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- PDB-7lxb: HeLa-tubulin in complex with cryptophycin 52 -

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Basic information

Entry
Database: PDB / ID: 7lxb
TitleHeLa-tubulin in complex with cryptophycin 52
Components
  • Tubulin alpha-1B chain
  • Tubulin beta-3 chain
KeywordsCELL CYCLE / anti-tumor / microtubule / tubulin-rings / polymerization-inhibitor
Function / homology
Function and homology information


netrin receptor binding / Post-chaperonin tubulin folding pathway / dorsal root ganglion development / Carboxyterminal post-translational modifications of tubulin / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Cilium Assembly / Sealing of the nuclear envelope (NE) by ESCRT-III / Intraflagellar transport / cytoskeleton-dependent intracellular transport / Formation of tubulin folding intermediates by CCT/TriC ...netrin receptor binding / Post-chaperonin tubulin folding pathway / dorsal root ganglion development / Carboxyterminal post-translational modifications of tubulin / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Cilium Assembly / Sealing of the nuclear envelope (NE) by ESCRT-III / Intraflagellar transport / cytoskeleton-dependent intracellular transport / Formation of tubulin folding intermediates by CCT/TriC / Gap junction assembly / COPI-independent Golgi-to-ER retrograde traffic / Prefoldin mediated transfer of substrate to CCT/TriC / Assembly and cell surface presentation of NMDA receptors / Kinesins / COPI-dependent Golgi-to-ER retrograde traffic / intercellular bridge / Recycling pathway of L1 / cytoplasmic microtubule / microtubule-based process / RHOH GTPase cycle / RHO GTPases activate IQGAPs / cellular response to interleukin-4 / Hedgehog 'off' state / COPI-mediated anterograde transport / Activation of AMPK downstream of NMDARs / Mitotic Prometaphase / EML4 and NUDC in mitotic spindle formation / Recruitment of NuMA to mitotic centrosomes / Resolution of Sister Chromatid Cohesion / HSP90 chaperone cycle for steroid hormone receptors (SHR) in the presence of ligand / MHC class II antigen presentation / filopodium / cell periphery / Translocation of SLC2A4 (GLUT4) to the plasma membrane / RHO GTPases Activate Formins / peptide binding / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / axon guidance / PKR-mediated signaling / structural constituent of cytoskeleton / mitotic spindle / Aggrephagy / microtubule cytoskeleton organization / HCMV Early Events / Separation of Sister Chromatids / The role of GTSE1 in G2/M progression after G2 checkpoint / microtubule cytoskeleton / double-stranded RNA binding / lamellipodium / mitotic cell cycle / growth cone / microtubule / cell division / axon / GTPase activity / neuronal cell body / ubiquitin protein ligase binding / dendrite / GTP binding / structural molecule activity / extracellular exosome / nucleus / metal ion binding / cytoplasm
Similarity search - Function
Tubulin-beta mRNA autoregulation signal. / Alpha tubulin / 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-beta mRNA autoregulation signal. / Alpha tubulin / 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
Similarity search - Domain/homology
GUANOSINE-5'-DIPHOSPHATE / GUANOSINE-5'-TRIPHOSPHATE / Cryptophycin 52 / Tubulin alpha-1B chain / Tubulin beta-3 chain
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.26 Å
AuthorsEren, E.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIH/NIAMS)Intramural Research United States
CitationJournal: J Biol Chem / Year: 2021
Title: Conformational changes in tubulin upon binding cryptophycin-52 reveal its mechanism of action.
Authors: Elif Eren / Norman R Watts / Dan L Sackett / Paul T Wingfield /
Abstract: Cryptophycin-52 (Cp-52) is potentially the most potent anticancer drug known, with IC values in the low picomolar range, but its binding site on tubulin and mechanism of action are unknown. Here, we ...Cryptophycin-52 (Cp-52) is potentially the most potent anticancer drug known, with IC values in the low picomolar range, but its binding site on tubulin and mechanism of action are unknown. Here, we have determined the binding site of Cp-52, and its parent compound, cryptophycin-1, on HeLa tubulin, to a resolution of 3.3 Å and 3.4 Å, respectively, by cryo-EM and characterized this binding further by molecular dynamics simulations. The binding site was determined to be located at the tubulin interdimer interface and partially overlap that of maytansine, another cytotoxic tubulin inhibitor. Binding induces curvature both within and between tubulin dimers that is incompatible with the microtubule lattice. Conformational changes occur in both α-tubulin and β-tubulin, particularly in helices H8 and H10, with distinct differences between α and β monomers and between Cp-52-bound and cryptophycin-1-bound tubulin. From these results, we have determined: (i) the mechanism of action of inhibition of both microtubule polymerization and depolymerization, (ii) how the affinity of Cp-52 for tubulin may be enhanced, and (iii) where linkers for targeted delivery can be optimally attached to this molecule.
History
DepositionMar 3, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 8, 2021Provider: repository / Type: Initial release
Revision 1.1Sep 15, 2021Group: Database references / Category: citation
Item: _citation.page_first / _citation.page_last ..._citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Oct 6, 2021Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.title
Revision 1.3May 29, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure viewerMolecule:
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Assembly

Deposited unit
A: Tubulin alpha-1B chain
B: Tubulin beta-3 chain
C: Tubulin alpha-1B chain
D: Tubulin beta-3 chain
E: Tubulin alpha-1B chain
F: Tubulin beta-3 chain
G: Tubulin alpha-1B chain
H: Tubulin beta-3 chain
I: Tubulin alpha-1B chain
J: Tubulin beta-3 chain
K: Tubulin alpha-1B chain
L: Tubulin beta-3 chain
M: Tubulin alpha-1B chain
N: Tubulin beta-3 chain
O: Tubulin alpha-1B chain
P: Tubulin beta-3 chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)818,57240
Polymers805,48816
Non-polymers13,08524
Water00
1


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

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Components

#1: Protein
Tubulin alpha-1B chain / Alpha-tubulin ubiquitous / Tubulin K-alpha-1 / Tubulin alpha-ubiquitous chain


Mass: 50204.445 Da / Num. of mol.: 8 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: HeLa / References: UniProt: P68363
#2: Protein
Tubulin beta-3 chain / Tubulin beta-4 chain / Tubulin beta-III


Mass: 50481.520 Da / Num. of mol.: 8 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: HeLa / References: UniProt: Q13509
#3: Chemical
ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE


Mass: 523.180 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C10H16N5O14P3 / Comment: GTP, energy-carrying molecule*YM
#4: Chemical
ChemComp-YGY / Cryptophycin 52 / (3S,10S,13Z,16S)-10-[(3-chloro-4-methoxyphenyl)methyl]-6,6-dimethyl-3-(2-methylpropyl)-16-{(1S)-1-[(2R,3R)-3-phenyloxiran-2-yl]ethyl}-1,4-dioxa-8,11-diazacyclohexadec-13-ene-2,5,9,12-tetrone


Mass: 669.204 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C36H45ClN2O8 / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical
ChemComp-GDP / GUANOSINE-5'-DIPHOSPHATE


Type: RNA linking / Mass: 443.201 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C10H15N5O11P2 / Comment: GDP, 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: complex of tubulin with cryptophycin 52 / Type: COMPLEX / Entity ID: #1-#2 / Source: NATURAL
Molecular weightValue: 0.88 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Buffer solutionpH: 6.9
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 277.15 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: 130000 X / Cs: 2.7 mm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 66 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
EM softwareName: SerialEM / Category: image acquisition
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 40746
SymmetryPoint symmetry: C8 (8 fold cyclic)
3D reconstructionResolution: 3.26 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 31098 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 6S8L

6s8l


Accession code: 6S8L / Source name: PDB / Type: experimental model
RefinementHighest resolution: 3.26 Å
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00955528
ELECTRON MICROSCOPYf_angle_d0.97775552
ELECTRON MICROSCOPYf_dihedral_angle_d10.6297672
ELECTRON MICROSCOPYf_chiral_restr0.0568248
ELECTRON MICROSCOPYf_plane_restr0.0059832

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