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

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Basic information

Entry
Database: PDB / ID: 5ocu
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 / homologyTubulin, C-terminal / Tubulin/FtsZ, 2-layer sandwich domain / Tubulin/FtsZ, C-terminal domain superfamily / Kinesin motor domain superfamily / Tubulin/FtsZ, GTPase domain superfamily / Kinesin-like protein / P-loop containing nucleoside triphosphate hydrolase / Kinesin motor domain, conserved site / Tubulin, conserved site / Kinesin motor domain ...Tubulin, C-terminal / Tubulin/FtsZ, 2-layer sandwich domain / Tubulin/FtsZ, C-terminal domain superfamily / Kinesin motor domain superfamily / Tubulin/FtsZ, GTPase domain superfamily / Kinesin-like protein / P-loop containing nucleoside triphosphate hydrolase / Kinesin motor domain, conserved site / Tubulin, conserved site / Kinesin motor domain / Beta tubulin, autoregulation binding site / Tubulin/FtsZ, C-terminal / Tubulin/FtsZ, GTPase domain / Beta tubulin / Alpha tubulin / Kinesin motor domain / Tubulin/FtsZ family, GTPase domain / Tubulin C-terminal domain / Anchoring of the basal body to the plasma membrane / Regulation of PLK1 Activity at G2/M Transition / Hedgehog 'off' state / Recycling pathway of L1 / Recruitment of NuMA to mitotic centrosomes / Recruitment of mitotic centrosome proteins and complexes / Loss of Nlp from mitotic centrosomes / HSP90 chaperone cycle for steroid hormone receptors (SHR) / Resolution of Sister Chromatid Cohesion / Tubulin subunits alpha, beta, and gamma signature. / Separation of Sister Chromatids / MHC class II antigen presentation / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Kinesin motor domain profile. / Kinesin motor domain signature. / Tubulin-beta mRNA autoregulation signal. / Tubulin / Intraflagellar transport / Cilium Assembly / COPI-dependent Golgi-to-ER retrograde traffic / Kinesins / Carboxyterminal post-translational modifications of tubulin / AURKA Activation by TPX2 / The role of GTSE1 in G2/M progression after G2 checkpoint / Separation of Sister Chromatids / Mitotic Prometaphase / Resolution of Sister Chromatid Cohesion / RHO GTPases Activate Formins / MHC class II antigen presentation / COPI-independent Golgi-to-ER retrograde traffic / COPI-dependent Golgi-to-ER retrograde traffic / RHO GTPases Activate Formins / Kinesins / RHO GTPases activate IQGAPs / Hedgehog 'on' state / Amplification of signal from unattached kinetochores via a MAD2 inhibitory signal / COPI-mediated anterograde transport / Mitotic Prometaphase / tubulin-dependent ATPase activity / kinetochore microtubule / mitotic spindle midzone / male meiotic nuclear division / microtubule plus-end binding / seminiferous tubule development / microtubule depolymerization / ATP-dependent microtubule motor activity, plus-end-directed / mitotic metaphase plate congression / kinesin complex / retrograde vesicle-mediated transport, Golgi to ER / microtubule-based movement / regulation of microtubule cytoskeleton organization / cytoplasmic microtubule / recycling endosome / cellular response to interleukin-4 / microtubule-based process / antigen processing and presentation of exogenous peptide antigen via MHC class II / kinetochore / microtubule cytoskeleton organization / cytoplasmic ribonucleoprotein granule / structural constituent of cytoskeleton / ruffle / cellular response to estradiol stimulus / microtubule cytoskeleton / microtubule organizing center / caveola / double-stranded RNA binding / protein transport / actin binding / microtubule binding / microtubule / GTPase activity / myelin sheath / GTP binding / ubiquitin protein ligase binding / ATP binding / nucleus / cytosol / cytoplasm / Tubulin alpha chain / Tubulin beta chain / Tubulin alpha-1B chain / Kinesin-like protein KIF18A
Function and homology information
Specimen sourceHomo sapiens (human)
Bos taurus (cattle)
Sus scrofa (pig)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / 5.2 Å resolution
AuthorsLocke, J. / Joseph, A.P. / Topf, M. / Moores, C.A.
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.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Jul 3, 2017 / Release: Oct 25, 2017
RevisionDateData content typeGroupCategoryItemProviderType
1.0Oct 25, 2017Structure modelrepositoryInitial release
1.1Nov 8, 2017Structure modelDatabase referencescitation / citation_author_citation.journal_abbrev / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.name
1.2Nov 15, 2017Structure modelDatabase referencescitation_citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

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  • Deposited structure unit
  • Imaged by Jmol
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  • Superimposition on EM map
  • EMDB-3780
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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)144,66910
Polyers142,2293
Non-polymers2,4417
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein/peptide , 3 types, 3 molecules KAB

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


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


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


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

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Non-polymers , 6 types, 7 molecules

#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Formula: Mg / Magnesium
#5: Chemical ChemComp-ANP / PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER


Mass: 506.196 Da / Num. of mol.: 1 / Formula: C10H17N6O12P3 / Comment: AMP-PNP (energy-carrying molecule analogue) *YM
#6: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Formula: Zn / Zinc
#7: Chemical ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE


Mass: 523.180 Da / Num. of mol.: 1 / Formula: C10H16N5O14P3 / Guanosine triphosphate / Comment: GTP (energy-carrying molecule) *YM
#8: Chemical ChemComp-GDP / GUANOSINE-5'-DIPHOSPHATE


Mass: 443.201 Da / Num. of mol.: 1 / Formula: C10H15N5O11P2 / Guanosine diphosphate / Comment: GDP (energy-carrying molecule) *YM
#9: Chemical ChemComp-TA1 / TAXOL


Mass: 853.906 Da / Num. of mol.: 1 / Formula: C47H51NO14 / Paclitaxel

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 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 AMPPNPCOMPLEX1, 2, 30MULTIPLE SOURCES
2Kinesin-8 motor domain neck-linkerCOMPLEX11RECOMBINANT
3Tubulin, alphaCOMPLEX21NATURAL
4Tubulin, betaCOMPLEX31NATURAL
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly IDNcbi tax IDOrganism
129606Homo sapiens (human)
139913Bos taurus (cattle)
249823Sus scrofa (pig)
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 kelvins

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
MicroscopyMicroscope model: FEI POLARA 300 / Details: FEI Polara
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / 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
7Chimeramodel 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 deg. / Axial rise/subunit: 81 Å / Axial symmetry: C1
3D reconstructionResolution: 5.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 70473 / Algorithm: FOURIER SPACE / Symmetry type: HELICAL
Atomic model buildingRef protocol: FLEXIBLE FIT / Ref space: REAL

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