladenov / Marian Seda / Dagan Jenkins / David J Stephens / Anthony J Roberts /
Abstract: Dynein-2 is a large multiprotein complex that powers retrograde intraflagellar transport (IFT) of cargoes within cilia/flagella, but the molecular mechanism underlying this function is still emerging. ...Dynein-2 is a large multiprotein complex that powers retrograde intraflagellar transport (IFT) of cargoes within cilia/flagella, but the molecular mechanism underlying this function is still emerging. Distinctively, dynein-2 contains two identical force-generating heavy chains that interact with two different intermediate chains (WDR34 and WDR60). Here, we dissect regulation of dynein-2 function by WDR34 and WDR60 using an integrative approach including cryo-electron microscopy and CRISPR/Cas9-enabled cell biology. A 3.9 Å resolution structure shows how WDR34 and WDR60 use surprisingly different interactions to engage equivalent sites of the two heavy chains. We show that cilia can assemble in the absence of either WDR34 or WDR60 individually, but not both subunits. Dynein-2-dependent distribution of cargoes depends more strongly on WDR60, because the unique N-terminal extension of WDR60 facilitates dynein-2 targeting to cilia. Strikingly, this N-terminal extension can be transplanted onto WDR34 and retain function, suggesting it acts as a flexible tether to the IFT "trains" that assemble at the ciliary base. We discuss how use of unstructured tethers represents an emerging theme in IFT train interactions.
History
DepositionDec 13, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 20, 2024Provider: repository / Type: Initial release
Revision 1.1Apr 10, 2024Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Methylated-DNA--protein-cysteine methyltransferase,Cytoplasmic dynein 2 heavy chain 1
C: Cytoplasmic dynein 2 intermediate chain 1
D: Cytoplasmic dynein 2 intermediate chain 2
E: Cytoplasmic dynein 2 light intermediate chain 1
G: Dynein light chain roadblock-type 1
H: Dynein light chain roadblock-type 1


Theoretical massNumber of molelcules
Total (without water)760,2786
Polymers760,2786
Non-polymers00
Water0
1


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

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Components

#1: Protein Methylated-DNA--protein-cysteine methyltransferase,Cytoplasmic dynein 2 heavy chain 1 / 6-O-methylguanine-DNA methyltransferase / MGMT / O-6-methylguanine-DNA-alkyltransferase / ...6-O-methylguanine-DNA methyltransferase / MGMT / O-6-methylguanine-DNA-alkyltransferase / Cytoplasmic dynein 2 heavy chain / Dynein cytoplasmic heavy chain 2 / Dynein heavy chain 11 / hDHC11 / Dynein heavy chain isotype 1B


Mass: 515223.031 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: DYNC2H1 with N-terminal SNAPf tag / Source: (gene. exp.) Homo sapiens (human) / Gene: MGMT, DYNC2H1, DHC1B, DHC2, DNCH2, DYH1B, KIAA1997 / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: P16455, UniProt: Q8NCM8, methylated-DNA-[protein]-cysteine S-methyltransferase
#2: Protein Cytoplasmic dynein 2 intermediate chain 1 / Dynein 2 intermediate chain 1 / WD repeat-containing protein 60


Mass: 122865.156 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: DYNC2I1, WDR60 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q8WVS4
#3: Protein Cytoplasmic dynein 2 intermediate chain 2 / Dynein 2 intermediate chain 2 / WD repeat-containing protein 34


Mass: 60639.129 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: DYNC2I2 (also known as WDR34) with C-terminal Strep tag
Source: (gene. exp.) Homo sapiens (human) / Gene: DYNC2I2, WDR34 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q96EX3
#4: Protein Cytoplasmic dynein 2 light intermediate chain 1 / Dynein 2 light intermediate chain


Mass: 39681.621 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: DYNC2LI1, D2LIC, LIC3, CGI-60 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q8TCX1
#5: Protein Dynein light chain roadblock-type 1 / Bithoraxoid-like protein / BLP / Dynein light chain 2A / cytoplasmic / Dynein-associated protein ...Bithoraxoid-like protein / BLP / Dynein light chain 2A / cytoplasmic / Dynein-associated protein Km23 / Roadblock domain-containing protein 1


Mass: 10934.576 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: DYNLRB1, BITH, DNCL2A, DNLC2A, ROBLD1, HSPC162 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q9NP97

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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: Dynein-2 complex / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.5
SpecimenEmbedding 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 FIELDBright-field microscopy / Nominal defocus max: 3500 nm / Nominal defocus min: 1500 nm
Image recordingElectron dose: 50.6 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 113479 / Details: Global resolution 3.9A / Symmetry type: POINT

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