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- PDB-8v4k: CCP5 in complex with microtubules class1 -

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Basic information

Entry
Database: PDB / ID: 8v4k
TitleCCP5 in complex with microtubules class1
Components
  • Cytosolic carboxypeptidase-like protein 5
  • Tubulin alpha-1B chain
  • Tubulin beta chain
KeywordsHYDROLASE/SUBSTRATE / STRUCTURAL PROTEIN / carboxypeptidase deglutamylation branch glutamate removal microtubule / HYDROLASE / HYDROLASE-SUBSTRATE complex / HYDROLASE-SUBSTRATE / STRUCTURAL PROTEIN complex
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 / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Resolution of Sister Chromatid Cohesion / Hedgehog 'off' state / Cilium Assembly ...tubulin-glutamate carboxypeptidase / protein deglutamylation / protein side chain deglutamylation / protein branching point deglutamylation / C-terminal protein deglutamylation / Carboxyterminal post-translational modifications of tubulin / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Resolution of Sister Chromatid Cohesion / Hedgehog 'off' state / Cilium Assembly / Intraflagellar transport / COPI-dependent Golgi-to-ER retrograde traffic / Mitotic Prometaphase / Carboxyterminal post-translational modifications of tubulin / RHOH GTPase cycle / EML4 and NUDC in mitotic spindle formation / Sealing of the nuclear envelope (NE) by ESCRT-III / Kinesins / PKR-mediated signaling / Separation of Sister Chromatids / The role of GTSE1 in G2/M progression after G2 checkpoint / Aggrephagy / metallocarboxypeptidase activity / RHO GTPases activate IQGAPs / RHO GTPases Activate Formins / HSP90 chaperone cycle for steroid hormone receptors (SHR) in the presence of ligand / MHC class II antigen presentation / Recruitment of NuMA to mitotic centrosomes / COPI-mediated anterograde transport / intercellular bridge / Hydrolases; Acting on peptide bonds (peptidases); Metallocarboxypeptidases / tubulin binding / structural constituent of cytoskeleton / mitotic spindle / microtubule cytoskeleton organization / microtubule cytoskeleton / mitotic cell cycle / midbody / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / defense response to virus / microtubule / GTPase activity / GTP binding / proteolysis / 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 / Alpha tubulin / Tubulin-beta mRNA autoregulation signal. / Beta tubulin, autoregulation binding site ...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 / Alpha tubulin / 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
PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER / GLUTAMIC ACID / GUANOSINE-5'-TRIPHOSPHATE / Tubulin beta chain / Tubulin alpha-1B chain / Cytosolic carboxypeptidase-like protein 5
Similarity search - Component
Biological speciesHomo sapiens (human)
Sus scrofa (pig)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 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 29, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 17, 2024Provider: repository / Type: Initial release
Revision 1.1Jul 31, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.pdbx_database_id_PubMed / _citation.title ..._citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _em_admin.last_update
Revision 1.2Aug 7, 2024Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _em_admin.last_update
Revision 1.3Oct 16, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Tubulin alpha-1B chain
B: Tubulin beta chain
C: Tubulin alpha-1B chain
D: Tubulin beta chain
E: Cytosolic carboxypeptidase-like protein 5
hetero molecules


Theoretical massNumber of molelcules
Total (without water)270,04715
Polymers267,6485
Non-polymers2,39910
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 3 types, 5 molecules ACBDE

#1: Protein Tubulin alpha-1B chain / Alpha-tubulin ubiquitous / Tubulin K-alpha-1 / Tubulin alpha-ubiquitous chain


Mass: 50204.445 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Sus scrofa (pig) / Organ: brain / References: UniProt: Q2XVP4
#2: Protein Tubulin beta chain / Beta-tubulin


Mass: 49504.801 Da / Num. of mol.: 2 / Source method: isolated from a natural source
Details: Beta tubulin from porcine brain microtubules, heterogeneous in tail sequence
Source: (natural) Sus scrofa (pig) / Organ: brain / References: UniProt: P02554
#3: Protein Cytosolic carboxypeptidase-like protein 5


Mass: 68229.547 Da / Num. of mol.: 1 / Fragment: residues 2-605 / Mutation: E516A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: AGBL5 / Plasmid: pFastBac / Details (production host): His_MBP_Asn10_TEV / Production host: Spodoptera frugiperda (fall armyworm) / Strain (production host): IPLB-Sf-21-AE / References: UniProt: Q8NDL9

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Non-polymers , 5 types, 10 molecules

#4: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg
#5: Chemical ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE


Mass: 523.180 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H16N5O14P3 / Comment: GTP, energy-carrying molecule*YM
#6: Chemical ChemComp-G2P / PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER


Mass: 521.208 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C11H18N5O13P3 / Comment: GMP-CPP, energy-carrying molecule analogue*YM
#7: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn
#8: Chemical ChemComp-GLU / GLUTAMIC ACID


Type: L-peptide linking / Mass: 147.129 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Formula: C5H9NO4
Details: enzymatically linked to the side chain of GLU B 435 of beta-tubulin

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Details

Has ligand of interestY
Has protein modificationY
Sequence detailsBrain tubulin contains multiple tubulin isotypes. The tubulin body was modeled using the sequence ...Brain tubulin contains multiple tubulin isotypes. The tubulin body was modeled using the sequence of betaII, the most abundant isotype in brain. The residues in the variable tails are modeled as UNK.

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: CCP5 in complex with microtubules / Type: COMPLEX / Details: Composite structure of CCP5:microtubule class#1 / Entity ID: #1, #3 / Source: MULTIPLE SOURCES
Molecular weightExperimental value: NO
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPESC8H18N2O4S1
2150 mMpotassium chlorideKCl1
31 mMTCEPC9H15O6P1
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid type: Au-flat 1.2/1.3
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 303 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 135000 X / Nominal defocus max: 2200 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 1.651 sec. / Electron dose: 53.34 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)
EM imaging opticsEnergyfilter slit width: 20 eV

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Processing

EM softwareName: PHENIX / Category: model refinement
CTF correctionType: NONE
Particle selectionNum. of particles selected: 162521
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 239209 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL / Target criteria: Cross-correlation coefficient
Atomic model building
IDPDB-ID 3D fitting-IDDetailsSource nameTypeAccession code
11crystal structure of apo CCP5Otherexperimental model
26DPU

6dpu

1GMPCPP microtubulePDBexperimental model6DPU

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