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- PDB-8v3m: CCP5 apo structure -

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Basic information

Entry
Database: PDB / ID: 8v3m
TitleCCP5 apo structure
ComponentsCytosolic carboxypeptidase-like protein 5
KeywordsHYDROLASE / carboxypeptidase deglutamylation branch glutamate removal microtubule
Function / homology
Function and homology information


tubulin-glutamate carboxypeptidase / protein deglutamylation / protein side chain deglutamylation / protein branching point deglutamylation / C-terminal protein deglutamylation / Carboxyterminal post-translational modifications of tubulin / Hydrolases; Acting on peptide bonds (peptidases); Metallocarboxypeptidases / metallocarboxypeptidase activity / tubulin binding / mitotic spindle ...tubulin-glutamate carboxypeptidase / protein deglutamylation / protein side chain deglutamylation / protein branching point deglutamylation / C-terminal protein deglutamylation / Carboxyterminal post-translational modifications of tubulin / Hydrolases; Acting on peptide bonds (peptidases); Metallocarboxypeptidases / metallocarboxypeptidase activity / tubulin binding / mitotic spindle / microtubule cytoskeleton / midbody / defense response to virus / proteolysis / zinc ion binding / nucleus / cytoplasm / cytosol
Similarity search - Function
Cytosolic carboxypeptidase-like protein 5 catalytic domain / Cytosolic carboxypeptidase, N-terminal / : / Cytosolic carboxypeptidase N-terminal domain / Zinc carboxypeptidase / Peptidase family M14 domain profile. / Peptidase M14, carboxypeptidase A
Similarity search - Domain/homology
IMIDAZOLE / D-MALATE / Cytosolic carboxypeptidase-like protein 5
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.8 Å
AuthorsChen, J. / Zehr, E.A. / Gruschus, J.M. / Szyk, A. / Liu, Y. / Tanner, M.E. / Tjandra, N. / Roll-Mecak, A.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)1ZIANS003163 United States
CitationJournal: Nature / Year: 2024
Title: Tubulin code eraser CCP5 binds branch glutamates by substrate deformation.
Authors: Jiayi Chen / Elena A Zehr / James M Gruschus / Agnieszka Szyk / Yanjie Liu / Martin E Tanner / Nico Tjandra / Antonina Roll-Mecak /
Abstract: Microtubule function is modulated by the tubulin code, diverse posttranslational modifications that are altered dynamically by writer and eraser enzymes. Glutamylation-the addition of branched ...Microtubule function is modulated by the tubulin code, diverse posttranslational modifications that are altered dynamically by writer and eraser enzymes. Glutamylation-the addition of branched (isopeptide-linked) glutamate chains-is the most evolutionarily widespread tubulin modification. It is introduced by tubulin tyrosine ligase-like enzymes and erased by carboxypeptidases of the cytosolic carboxypeptidase (CCP) family. Glutamylation homeostasis, achieved through the balance of writers and erasers, is critical for normal cell function, and mutations in CCPs lead to human disease. Here we report cryo-electron microscopy structures of the glutamylation eraser CCP5 in complex with the microtubule, and X-ray structures in complex with transition-state analogues. Combined with NMR analysis, these analyses show that CCP5 deforms the tubulin main chain into a unique turn that enables lock-and-key recognition of the branch glutamate in a cationic pocket that is unique to CCP family proteins. CCP5 binding of the sequences flanking the branch point primarily through peptide backbone atoms enables processing of diverse tubulin isotypes and non-tubulin substrates. Unexpectedly, CCP5 exhibits inefficient processing of an abundant β-tubulin isotype in the brain. This work provides an atomistic view into glutamate branch recognition and resolution, and sheds light on homeostasis of the tubulin glutamylation syntax.
History
DepositionNov 28, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 17, 2024Provider: repository / Type: Initial release
Revision 1.1Jul 31, 2024Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Aug 7, 2024Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Cytosolic carboxypeptidase-like protein 5
hetero molecules


Theoretical massNumber of molelcules
Total (without water)59,6426
Polymers59,1711
Non-polymers4725
Water4,612256
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)65.224, 80.925, 103.383
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number18
Space group name H-MP22121
Symmetry operation#1: x,y,z
#2: x,-y,-z
#3: -x,y+1/2,-z+1/2
#4: -x,-y+1/2,z+1/2

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Components

#1: Protein Cytosolic carboxypeptidase-like protein 5 / ATP/GTP-binding protein-like 5 / Protein deglutamylase CCP5


Mass: 59170.668 Da / Num. of mol.: 1 / Mutation: E516A,residues 339-424 replaced with a SGSGG loop
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: AGBL5, CCP5 / Plasmid: pFastBac / Details (production host): His6_MBP_Asn10_TEV / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / Strain (production host): IPLB-Sf-21-AE
References: UniProt: Q8NDL9, Hydrolases; Acting on peptide bonds (peptidases); Metallocarboxypeptidases, tubulin-glutamate carboxypeptidase
#2: Chemical ChemComp-MLT / D-MALATE / (2R)-2-HYDROXYBUTANEDIOIC ACID / 2-HYDROXY-SUCCINIC ACID


Mass: 134.087 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C4H6O5
#3: Chemical ChemComp-IMD / IMIDAZOLE


Mass: 69.085 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C3H5N2
#4: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 256 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN

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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: 48.48 %
Crystal growTemperature: 294 K / Method: vapor diffusion, hanging drop / pH: 7
Details: 0.15 M DL-Malic acid, pH7.0, 0.1 M imidazole, pH7.0, 16% PEG MME 550

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Data collection

DiffractionMean temperature: 93 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 24-ID-E / Wavelength: 0.9792 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Feb 10, 2022
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9792 Å / Relative weight: 1
ReflectionResolution: 1.8→80.93 Å / Num. obs: 51328 / % possible obs: 99.8 % / Redundancy: 13.2 % / Biso Wilson estimate: 32.95 Å2 / CC1/2: 0.999 / Rmerge(I) obs: 0.085 / Rpim(I) all: 0.034 / Rrim(I) all: 0.091 / Net I/σ(I): 15.4
Reflection shellResolution: 1.8→1.84 Å / Redundancy: 12.8 % / Rmerge(I) obs: 1.658 / Mean I/σ(I) obs: 1.6 / Num. unique obs: 2975 / CC1/2: 0.669 / Rrim(I) all: 1.801 / % possible all: 99.6

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Processing

Software
NameVersionClassification
PHENIX1.20.1_4487refinement
XDSdata reduction
Aimlessdata scaling
PHENIXphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.8→55.16 Å / SU ML: 0.22 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 21.41 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2009 2556 4.99 %
Rwork0.1858 --
obs0.1866 51269 99.69 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 1.8→55.16 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3957 0 29 256 4242
Refine LS restraints
Refine-IDTypeDev ideal
X-RAY DIFFRACTIONf_bond_d0.002
X-RAY DIFFRACTIONf_angle_d0.543
X-RAY DIFFRACTIONf_dihedral_angle_d13.462
X-RAY DIFFRACTIONf_chiral_restr0.039
X-RAY DIFFRACTIONf_plane_restr0.005
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.8-1.830.35641320.33762629X-RAY DIFFRACTION99
1.83-1.870.31051320.28472694X-RAY DIFFRACTION100
1.87-1.910.28391680.24182661X-RAY DIFFRACTION100
1.91-1.960.22771380.23152659X-RAY DIFFRACTION100
1.96-2.010.25261360.2192666X-RAY DIFFRACTION100
2.01-2.060.24061400.23012664X-RAY DIFFRACTION99
2.06-2.120.25151400.22652665X-RAY DIFFRACTION99
2.12-2.190.211550.19512686X-RAY DIFFRACTION100
2.19-2.270.19971240.20572696X-RAY DIFFRACTION100
2.27-2.360.24061500.20032691X-RAY DIFFRACTION100
2.36-2.470.24141520.20322687X-RAY DIFFRACTION100
2.47-2.60.23741490.20452707X-RAY DIFFRACTION100
2.6-2.760.22931300.21052706X-RAY DIFFRACTION100
2.76-2.970.25391500.20542718X-RAY DIFFRACTION100
2.97-3.270.17691360.19242739X-RAY DIFFRACTION100
3.27-3.740.22291250.17122763X-RAY DIFFRACTION100
3.74-4.720.1411410.14242802X-RAY DIFFRACTION100
4.72-55.160.15741580.16362880X-RAY DIFFRACTION99

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