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- PDB-8v3p: CCP5 in complex with Glu-P-peptide 2 transition state analog -

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Basic information

Entry
Database: PDB / ID: 8v3p
TitleCCP5 in complex with Glu-P-peptide 2 transition state analog
Components
  • Cytosolic carboxypeptidase-like protein 5
  • Tubulin beta-2A chain
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 / Post-chaperonin tubulin folding pathway / C-terminal protein deglutamylation / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Cilium Assembly / Carboxyterminal post-translational modifications of tubulin / Sealing of the nuclear envelope (NE) by ESCRT-III ...tubulin-glutamate carboxypeptidase / protein deglutamylation / protein side chain deglutamylation / protein branching point deglutamylation / Post-chaperonin tubulin folding pathway / C-terminal protein deglutamylation / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Cilium Assembly / Carboxyterminal post-translational modifications of tubulin / Sealing of the nuclear envelope (NE) by ESCRT-III / Intraflagellar transport / Formation of tubulin folding intermediates by CCT/TriC / COPI-independent Golgi-to-ER retrograde traffic / Gap junction assembly / Prefoldin mediated transfer of substrate to CCT/TriC / Kinesins / Assembly and cell surface presentation of NMDA receptors / metallocarboxypeptidase activity / COPI-dependent Golgi-to-ER retrograde traffic / intercellular bridge / Recycling pathway of L1 / Hydrolases; Acting on peptide bonds (peptidases); Metallocarboxypeptidases / RHO GTPases activate IQGAPs / 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 / HSP90 chaperone cycle for steroid hormone receptors (SHR) in the presence of ligand / MHC class II antigen presentation / Resolution of Sister Chromatid Cohesion / tubulin binding / Translocation of SLC2A4 (GLUT4) to the plasma membrane / RHO GTPases Activate Formins / PKR-mediated signaling / cerebral cortex development / mitotic spindle / structural constituent of cytoskeleton / microtubule cytoskeleton organization / HCMV Early Events / Aggrephagy / Separation of Sister Chromatids / The role of GTSE1 in G2/M progression after G2 checkpoint / microtubule cytoskeleton / extracellular vesicle / mitotic cell cycle / midbody / defense response to virus / microtubule / GTPase activity / GTP binding / proteolysis / extracellular exosome / zinc ion binding / nucleus / metal ion binding / cytosol / cytoplasm
Similarity search - Function
Cytosolic carboxypeptidase-like protein 5 catalytic domain / Cytosolic carboxypeptidase, N-terminal / : / Cytosolic carboxypeptidase N-terminal domain / Peptidase family M14 domain profile. / Peptidase M14, carboxypeptidase A / Zinc carboxypeptidase / Tubulin-beta mRNA autoregulation signal. / Beta tubulin, autoregulation binding site / Beta tubulin ...Cytosolic carboxypeptidase-like protein 5 catalytic domain / Cytosolic carboxypeptidase, N-terminal / : / Cytosolic carboxypeptidase N-terminal domain / Peptidase family M14 domain profile. / Peptidase M14, carboxypeptidase A / Zinc carboxypeptidase / Tubulin-beta mRNA autoregulation signal. / 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
Tubulin beta-2A chain / Cytosolic carboxypeptidase-like protein 5
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.36 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
Revision 1.3Nov 6, 2024Group: Advisory / Structure summary / Category: pdbx_entry_details / pdbx_unobs_or_zero_occ_atoms / Item: _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Cytosolic carboxypeptidase-like protein 5
I: Tubulin beta-2A chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)61,1423
Polymers61,0762
Non-polymers651
Water1,24369
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)65.154, 78.299, 103.373
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number18
Space group name H-MP22121

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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,construct is from residues 2-605,with 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): His_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: Protein/peptide Tubulin beta-2A chain / Tubulin beta class IIa


Mass: 1905.682 Da / Num. of mol.: 1 / Fragment: C-terminal tail / Mutation: E435 replaced with transition state analog / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human) / References: UniProt: Q13885
#3: Chemical ChemComp-ZN / ZINC ION


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


Mass: 18.015 Da / Num. of mol.: 69 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY
Has protein modificationN

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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: 2.23 Å3/Da / Density % sol: 44.8 %
Crystal growTemperature: 294 K / Method: vapor diffusion, hanging drop
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: ALS / Beamline: 5.0.2 / Wavelength: 1 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Jun 21, 2023
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 2.36→43.14 Å / Num. obs: 22434 / % possible obs: 100 % / Redundancy: 6.4 % / CC1/2: 0.998 / Rmerge(I) obs: 0.097 / Rrim(I) all: 0.115 / Net I/σ(I): 12.8
Reflection shellResolution: 2.36→2.45 Å / Redundancy: 6.4 % / Rmerge(I) obs: 0.782 / Mean I/σ(I) obs: 2.4 / Num. unique obs: 2327 / CC1/2: 0.724 / Rrim(I) all: 0.93 / % possible all: 100

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Processing

Software
NameVersionClassification
PHENIX1.20.1_4487refinement
XDSdata reduction
Aimlessdata scaling
PHENIXphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.36→43.14 Å / SU ML: 0.26 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 23.29 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2253 1117 4.99 %
Rwork0.2146 --
obs0.2152 22392 99.93 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 2.36→43.14 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3641 0 20 69 3730
Refine LS restraints
Refine-IDTypeDev ideal
X-RAY DIFFRACTIONf_bond_d0.008
X-RAY DIFFRACTIONf_angle_d1.135
X-RAY DIFFRACTIONf_dihedral_angle_d7.593
X-RAY DIFFRACTIONf_chiral_restr0.065
X-RAY DIFFRACTIONf_plane_restr0.008
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.36-2.470.29711600.29012579X-RAY DIFFRACTION100
2.47-2.60.31341360.27692652X-RAY DIFFRACTION100
2.6-2.760.28221240.26342618X-RAY DIFFRACTION100
2.76-2.970.28451370.24142641X-RAY DIFFRACTION100
2.97-3.270.23591550.22942604X-RAY DIFFRACTION100
3.27-3.750.24011290.20322665X-RAY DIFFRACTION100
3.75-4.720.19111350.18092693X-RAY DIFFRACTION100
4.72-43.140.17691410.20192823X-RAY DIFFRACTION100

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