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- PDB-6ob1: Structure of WHB in complex with Ubiquitin Variant -

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Basic information

Entry
Database: PDB / ID: 6ob1
TitleStructure of WHB in complex with Ubiquitin Variant
Components
  • (Ubiquitin) x 2
  • Anaphase-promoting complex subunit 2
KeywordsPROTEIN BINDING / Ubiquitin / WHB
Function / homology
Function and homology information


positive regulation of synapse maturation / Conversion from APC/C:Cdc20 to APC/C:Cdh1 in late anaphase / Inactivation of APC/C via direct inhibition of the APC/C complex / APC/C:Cdc20 mediated degradation of mitotic proteins / anaphase-promoting complex / Aberrant regulation of mitotic exit in cancer due to RB1 defects / regulation of meiotic cell cycle / anaphase-promoting complex-dependent catabolic process / metaphase/anaphase transition of mitotic cell cycle / positive regulation of synaptic plasticity ...positive regulation of synapse maturation / Conversion from APC/C:Cdc20 to APC/C:Cdh1 in late anaphase / Inactivation of APC/C via direct inhibition of the APC/C complex / APC/C:Cdc20 mediated degradation of mitotic proteins / anaphase-promoting complex / Aberrant regulation of mitotic exit in cancer due to RB1 defects / regulation of meiotic cell cycle / anaphase-promoting complex-dependent catabolic process / metaphase/anaphase transition of mitotic cell cycle / positive regulation of synaptic plasticity / Phosphorylation of the APC/C / protein K11-linked ubiquitination / positive regulation of dendrite morphogenesis / Regulation of APC/C activators between G1/S and early anaphase / Transcriptional Regulation by VENTX / positive regulation of axon extension / APC/C:Cdc20 mediated degradation of Cyclin B / regulation of mitotic cell cycle / APC-Cdc20 mediated degradation of Nek2A / Autodegradation of Cdh1 by Cdh1:APC/C / APC/C:Cdc20 mediated degradation of Securin / Assembly of the pre-replicative complex / Cdc20:Phospho-APC/C mediated degradation of Cyclin A / APC/C:Cdh1 mediated degradation of Cdc20 and other APC/C:Cdh1 targeted proteins in late mitosis/early G1 / CDK-mediated phosphorylation and removal of Cdc6 / Separation of Sister Chromatids / Antigen processing: Ubiquitination & Proteasome degradation / nervous system development / Senescence-Associated Secretory Phenotype (SASP) / cell differentiation / cell division / negative regulation of gene expression / ubiquitin protein ligase binding / nucleoplasm / cytosol
Similarity search - Function
Anaphase-promoting complex subunit 2, C-terminal / Anaphase-promoting complex subunit 2 / Anaphase promoting complex (APC) subunit 2 / Anaphase promoting complex (APC) subunit 2 / Cullin / Cullin, N-terminal / Cullin homology domain / Cullin homology domain superfamily / Cullin family / Cullin family profile. ...Anaphase-promoting complex subunit 2, C-terminal / Anaphase-promoting complex subunit 2 / Anaphase promoting complex (APC) subunit 2 / Anaphase promoting complex (APC) subunit 2 / Cullin / Cullin, N-terminal / Cullin homology domain / Cullin homology domain superfamily / Cullin family / Cullin family profile. / Winged helix-like DNA-binding domain superfamily/Winged helix DNA-binding domain / Arc Repressor Mutant, subunit A / Winged helix DNA-binding domain superfamily / Winged helix-like DNA-binding domain superfamily / Orthogonal Bundle / Mainly Alpha
Similarity search - Domain/homology
Anaphase-promoting complex subunit 2
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodSOLUTION NMR / torsion angle dynamics
AuthorsEdmond, R.W. / Grace, C.R.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)R35GM128855 United States
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)R37GM065930 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2019
Title: Protein engineering of a ubiquitin-variant inhibitor of APC/C identifies a cryptic K48 ubiquitin chain binding site.
Authors: Edmond R Watson / Christy R R Grace / Wei Zhang / Darcie J Miller / Iain F Davidson / J Rajan Prabu / Shanshan Yu / Derek L Bolhuis / Elizaveta T Kulko / Ronnald Vollrath / David Haselbach / ...Authors: Edmond R Watson / Christy R R Grace / Wei Zhang / Darcie J Miller / Iain F Davidson / J Rajan Prabu / Shanshan Yu / Derek L Bolhuis / Elizaveta T Kulko / Ronnald Vollrath / David Haselbach / Holger Stark / Jan-Michael Peters / Nicholas G Brown / Sachdev S Sidhu / Brenda A Schulman /
Abstract: Ubiquitin (Ub)-mediated proteolysis is a fundamental mechanism used by eukaryotic cells to maintain homeostasis and protein quality, and to control timing in biological processes. Two essential ...Ubiquitin (Ub)-mediated proteolysis is a fundamental mechanism used by eukaryotic cells to maintain homeostasis and protein quality, and to control timing in biological processes. Two essential aspects of Ub regulation are conjugation through E1-E2-E3 enzymatic cascades and recognition by Ub-binding domains. An emerging theme in the Ub field is that these 2 properties are often amalgamated in conjugation enzymes. In addition to covalent thioester linkage to Ub's C terminus for Ub transfer reactions, conjugation enzymes often bind noncovalently and weakly to Ub at "exosites." However, identification of such sites is typically empirical and particularly challenging in large molecular machines. Here, studying the 1.2-MDa E3 ligase anaphase-promoting complex/cyclosome (APC/C), which controls cell division and many aspects of neurobiology, we discover a method for identifying unexpected Ub-binding sites. Using a panel of Ub variants (UbVs), we identify a protein-based inhibitor that blocks Ub ligation to APC/C substrates in vitro and ex vivo. Biochemistry, NMR, and cryo-electron microscopy (cryo-EM) structurally define the UbV interaction, explain its inhibitory activity through binding the surface on the APC2 subunit that recruits the E2 enzyme UBE2C, and ultimately reveal that this APC2 surface is also a Ub-binding exosite with preference for K48-linked chains. The results provide a tool for probing APC/C activity, have implications for the coordination of K48-linked Ub chain binding by APC/C with the multistep process of substrate polyubiquitylation, and demonstrate the power of UbV technology for identifying cryptic Ub-binding sites within large multiprotein complexes.
History
DepositionMar 19, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 14, 2019Provider: repository / Type: Initial release
Revision 1.1Sep 4, 2019Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.2Dec 18, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.3May 1, 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

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Ubiquitin
B: Ubiquitin
C: Anaphase-promoting complex subunit 2


Theoretical massNumber of molelcules
Total (without water)27,3633
Polymers27,3633
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: light scattering
TypeNameSymmetry operationNumber
identity operation1_5551
NMR ensembles
DataCriteria
Number of conformers (submitted / calculated)20 / 400structures with the lowest energy
RepresentativeModel #1lowest energy

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Components

#1: Protein Ubiquitin /


Mass: 8550.763 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human)
Production host: Escherichia coli-Pichia pastoris shuttle vector pPpARG4 (others)
#2: Protein Ubiquitin /


Mass: 8552.692 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human)
Production host: Escherichia coli-Pichia pastoris shuttle vector pPpARG4 (others)
#3: Protein Anaphase-promoting complex subunit 2 / / APC2 / Cyclosome subunit 2


Mass: 10259.662 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ANAPC2, APC2, KIAA1406
Production host: Escherichia coli-Pichia pastoris shuttle vector pPpARG4 (others)
References: UniProt: Q9UJX6

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Experimental details

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Experiment

ExperimentMethod: SOLUTION NMR
NMR experiment
Conditions-IDExperiment-IDSolution-IDSample stateSpectrometer-IDType
111isotropic22D 1H-15N HSQC
121isotropic32D 1H-13C HSQC aliphatic
131isotropic32D 1H-13C HSQC aromatic
141isotropic13D CBCA(CO)NH
151isotropic13D HN(CA)CB
171isotropic23D HNCA
161isotropic13D (H)CCH-TOCSY
181isotropic13D 1H-15N NOESY
191isotropic23D 1H-13C NOESY aliphatic
1101isotropic13D 1H-13C NOESY aromatic
1111isotropic13D 1H-13C filtered NOESY
1121isotropic13D HBHA(CO)NH
2132isotropic22D 1H-15N HSQC
2142isotropic22D 1H-13C HSQC aliphatic
2152isotropic22D 1H-13C HSQC aromatic
2172isotropic13D HNCA
2162isotropic13D HN(CA)CB
2182isotropic33D HBHA(CO)NH
2192isotropic13D 1H-15N NOESY
2202isotropic23D 1H-13C NOESY aliphatic
2212isotropic23D 1H-13C NOESY aromatic
2222isotropic13D H(CCO)NH
2232isotropic23D 1H-13C filtered NOESY

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Sample preparation

DetailsType: solution
Contents: 0.5 mM [U-99% 13C; U-99% 15N] WHB, 0.6 mM Ubiquitin Variant, 90% H2O/10% D2O
Details: 0.5mM, 10mM HEPES, pH7, 100mM NaCl, 10mM DTT / Label: 15N_13C_WHB+UbVw / Solvent system: 90% H2O/10% D2O
Sample
Conc. (mg/ml)ComponentIsotopic labelingSolution-ID
0.5 mMWHB[U-99% 13C; U-99% 15N]1
0.6 mMUbiquitin Variantnatural abundance1
Sample conditionsDetails: 0.5mM, 10mM HEPES, pH7, 100mM NaCl, 10mM DTT / Ionic strength: 100 mM / Label: 15N_13C_WHB+UbVw / pH: 7 / Pressure: atmospheric atm / Temperature: 308 K

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NMR measurement

NMR spectrometer
TypeManufacturerModelField strength (MHz)Spectrometer-IDDetails
Bruker AVANCE IIIBrukerAVANCE III6001TCI probe
Bruker AVANCE IIIBrukerAVANCE III7002TCI probe
Bruker AVANCE IIIBrukerAVANCE III8003TCI probe

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Processing

NMR software
NameDeveloperClassification
TopSpinBruker Biospinprocessing
CARAKeller and Wuthrichchemical shift assignment
CARAKeller and Wuthrichpeak picking
CYANAGuntert, Mumenthaler and Wuthrichstructure calculation
CYANAGuntert, Mumenthaler and Wuthrichrefinement
CNSrefinement
RefinementMethod: torsion angle dynamics / Software ordinal: 1 / Details: Structures from cyana were minimized with CNS
NMR representativeSelection criteria: lowest energy
NMR ensembleConformer selection criteria: structures with the lowest energy
Conformers calculated total number: 400 / Conformers submitted total number: 20

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