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- PDB-7k5k: Plasmodium vivax M17 leucyl aminopeptidase Pv-M17 -

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Basic information

Entry
Database: PDB / ID: 7k5k
TitlePlasmodium vivax M17 leucyl aminopeptidase Pv-M17
ComponentsM17 leucyl aminopeptidase, putative
KeywordsHYDROLASE / M17 aminopeptidase / leucyl aminopeptidase
Function / homology
Function and homology information


leucyl aminopeptidase / metalloaminopeptidase activity / manganese ion binding / proteolysis / cytoplasm
Similarity search - Function
Peptidase M17, leucine aminopeptidase / Cytosol aminopeptidase signature. / Peptidase M17, leucyl aminopeptidase, C-terminal / Peptidase M17, leucine aminopeptidase/peptidase B / Cytosol aminopeptidase family, catalytic domain / Macro domain-like
Similarity search - Domain/homology
CARBONATE ION / : / leucyl aminopeptidase / Leucine aminopeptidase, putative
Similarity search - Component
Biological speciesPlasmodium vivax (malaria parasite P. vivax)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.66 Å
AuthorsMalcolm, T.R. / McGowan, S. / Belousoff, M.J.
Funding support Australia, 1items
OrganizationGrant numberCountry
National Health and Medical Research Council (NHMRC, Australia)APP1185354 Australia
CitationJournal: J Biol Chem / Year: 2021
Title: Active site metals mediate an oligomeric equilibrium in Plasmodium M17 aminopeptidases.
Authors: Tess R Malcolm / Matthew J Belousoff / Hariprasad Venugopal / Natalie A Borg / Nyssa Drinkwater / Sarah C Atkinson / Sheena McGowan /
Abstract: M17 leucyl aminopeptidases are metal-dependent exopeptidases that rely on oligomerization to diversify their functional roles. The M17 aminopeptidases from Plasmodium falciparum (PfA-M17) and ...M17 leucyl aminopeptidases are metal-dependent exopeptidases that rely on oligomerization to diversify their functional roles. The M17 aminopeptidases from Plasmodium falciparum (PfA-M17) and Plasmodium vivax (Pv-M17) function as catalytically active hexamers to generate free amino acids from human hemoglobin and are drug targets for the design of novel antimalarial agents. However, the molecular basis for oligomeric assembly is not fully understood. In this study, we found that the active site metal ions essential for catalytic activity have a secondary structural role mediating the formation of active hexamers. We found that PfA-M17 and Pv-M17 exist in a metal-dependent dynamic equilibrium between active hexameric species and smaller inactive species that can be controlled by manipulating the identity and concentration of metals available. Mutation of residues involved in metal ion binding impaired catalytic activity and the formation of active hexamers. Structural resolution of Pv-M17 by cryoelectron microscopy and X-ray crystallography together with solution studies revealed that PfA-M17 and Pv-M17 bind metal ions and substrates in a conserved fashion, although Pv-M17 forms the active hexamer more readily and processes substrates faster than PfA-M17. On the basis of these studies, we propose a dynamic equilibrium between monomer ↔ dimer ↔ tetramer ↔ hexamer, which becomes directional toward the large oligomeric states with the addition of metal ions. This sophisticated metal-dependent dynamic equilibrium may apply to other M17 aminopeptidases and underpin the moonlighting capabilities of this enzyme family.
History
DepositionSep 17, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 16, 2020Provider: repository / Type: Initial release
Revision 1.1Dec 23, 2020Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed ..._citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Jul 21, 2021Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.3Mar 6, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Assembly

Deposited unit
A: M17 leucyl aminopeptidase, putative
B: M17 leucyl aminopeptidase, putative
C: M17 leucyl aminopeptidase, putative
D: M17 leucyl aminopeptidase, putative
E: M17 leucyl aminopeptidase, putative
F: M17 leucyl aminopeptidase, putative
hetero molecules


Theoretical massNumber of molelcules
Total (without water)363,67324
Polymers362,6546
Non-polymers1,01918
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, Analytical ultracentrifugation
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
M17 leucyl aminopeptidase, putative


Mass: 60442.328 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Plasmodium vivax (malaria parasite P. vivax)
Gene: PVC01_120064700, PVP01_1260800 / Production host: Escherichia coli BL21 (bacteria)
References: UniProt: A0A1G4HHP8, UniProt: A5K3U9*PLUS, leucyl aminopeptidase
#2: Chemical
ChemComp-CO3 / CARBONATE ION


Mass: 60.009 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: CO3
#3: Chemical
ChemComp-MN / MANGANESE (II) ION


Mass: 54.938 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: Mn
Has ligand of interestN

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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: PvM17-hexamer / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Plasmodium vivax (malaria parasite P. vivax)
Source (recombinant)Organism: Escherichia coli BL21 (bacteria)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMHEPESC8H18N2O4S1
20.3 Msodium cholorideNaCl1
31 mMmanganese chlorideMnCl21
SpecimenConc.: 2.9 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: Quantifoil
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K
Details: Blot for 3 seconds with blot force of -1. Drain time of 1 second.

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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
Image recordingElectron dose: 63 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.17.1_3660: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: D3 (2x3 fold dihedral)
3D reconstructionResolution: 2.66 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 32326 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00724674
ELECTRON MICROSCOPYf_angle_d0.66533500
ELECTRON MICROSCOPYf_dihedral_angle_d6.0313468
ELECTRON MICROSCOPYf_chiral_restr0.0483878
ELECTRON MICROSCOPYf_plane_restr0.0044288

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