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- PDB-5ogc: Molecular basis of human kinesin-8 function and inhibition -

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Basic information

Entry
Database: PDB / ID: 5ogc
TitleMolecular basis of human kinesin-8 function and inhibition
Components
  • Kinesin-like protein KIF18A
  • Tubulin alpha chain
  • Tubulin beta chain
KeywordsTRANSPORT PROTEIN / Cytoskeleton / Motor protein / Kinesin
Function / homology
Function and homology information


tubulin-dependent ATPase activity / mitotic spindle astral microtubule / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Cilium Assembly / Intraflagellar transport / Carboxyterminal post-translational modifications of tubulin / Sealing of the nuclear envelope (NE) by ESCRT-III / Kinesins / Resolution of Sister Chromatid Cohesion / Mitotic Prometaphase ...tubulin-dependent ATPase activity / mitotic spindle astral microtubule / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Cilium Assembly / Intraflagellar transport / Carboxyterminal post-translational modifications of tubulin / Sealing of the nuclear envelope (NE) by ESCRT-III / Kinesins / Resolution of Sister Chromatid Cohesion / Mitotic Prometaphase / EML4 and NUDC in mitotic spindle formation / COPI-dependent Golgi-to-ER retrograde traffic / COPI-independent Golgi-to-ER retrograde traffic / COPI-mediated anterograde transport / RHO GTPases activate IQGAPs / RHO GTPases Activate Formins / MHC class II antigen presentation / mitotic spindle midzone / HSP90 chaperone cycle for steroid hormone receptors (SHR) in the presence of ligand / Aggrephagy / kinetochore microtubule / The role of GTSE1 in G2/M progression after G2 checkpoint / Separation of Sister Chromatids / Loss of Nlp from mitotic centrosomes / Recruitment of mitotic centrosome proteins and complexes / Loss of proteins required for interphase microtubule organization from the centrosome / Anchoring of the basal body to the plasma membrane / AURKA Activation by TPX2 / male meiotic nuclear division / Recruitment of NuMA to mitotic centrosomes / Regulation of PLK1 Activity at G2/M Transition / Hedgehog 'off' state / microtubule plus-end binding / Kinesins / plus-end-directed microtubule motor activity / microtubule depolymerization / COPI-dependent Golgi-to-ER retrograde traffic / kinesin complex / mitotic metaphase chromosome alignment / microtubule-based movement / seminiferous tubule development / mitotic sister chromatid segregation / Amplification of signal from unattached kinetochores via a MAD2 inhibitory signal / regulation of microtubule cytoskeleton organization / Mitotic Prometaphase / EML4 and NUDC in mitotic spindle formation / ruffle / MHC class II antigen presentation / Resolution of Sister Chromatid Cohesion / cellular response to estradiol stimulus / RHO GTPases Activate Formins / caveola / structural constituent of cytoskeleton / kinetochore / microtubule cytoskeleton organization / Separation of Sister Chromatids / microtubule cytoskeleton / protein transport / mitotic cell cycle / actin binding / microtubule binding / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / microtubule / hydrolase activity / GTPase activity / centrosome / GTP binding / ATP hydrolysis activity / ATP binding / nucleus / metal ion binding / cytosol / cytoplasm
Similarity search - Function
Kinesin-like protein / Kinesin motor domain signature. / Kinesin motor domain, conserved site / Kinesin motor domain profile. / Kinesin motor, catalytic domain. ATPase. / Kinesin motor domain / Kinesin motor domain / Kinesin motor domain superfamily / Alpha tubulin / Tubulin-beta mRNA autoregulation signal. ...Kinesin-like protein / Kinesin motor domain signature. / Kinesin motor domain, conserved site / Kinesin motor domain profile. / Kinesin motor, catalytic domain. ATPase. / Kinesin motor domain / Kinesin motor domain / Kinesin motor domain superfamily / Alpha tubulin / Tubulin-beta mRNA autoregulation signal. / Beta tubulin, autoregulation binding site / Beta tubulin / Tubulin / Tubulin, C-terminal / Tubulin C-terminal domain / Tubulin, conserved site / Tubulin subunits alpha, beta, and gamma signature. / Tubulin/FtsZ family, C-terminal domain / Tubulin/FtsZ-like, C-terminal domain / Tubulin/FtsZ, C-terminal / Tubulin/FtsZ, 2-layer sandwich domain / Tubulin/FtsZ family, GTPase domain / Tubulin/FtsZ family, GTPase domain / Tubulin/FtsZ, GTPase domain / Tubulin/FtsZ, GTPase domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
4-chloranyl-2-nitro-1-(phenylsulfonyl)benzene / GUANOSINE-5'-DIPHOSPHATE / GUANOSINE-5'-TRIPHOSPHATE / TAXOL / Tubulin alpha chain / Tubulin beta chain / Tubulin alpha-1B chain / Kinesin-like protein KIF18A
Similarity search - Component
Biological speciesHomo sapiens (human)
Bos taurus (cattle)
Sus scrofa (pig)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 4.8 Å
AuthorsLocke, J. / Joseph, A.P. / Topf, M. / Moores, C.A.
Funding support United Kingdom, 2items
OrganizationGrant numberCountry
Cancer Research UKC33336/A13177 United Kingdom
Worldwide Cancer Research16-0037 United Kingdom
CitationJournal: Proc Natl Acad Sci U S A / Year: 2017
Title: Structural basis of human kinesin-8 function and inhibition.
Authors: Julia Locke / Agnel Praveen Joseph / Alejandro Peña / Martin M Möckel / Thomas U Mayer / Maya Topf / Carolyn A Moores /
Abstract: Kinesin motors play diverse roles in mitosis and are targets for antimitotic drugs. The clinical significance of these motors emphasizes the importance of understanding the molecular basis of their ...Kinesin motors play diverse roles in mitosis and are targets for antimitotic drugs. The clinical significance of these motors emphasizes the importance of understanding the molecular basis of their function. Equally important, investigations into the modes of inhibition of these motors provide crucial information about their molecular mechanisms. Kif18A regulates spindle microtubules through its dual functionality, with microtubule-based stepping and regulation of microtubule dynamics. We investigated the mechanism of Kif18A and its inhibition by the small molecule BTB-1. The Kif18A motor domain drives ATP-dependent plus-end microtubule gliding, and undergoes conformational changes consistent with canonical mechanisms of plus-end-directed motility. The Kif18A motor domain also depolymerizes microtubule plus and minus ends. BTB-1 inhibits both of these microtubule-based Kif18A activities. A reconstruction of BTB-1-bound, microtubule-bound Kif18A, in combination with computational modeling, identified an allosteric BTB-1-binding site near loop5, where it blocks the ATP-dependent conformational changes that we characterized. Strikingly, BTB-1 binding is close to that of well-characterized Kif11 inhibitors that block tight microtubule binding, whereas BTB-1 traps Kif18A on the microtubule. Our work highlights a general mechanism of kinesin inhibition in which small-molecule binding near loop5 prevents a range of conformational changes, blocking motor function.
History
DepositionJul 12, 2017Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 25, 2017Provider: repository / Type: Initial release
Revision 1.1Nov 8, 2017Group: Database references / Category: citation / citation_author
Item: _citation.journal_abbrev / _citation.pdbx_database_id_DOI ..._citation.journal_abbrev / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.name
Revision 1.2Nov 15, 2017Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Aug 21, 2019Group: Data collection / Category: em_software / Item: _em_software.name
Revision 1.4May 15, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
K: Kinesin-like protein KIF18A
A: Tubulin alpha chain
B: Tubulin beta chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)144,4379
Polymers142,2293
Non-polymers2,2086
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area9170 Å2
ΔGint-70 kcal/mol
Surface area42980 Å2
MethodPISA
Number of models2

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Components

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Protein , 3 types, 3 molecules KAB

#1: Protein Kinesin-like protein KIF18A / Marrow stromal KIF18A / MS-KIF18A


Mass: 42213.938 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: KIF18A, OK/SW-cl.108 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q8NI77
#2: Protein Tubulin alpha chain


Mass: 50107.238 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Bos taurus (cattle) / References: UniProt: F2Z4C1, UniProt: P81947*PLUS
#3: Protein Tubulin beta chain / Beta-tubulin


Mass: 49907.770 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Sus scrofa (pig) / References: UniProt: P02554

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Non-polymers , 6 types, 6 molecules

#4: Chemical ChemComp-9V5 / 4-chloranyl-2-nitro-1-(phenylsulfonyl)benzene / BIS-TRIS BUFFER


Mass: 297.714 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C12H8ClNO4S
#5: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn
#6: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#7: Chemical ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE


Mass: 523.180 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O14P3 / Comment: GTP, energy-carrying molecule*YM
#8: Chemical ChemComp-GDP / GUANOSINE-5'-DIPHOSPHATE


Type: RNA linking / Mass: 443.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O11P2 / Comment: GDP, energy-carrying molecule*YM
#9: Chemical ChemComp-TA1 / TAXOL


Mass: 853.906 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C47H51NO14 / Comment: medication, chemotherapy*YM

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: helical reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
113-Protofilament microtuble-bound human Kinesin-8 motor domain neck-linker with its specific inhibitor BTB-1COMPLEX#1-#30MULTIPLE SOURCES
2Kinesin-8 motor domain neck-linkerCOMPLEX#11RECOMBINANT
3Tubulin alpha chainCOMPLEX#21NATURAL
4Tubulin beta chainCOMPLEX#31NATURAL
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23Bos taurus (cattle)9913
34sus scrofa (pig)9823
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: C-flat-2/2
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 298 K

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
MicroscopyModel: FEI POLARA 300 / Details: FEI Polara
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus min: 500 nm / Calibrated defocus max: 3500 nm / Cs: 2.3 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: OTHER
Image recordingAverage exposure time: 2 sec. / Electron dose: 5 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
EM imaging opticsEnergyfilter name: GIF Quantum
Image scansWidth: 3840 / Height: 3712 / Movie frames/image: 5

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Processing

EM software
IDNameVersionCategory
2SerialEMimage acquisition
4CTFFIND3CTF correction
7UCSF Chimeramodel fitting
9SPIDERinitial Euler assignment
10SPIDERfinal Euler assignment
11SPIDERclassification
12FREALIGN3D reconstruction
13Flex-EMmodel refinement
14Flex-EMmodel fitting
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: 0 ° / Axial rise/subunit: 81 Å / Axial symmetry: C1
3D reconstructionResolution: 4.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 135382 / Algorithm: FOURIER SPACE / Symmetry type: HELICAL
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
RefinementHighest resolution: 4.8 Å

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