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- PDB-8v3o: CCP5 in complex with Glu-P-peptide 1 transition state analog -

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Basic information

Entry
Database: PDB / ID: 8v3o
TitleCCP5 in complex with Glu-P-peptide 1 transition state analog
Components
  • Cytosolic carboxypeptidase-like protein 5
  • Tubulin beta-2A chain
KeywordsHYDROLASE/INHIBITOR / carboxypeptidase deglutamylation branch glutamate removal microtubule / HYDROLASE / HYDROLASE-INHIBITOR complex
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 / Cilium Assembly / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Carboxyterminal post-translational modifications of tubulin / Intraflagellar transport ...tubulin-glutamate carboxypeptidase / protein deglutamylation / protein side chain deglutamylation / protein branching point deglutamylation / Post-chaperonin tubulin folding pathway / C-terminal protein deglutamylation / Cilium Assembly / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Carboxyterminal post-translational modifications of tubulin / Intraflagellar transport / Sealing of the nuclear envelope (NE) by ESCRT-III / Formation of tubulin folding intermediates by CCT/TriC / Gap junction assembly / Prefoldin mediated transfer of substrate to CCT/TriC / COPI-independent Golgi-to-ER retrograde traffic / Kinesins / Assembly and cell surface presentation of NMDA receptors / COPI-dependent Golgi-to-ER retrograde traffic / Recycling pathway of L1 / Hydrolases; Acting on peptide bonds (peptidases); Metallocarboxypeptidases / RHO GTPases activate IQGAPs / intercellular bridge / Hedgehog 'off' state / Activation of AMPK downstream of NMDARs / COPI-mediated anterograde transport / metallocarboxypeptidase activity / Mitotic Prometaphase / EML4 and NUDC in mitotic spindle formation / MHC class II antigen presentation / tubulin binding / Recruitment of NuMA to mitotic centrosomes / Resolution of Sister Chromatid Cohesion / HSP90 chaperone cycle for steroid hormone receptors (SHR) in the presence of ligand / Translocation of SLC2A4 (GLUT4) to the plasma membrane / RHO GTPases Activate Formins / PKR-mediated signaling / cerebral cortex development / structural constituent of cytoskeleton / microtubule cytoskeleton organization / HCMV Early Events / neuron migration / Aggrephagy / mitotic spindle / Separation of Sister Chromatids / The role of GTSE1 in G2/M progression after G2 checkpoint / extracellular vesicle / mitotic cell cycle / microtubule cytoskeleton / midbody / defense response to virus / microtubule / GTPase activity / GTP binding / proteolysis / extracellular exosome / zinc ion binding / metal ion binding / nucleus / 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
: / D-MALATE / Tubulin beta-2A chain / Cytosolic carboxypeptidase-like protein 5
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.3 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 1.3Oct 30, 2024Group: Derived calculations / Structure summary
Category: pdbx_entry_details / pdbx_modification_feature / struct_conn
Item: _pdbx_entry_details.has_protein_modification / _struct_conn.pdbx_leaving_atom_flag

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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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)61,4496
Polymers61,0762
Non-polymers3734
Water2,504139
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1970 Å2
ΔGint-34 kcal/mol
Surface area18960 Å2
MethodPISA
Unit cell
Length a, b, c (Å)65.429, 81.372, 104.000
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number18
Space group name H-MP22121

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Components

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Protein / Protein/peptide , 2 types, 2 molecules AI

#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): 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 / Mutation: One of the glutamates replaced with BIX / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human) / References: UniProt: Q13885

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Non-polymers , 4 types, 143 molecules

#3: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn
#4: 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
#5: Chemical ChemComp-K / POTASSIUM ION


Mass: 39.098 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: K
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 139 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY
Has protein modificationY

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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: 2.26 Å3/Da / Density % sol: 45.49 %
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: 8.2.1 / Wavelength: 1 Å
DetectorType: DECTRIS EIGER X 9M / Detector: PIXEL / Date: May 17, 2022
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 2.3→34.55 Å / Num. obs: 25384 / % possible obs: 100 % / Redundancy: 14.3 % / CC1/2: 0.998 / Rmerge(I) obs: 0.175 / Rrim(I) all: 0.187 / Net I/σ(I): 13.8
Reflection shellResolution: 2.3→2.38 Å / Redundancy: 14.7 % / Rmerge(I) obs: 1.455 / Mean I/σ(I) obs: 2 / Num. unique obs: 2454 / CC1/2: 0.712 / Rrim(I) all: 1.56 / % 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: 2.3→34.55 Å / SU ML: 0.21 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 23.91 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2269 1187 4.68 %
Rwork0.2031 --
obs0.2043 25340 99.97 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 2.3→34.55 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3765 0 20 139 3924
Refine LS restraints
Refine-IDTypeDev ideal
X-RAY DIFFRACTIONf_bond_d0.004
X-RAY DIFFRACTIONf_angle_d0.642
X-RAY DIFFRACTIONf_dihedral_angle_d10.252
X-RAY DIFFRACTIONf_chiral_restr0.04
X-RAY DIFFRACTIONf_plane_restr0.005
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.3-2.40.2891340.26422993X-RAY DIFFRACTION100
2.4-2.530.31591400.25792970X-RAY DIFFRACTION100
2.53-2.690.29931570.25692965X-RAY DIFFRACTION100
2.69-2.90.23541480.24022982X-RAY DIFFRACTION100
2.9-3.190.25131210.23083016X-RAY DIFFRACTION100
3.19-3.650.21151440.2013026X-RAY DIFFRACTION100
3.65-4.60.19321690.17043022X-RAY DIFFRACTION100
4.6-34.550.2121740.17323179X-RAY DIFFRACTION100

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