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- EMDB-22682: Plasmodium vivax M17 leucyl aminopeptidase Pv-M17 -

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Basic information

Entry
Database: EMDB / ID: EMD-22682
TitlePlasmodium vivax M17 leucyl aminopeptidase Pv-M17
Map dataPlasmodium vivax M17 leucyl aminopeptidase Pv-M17
Sample
  • Complex: PvM17-hexamer
    • Protein or peptide: M17 leucyl aminopeptidase, putative
  • Ligand: CARBONATE IONCarbonate
  • Ligand: MANGANESE (II) ION
KeywordsM17 aminopeptidase / leucyl aminopeptidase / HYDROLASE
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
leucyl aminopeptidase / Leucine aminopeptidase, putative
Similarity search - Component
Biological speciesPlasmodium vivax (malaria parasite P. vivax)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.66 Å
AuthorsMalcolm TR / McGowan S
Funding support Australia, 1 items
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, 2020-
Header (metadata) releaseDec 16, 2020-
Map releaseDec 16, 2020-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.6
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.6
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7k5k
  • Surface level: 0.6
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22682.png

Downloads & links

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Map

FileDownload / File: emd_22682.map.gz / Format: CCP4 / Size: 38.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPlasmodium vivax M17 leucyl aminopeptidase Pv-M17
Voxel sizeX=Y=Z: 1.06 Å
Density
Contour LevelBy EMDB: 0.4 / Movie #1: 0.6
Minimum - Maximum-2.1395714 - 4.107372
Average (Standard dev.)0.015726423 (±0.1560487)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions216216216
Spacing216216216
CellA=B=C: 228.95999 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.061.061.06
M x/y/z216216216
origin x/y/z0.0000.0000.000
length x/y/z228.960228.960228.960
α/β/γ90.00090.00090.000
start NX/NY/NZ1081340
NX/NY/NZ13584349
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS216216216
D min/max/mean-2.1404.1070.016

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Supplemental data

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Mask #1

Fileemd_22682_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Local resolution map

Fileemd_22682_additional_1.map
AnnotationLocal resolution map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Unfiltered map

Fileemd_22682_additional_2.map
AnnotationUnfiltered map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half-volume 1

Fileemd_22682_half_map_1.map
Annotationhalf-volume 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half-volume 2

Fileemd_22682_half_map_2.map
Annotationhalf-volume 2
Projections & Slices
AxesZYX

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Sample components

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Entire : PvM17-hexamer

EntireName: PvM17-hexamer
Components
  • Complex: PvM17-hexamer
    • Protein or peptide: M17 leucyl aminopeptidase, putative
  • Ligand: CARBONATE IONCarbonate
  • Ligand: MANGANESE (II) ION

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Supramolecule #1: PvM17-hexamer

SupramoleculeName: PvM17-hexamer / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Plasmodium vivax (malaria parasite P. vivax)

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Macromolecule #1: M17 leucyl aminopeptidase, putative

MacromoleculeName: M17 leucyl aminopeptidase, putative / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO / EC number: leucyl aminopeptidase
Source (natural)Organism: Plasmodium vivax (malaria parasite P. vivax)
Molecular weightTheoretical: 60.442328 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: MATTVPQVVS LDPTTIPIDY HTPIDDLSIE VKDISAEACP ADEGLIVFLL NSAPKHSSSG GSGGNGGSAG SSGNGEGGAQ IKINSSVKD NTINEFLKEG NMENFTGKLG TSKSFYIAND QKKYVSLAYV GCGPANEETE LEIRKVAYAL VTLLHDSKHK K VSIIFEIK ...String:
MATTVPQVVS LDPTTIPIDY HTPIDDLSIE VKDISAEACP ADEGLIVFLL NSAPKHSSSG GSGGNGGSAG SSGNGEGGAQ IKINSSVKD NTINEFLKEG NMENFTGKLG TSKSFYIAND QKKYVSLAYV GCGPANEETE LEIRKVAYAL VTLLHDSKHK K VSIIFEIK IEEALFRFFL EHLFYEYVTD ERFKSADKST ETDFIKNLSL HIANADAYKG QIDKARVYFY GTYYAAQLIA AP SNYCNPV SLSNAAVELA QKVNLECKIL DVKELEELKM GAYLSVGKGS MYPNKFIHLT YKGAQTGASQ NEKKKIALIG KGI TFDSGG YNLKAAPGSM IDLMKFDMSG CAAVLGCAYC IGTIKPDNVE VHFLSAVCEN MVSKNSYRPG DIITASNGKT IEVG NTDAE GRLTLADALV YAEKLGVDYI VDIATLTGAM LYSLGTSYAG VFGNNDQLIN KILSSSKTSN EPVWWLPIIN EYRSS LNSK YADLNNISSS VKASSVVASL FLKEFIENTP WAHIDIAGVS WNFKARKPKG FGVRLLTEFV LNDAVHHHHH H

UniProtKB: leucyl aminopeptidase

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Macromolecule #2: CARBONATE ION

MacromoleculeName: CARBONATE ION / type: ligand / ID: 2 / Number of copies: 6 / Formula: CO3
Molecular weightTheoretical: 60.009 Da
Chemical component information

ChemComp-CO3:
CARBONATE ION / Carbonate

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Macromolecule #3: MANGANESE (II) ION

MacromoleculeName: MANGANESE (II) ION / type: ligand / ID: 3 / Number of copies: 12 / Formula: MN
Molecular weightTheoretical: 54.938 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration2.9 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
50.0 mMC8H18N2O4SHEPES
0.3 MNaClSodium chloridesodium choloride
1.0 mMMnCl2manganese chloride
GridModel: Quantifoil / Material: COPPER / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details: Blot for 3 seconds with blot force of -1. Drain time of 1 second..

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 63.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: D3 (2x3 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 2.66 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 32326
FSC plot (resolution estimation)

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