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- PDB-6nxl: Ubiquitin binding variants -

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Basic information

Entry
Database: PDB / ID: 6nxl
TitleUbiquitin binding variants
ComponentsPolyubiquitin-B
KeywordsLIGASE / APC / Ubv / Ubiquitin / Inhibitor
Function / homologyUbiquitin conserved site / Ubiquitin domain / Ubiquitin domain signature. / Ubiquitin family / Ubiquitin homologues / Ubiquitin-like domain / Ubiquitin domain profile. / Ubiquitin-like domain superfamily / Polyubiquitin-B
Function and homology information
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.803 Å
AuthorsMiller, D.J. / Watson, E.R.
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
DepositionFeb 8, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 15, 2020Provider: repository / Type: Initial release
Revision 1.1Feb 3, 2021Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Oct 11, 2023Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
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: Polyubiquitin-B
B: Polyubiquitin-B
C: Polyubiquitin-B
D: Polyubiquitin-B
E: Polyubiquitin-B
F: Polyubiquitin-B
G: Polyubiquitin-B
H: Polyubiquitin-B


Theoretical massNumber of molelcules
Total (without water)71,1698
Polymers71,1698
Non-polymers00
Water0
1
A: Polyubiquitin-B
E: Polyubiquitin-B


Theoretical massNumber of molelcules
Total (without water)17,7922
Polymers17,7922
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area3290 Å2
ΔGint-22 kcal/mol
Surface area8560 Å2
MethodPISA
2
B: Polyubiquitin-B
F: Polyubiquitin-B


Theoretical massNumber of molelcules
Total (without water)17,7922
Polymers17,7922
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area3240 Å2
ΔGint-21 kcal/mol
Surface area8320 Å2
MethodPISA
3
C: Polyubiquitin-B
D: Polyubiquitin-B


Theoretical massNumber of molelcules
Total (without water)17,7922
Polymers17,7922
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area3300 Å2
ΔGint-22 kcal/mol
Surface area8400 Å2
MethodPISA
4
G: Polyubiquitin-B
H: Polyubiquitin-B


Theoretical massNumber of molelcules
Total (without water)17,7922
Polymers17,7922
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area3320 Å2
ΔGint-22 kcal/mol
Surface area8110 Å2
MethodPISA
Unit cell
Length a, b, c (Å)62.359, 62.359, 168.121
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number76
Space group name H-MP41

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Components

#1: Protein
Polyubiquitin-B


Mass: 8896.072 Da / Num. of mol.: 8
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: UBB
Production host: Escherichia coli-Pichia pastoris shuttle vector pPpARG4 (others)
Strain (production host): BL21(DE3)-RIL / References: UniProt: B4DV12

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.39 Å3/Da / Density % sol: 48.52 %
Crystal growTemperature: 277.15 K / Method: vapor diffusion, hanging drop
Details: 0.1M Sodium Acetate pH 4.6, 0.02 M Calcium Chloride, 30% MPD, 0.1M Potassium Sodium Tartrate Tetrahydrate and 56mg/mL protein co-sized Ubv and APC2 WHB (735-C)

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 22-ID / Wavelength: 1 Å
DetectorType: MARMOSAIC 300 mm CCD / Detector: CCD / Date: Aug 14, 2015
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 2.8→30 Å / Num. obs: 15140 / % possible obs: 96.1 % / Redundancy: 3.5 % / Rmerge(I) obs: 0.084 / Rpim(I) all: 0.051 / Rrim(I) all: 0.099 / Rsym value: 0.084 / Net I/av σ(I): 14.8 / Net I/σ(I): 14.8
Reflection shellResolution: 2.8→2.85 Å / Redundancy: 2.1 % / Rmerge(I) obs: 0.237 / Mean I/σ(I) obs: 2 / Num. unique obs: 529 / CC1/2: 0.935 / Rpim(I) all: 0.169 / Rrim(I) all: 0.293 / Rsym value: 0.237 / % possible all: 67

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Processing

Software
NameVersionClassification
PHENIX(1.11.1_2575: ???)refinement
DENZOdata reduction
SCALEPACKdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 4S1Z

4s1z


Resolution: 2.803→29.596 Å / SU ML: 0.46 / Cross valid method: THROUGHOUT / σ(F): 1.38 / Phase error: 32.43 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2654 777 5.15 %
Rwork0.2134 --
obs0.216 15083 95.91 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 2.803→29.596 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4490 0 0 0 4490
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0094546
X-RAY DIFFRACTIONf_angle_d1.0836186
X-RAY DIFFRACTIONf_dihedral_angle_d11.5422788
X-RAY DIFFRACTIONf_chiral_restr0.061775
X-RAY DIFFRACTIONf_plane_restr0.009785
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.8031-2.97850.38321330.30861933X-RAY DIFFRACTION79
2.9785-3.20830.33531330.27542381X-RAY DIFFRACTION97
3.2083-3.53060.34431230.25752502X-RAY DIFFRACTION100
3.5306-4.04040.30271490.21092457X-RAY DIFFRACTION100
4.0404-5.08630.17061040.1782515X-RAY DIFFRACTION100
5.0863-29.59760.23471350.19362518X-RAY DIFFRACTION100

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