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- PDB-5fwp: Atomic cryoEM structure of Hsp90-Cdc37-Cdk4 complex -

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Entry
Database: PDB / ID: 5fwp
TitleAtomic cryoEM structure of Hsp90-Cdc37-Cdk4 complex
Components
  • CYCLIN-DEPENDENT KINASE 4
  • HEAT SHOCK PROTEIN HSP 90 BETA
  • HSP90 CO-CHAPERONE CDC37
KeywordsCHAPERONE / HSP90 / CDC37 / CDK4 / KINASE / UNFOLDING
Function / homology
Function and homology information


cyclin D3-CDK4 complex / cyclin D1-CDK4 complex / cyclin D2-CDK4 complex / Evasion of Oncogene Induced Senescence Due to Defective p16INK4A binding to CDK4 / Evasion of Oxidative Stress Induced Senescence Due to Defective p16INK4A binding to CDK4 / cellular response to ionomycin / regulation of transcription initiation by RNA polymerase II / Drug-mediated inhibition of CDK4/CDK6 activity / Evasion of Oncogene Induced Senescence Due to Defective p16INK4A binding to CDK4 and CDK6 / Evasion of Oxidative Stress Induced Senescence Due to Defective p16INK4A binding to CDK4 and CDK6 ...cyclin D3-CDK4 complex / cyclin D1-CDK4 complex / cyclin D2-CDK4 complex / Evasion of Oncogene Induced Senescence Due to Defective p16INK4A binding to CDK4 / Evasion of Oxidative Stress Induced Senescence Due to Defective p16INK4A binding to CDK4 / cellular response to ionomycin / regulation of transcription initiation by RNA polymerase II / Drug-mediated inhibition of CDK4/CDK6 activity / Evasion of Oncogene Induced Senescence Due to Defective p16INK4A binding to CDK4 and CDK6 / Evasion of Oxidative Stress Induced Senescence Due to Defective p16INK4A binding to CDK4 and CDK6 / regulation of type II interferon-mediated signaling pathway / regulation of type B pancreatic cell proliferation / HSP90-CDC37 chaperone complex / receptor ligand inhibitor activity / negative regulation of proteasomal protein catabolic process / Aryl hydrocarbon receptor signalling / : / aryl hydrocarbon receptor complex / dynein axonemal particle / histone methyltransferase binding / Transcriptional regulation by RUNX2 / cellular response to phorbol 13-acetate 12-myristate / ATP-dependent protein binding / positive regulation of protein localization to cell surface / protein kinase regulator activity / protein folding chaperone complex / cyclin-dependent protein serine/threonine kinase regulator activity / telomerase holoenzyme complex assembly / post-transcriptional regulation of gene expression / Respiratory syncytial virus genome replication / Uptake and function of diphtheria toxin / regulation of cyclin-dependent protein serine/threonine kinase activity / Drug-mediated inhibition of ERBB2 signaling / Resistance of ERBB2 KD mutants to trastuzumab / Resistance of ERBB2 KD mutants to sapitinib / Resistance of ERBB2 KD mutants to tesevatinib / Resistance of ERBB2 KD mutants to neratinib / Resistance of ERBB2 KD mutants to osimertinib / Resistance of ERBB2 KD mutants to afatinib / Resistance of ERBB2 KD mutants to AEE788 / Resistance of ERBB2 KD mutants to lapatinib / Drug resistance in ERBB2 TMD/JMD mutants / TPR domain binding / PTK6 Regulates Cell Cycle / Assembly and release of respiratory syncytial virus (RSV) virions / positive regulation of transforming growth factor beta receptor signaling pathway / dendritic growth cone / Defective binding of RB1 mutants to E2F1,(E2F2, E2F3) / regulation of type I interferon-mediated signaling pathway / : / Sema3A PAK dependent Axon repulsion / The NLRP3 inflammasome / regulation of protein ubiquitination / telomere maintenance via telomerase / HSF1-dependent transactivation / response to unfolded protein / chaperone-mediated protein complex assembly / HSF1 activation / bicellular tight junction / Attenuation phase / cyclin-dependent kinase / RHOBTB2 GTPase cycle / protein targeting / cyclin-dependent protein serine/threonine kinase activity / cellular response to interleukin-4 / axonal growth cone / Purinergic signaling in leishmaniasis infection / DNA polymerase binding / cyclin-dependent protein kinase holoenzyme complex / supramolecular fiber organization / chaperone-mediated protein folding / Signaling by ERBB2 / negative regulation of proteasomal ubiquitin-dependent protein catabolic process / regulation of G2/M transition of mitotic cell cycle / heat shock protein binding / protein folding chaperone / HSP90 chaperone cycle for steroid hormone receptors (SHR) in the presence of ligand / positive regulation of G2/M transition of mitotic cell cycle / nitric-oxide synthase regulator activity / Constitutive Signaling by Overexpressed ERBB2 / cyclin binding / ESR-mediated signaling / Ubiquitin-dependent degradation of Cyclin D / positive regulation of cell differentiation / ATP-dependent protein folding chaperone / Signaling by ERBB2 TMD/JMD mutants / peptide binding / Constitutive Signaling by EGFRvIII / Hsp90 protein binding / Signaling by ERBB2 ECD mutants / DDX58/IFIH1-mediated induction of interferon-alpha/beta / Signaling by ERBB2 KD Mutants / placenta development / tau protein binding / Oncogene Induced Senescence / Regulation of necroptotic cell death / Regulation of actin dynamics for phagocytic cup formation / Meiotic recombination / kinase binding / Downregulation of ERBB2 signaling
Similarity search - Function
Cdc37, C-terminal / Cdc37, Hsp90 binding / Cdc37, Hsp90-binding domain superfamily / Cdc37 C terminal domain / Cdc37 Hsp90 binding domain / Cdc37 C terminal domain / Cdc37 Hsp90 binding domain / Cdc37 N terminal kinase binding / Cdc37 / Cdc37, N-terminal domain ...Cdc37, C-terminal / Cdc37, Hsp90 binding / Cdc37, Hsp90-binding domain superfamily / Cdc37 C terminal domain / Cdc37 Hsp90 binding domain / Cdc37 C terminal domain / Cdc37 Hsp90 binding domain / Cdc37 N terminal kinase binding / Cdc37 / Cdc37, N-terminal domain / Cdc37 N terminal kinase binding / Heat shock protein Hsp90, conserved site / Heat shock hsp90 proteins family signature. / HSP90, C-terminal domain / Heat shock protein Hsp90, N-terminal / Heat shock protein Hsp90 family / Hsp90 protein / Histidine kinase-, DNA gyrase B-, and HSP90-like ATPase / : / Histidine kinase-, DNA gyrase B-, and HSP90-like ATPase / Histidine kinase-like ATPases / Histidine kinase/HSP90-like ATPase / Histidine kinase/HSP90-like ATPase superfamily / Ribosomal protein S5 domain 2-type fold / Serine/threonine-protein kinase, active site / Serine/Threonine protein kinases active-site signature. / Protein kinase domain / Serine/Threonine protein kinases, catalytic domain / Protein kinase, ATP binding site / Protein kinases ATP-binding region signature. / Protein kinase domain profile. / Protein kinase domain / Protein kinase-like domain superfamily
Similarity search - Domain/homology
ADENOSINE-5'-TRIPHOSPHATE / Heat shock protein HSP 90-beta / Cyclin-dependent kinase 4 / Hsp90 co-chaperone Cdc37
Similarity search - Component
Biological speciesHOMO SAPIENS (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 7.2 Å
AuthorsVerba, K.A. / Wang, R.Y.R. / Arakawa, A. / Liu, Y. / Yokoyama, S. / Agard, D.A.
CitationJournal: Science / Year: 2016
Title: Atomic structure of Hsp90-Cdc37-Cdk4 reveals that Hsp90 traps and stabilizes an unfolded kinase.
Authors: Kliment A Verba / Ray Yu-Ruei Wang / Akihiko Arakawa / Yanxin Liu / Mikako Shirouzu / Shigeyuki Yokoyama / David A Agard /
Abstract: The Hsp90 molecular chaperone and its Cdc37 cochaperone help stabilize and activate more than half of the human kinome. However, both the mechanism by which these chaperones assist their "client" ...The Hsp90 molecular chaperone and its Cdc37 cochaperone help stabilize and activate more than half of the human kinome. However, both the mechanism by which these chaperones assist their "client" kinases and the reason why some kinases are addicted to Hsp90 while closely related family members are independent are unknown. Our structural understanding of these interactions is lacking, as no full-length structures of human Hsp90, Cdc37, or either of these proteins with a kinase have been elucidated. Here we report a 3.9 angstrom cryo-electron microscopy structure of the Hsp90-Cdc37-Cdk4 kinase complex. Surprisingly, the two lobes of Cdk4 are completely separated with the β4-β5 sheet unfolded. Cdc37 mimics part of the kinase N lobe, stabilizing an open kinase conformation by wedging itself between the two lobes. Finally, Hsp90 clamps around the unfolded kinase β5 strand and interacts with exposed N- and C-lobe interfaces, protecting the kinase in a trapped unfolded state. On the basis of this structure and an extensive amount of previously collected data, we propose unifying conceptual and mechanistic models of chaperone-kinase interactions.
History
DepositionFeb 18, 2016Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 26, 2016Provider: repository / Type: Initial release
Revision 1.1Aug 2, 2017Group: Data collection / Category: em_image_scans / em_software
Item: _em_software.fitting_id / _em_software.image_processing_id / _em_software.name
Revision 2.0Oct 23, 2019Group: Atomic model / Data collection / Derived calculations
Category: atom_site / struct_conn
Item: _atom_site.Cartn_x / _atom_site.Cartn_y ..._atom_site.Cartn_x / _atom_site.Cartn_y / _atom_site.Cartn_z / _struct_conn.pdbx_leaving_atom_flag
Revision 2.1Dec 18, 2019Group: Other / Category: atom_sites
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3]
Revision 2.2Nov 20, 2024Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_entry_details / pdbx_modification_feature / struct_conn / struct_conn_type / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_conn.conn_type_id / _struct_conn.id / _struct_conn.pdbx_dist_value / _struct_conn.pdbx_leaving_atom_flag / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_conn.ptnr2_label_seq_id / _struct_conn_type.id / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id

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Structure visualization

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  • Deposited structure unit
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  • EMDB-3340
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Assembly

Deposited unit
A: HEAT SHOCK PROTEIN HSP 90 BETA
B: HEAT SHOCK PROTEIN HSP 90 BETA
E: HSP90 CO-CHAPERONE CDC37
K: CYCLIN-DEPENDENT KINASE 4
hetero molecules


Theoretical massNumber of molelcules
Total (without water)247,4978
Polymers246,4344
Non-polymers1,0634
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein HEAT SHOCK PROTEIN HSP 90 BETA / HSP 90 / HEAT SHOCK 84 KDA / HSP 84 / HSP84 / HEAT SHOCK PROTEI N HSP 90 BETA


Mass: 83645.539 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: ATP / Source: (gene. exp.) HOMO SAPIENS (human) / Plasmid: PFASTBACHT / Production host: SPODOPTERA FRUGIPERDA (fall armyworm) / References: UniProt: P08238
#2: Protein HSP90 CO-CHAPERONE CDC37 / HSP90 CHAPERONE PROTEIN KINASE-TARGETING SUBUNIT / P50CDC37


Mass: 44622.363 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) HOMO SAPIENS (human) / Plasmid: PFASTBACHT / Production host: SPODOPTERA FRUGIPERDA (fall armyworm) / References: UniProt: Q16543
#3: Protein CYCLIN-DEPENDENT KINASE 4 / CELL DIVISION PROTEIN KINASE 4 / PSK-J3


Mass: 34520.629 Da / Num. of mol.: 1 / Fragment: KINASE DOMAIN
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) HOMO SAPIENS (human) / Plasmid: PFASTBACHT / Production host: SPODOPTERA FRUGIPERDA (fall armyworm) / References: UniProt: P11802
#4: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Comment: ATP, energy-carrying molecule*YM
#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
Has protein modificationY

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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

Component
IDNameParent-IDSource
1COMPLEX OF HUMAN HSP90BETA, HUMAN CDC37 AND HUMAN CDK40MULTIPLE SOURCES
2HUMAN HSP90BETA1RECOMBINANT
4HUMAN CDC371RECOMBINANT
3HUMAN CDC371RECOMBINANT
Molecular weightValue: 0.245 MDa / Experimental value: NO
Buffer solutionpH: 7.5
Details: 20mM Tris-HCl (pH 7.5), 150 mM NaCl, 10 mM KCl, 10 mM MgCl2, 20 mM Na2MoO4, 2mM DTT, 0.085mM DDM
SpecimenConc.: 0.27 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: HOLEY CARBON
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Temp: 95 K / Humidity: 90 % / Chamber temperature: 293 K / Details: LIQUID ETHANE / Method: Single blot from 4 to 6 seconds, at 20C

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS / Date: Nov 25, 2014
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELD / Nominal magnification: 22500 X / Nominal defocus max: 3800 nm / Nominal defocus min: 1400 nm / Cs: 2.7 mm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 7.6 sec. / Electron dose: 44 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 3718 / Details: 38 frames

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Processing

EM software
IDNameCategory
1Rosettamodel refinement
2UCSF Chimeramodel refinement
3RELION3D reconstruction
Image processingDetails: Image stacks were corrected for motion and summed as described previously, resulting in binned sums (1.315A/pix). For particle picking the images were binned to 5.2A/pix and Gaussian ...Details: Image stacks were corrected for motion and summed as described previously, resulting in binned sums (1.315A/pix). For particle picking the images were binned to 5.2A/pix and Gaussian bandpass filtered between 15A and 500A using EMAN2. SamViewer template based picking was then used to pick particles from all the micrographs, followed by manual review of all the picks. After such procedure 802877 particles were picked in total and extracted from images binned to 2.6A/pix. CTFFIND4 was used to estimate defocus parameters for all the images. Relion 1.4 was used for all the following steps unless noted otherwise. Reference free 2D classification into 300 classes for 75 iterations was performed followed by manual examination of the resulting class averages. Low resolution/signal to noise/feature class averages and contributing particles were discarded, resulting in 670000 particles left. The resulting particles were 3D classified into 4 classes resulting in two classes having high-resolution features (390000 particles). At this stage particles were extracted from 1.315A/pix micrographs and all the following processing was done with these particles. Using 3D Auto-refine in Relion 1.4, a reconstruction was obtained from 390000 particles resulting from 3D classification above (using highest resolution 3D class as initial model, low pass filtered to 20A). Using the resulting parameters, the particles were further drift corrected per particle and dose weighted using the Particle Polishing feature. The B-factor weighing curve was fit by a polynomial (with a rationale that such a curve should be smooth) and used to generate new weighting parameters for Particle Polishing, with which 390000 particles were then polished. All further data processing was done using the polished particles. Re-refinement of the 390000 particles after polishing yielded the map at about 4A resolution (determined using gold standard FSC in the PostProcessing tab). The 390000 particles were then 3D classified into four different classes without particle re-alignment, using the alignment parameters from 4A reconstruction. Particles contributing to each of the four classes were grouped and a full 3D refinement with a spherical 200A mask was performed with each of the four groups of particles using the same initial model, low pass filtered to 20A. This is one of the resulting reconstructions.
Particle selectionNum. of particles selected: 114683
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionMethod: FSC 0.143 CUT-OFF / Resolution: 7.2 Å / Num. of particles: 114683 / Actual pixel size: 1.315 Å
Details: SUBMISSION BASED ON EXPERIMENTAL DATA FROM EMDB EMD-3340. (DEPOSITION ID: 14281).
Refinement type: HALF-MAPS REFINED INDEPENDENTLY / Symmetry type: POINT
Atomic model buildingB value: 141 / Protocol: FLEXIBLE FIT / Space: REAL
Details: Cdc37 crystal structure from PDB:1US7 was fit into EMD-3340 manually and then with UCSF Chimera "Fit In Map" tool. The model was truncated at residue 260, as there was no reliable density ...Details: Cdc37 crystal structure from PDB:1US7 was fit into EMD-3340 manually and then with UCSF Chimera "Fit In Map" tool. The model was truncated at residue 260, as there was no reliable density for the rest of the crystal structure. PDB:5FWK and the above fit crystal structure for residues 148-260 were loaded in Coot. The residues 133-147 were built in by hand into the Cdc37 Reconstruction and residues 245-260 (helix) were rotated as a rigid body. To relieve atomic clashes or bond length/angle distortions at the linker regions, the resulting model was subjected to "Cartesian space relax" protocol within EMD-3340 map using Rosetta. Final model was selected using the combined score of Rosetta all-atom physically-realistic score and electron density score.
Atomic model building
ID 3D fitting-IDDetails
115FWK
211US7
RefinementHighest resolution: 7.2 Å
Refinement stepCycle: LAST / Highest resolution: 7.2 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms14123 0 64 0 14187

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