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- PDB-6wvv: Plasmodium vivax M17 leucyl aminopeptidase -

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Basic information

Entry
Database: PDB / ID: 6wvv
TitlePlasmodium vivax M17 leucyl aminopeptidase
ComponentsM17 leucyl aminopeptidase
KeywordsHYDROLASE / M17 Leucyl Aminopeptidase / Metalloprotease
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
DI(HYDROXYETHYL)ETHER / leucyl aminopeptidase / Leucine aminopeptidase, putative
Similarity search - Component
Biological speciesPlasmodium vivax (malaria parasite P. vivax)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.33 Å
AuthorsMalcolm, T.R. / Drinkwater, N. / McGowan, S.
Funding support Australia, 1items
OrganizationGrant numberCountry
National Health and Medical Research Council (NHMRC, Australia)1185354 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
DepositionMay 7, 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 1.3Oct 18, 2023Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: M17 leucyl aminopeptidase
B: M17 leucyl aminopeptidase
C: M17 leucyl aminopeptidase
D: M17 leucyl aminopeptidase
E: M17 leucyl aminopeptidase
F: M17 leucyl aminopeptidase
G: M17 leucyl aminopeptidase
H: M17 leucyl aminopeptidase
I: M17 leucyl aminopeptidase
J: M17 leucyl aminopeptidase
K: M17 leucyl aminopeptidase
L: M17 leucyl aminopeptidase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)706,57896
Polymers699,24212
Non-polymers7,33584
Water25,9601441
1
A: M17 leucyl aminopeptidase
B: M17 leucyl aminopeptidase
C: M17 leucyl aminopeptidase
D: M17 leucyl aminopeptidase
E: M17 leucyl aminopeptidase
F: M17 leucyl aminopeptidase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)354,45460
Polymers349,6216
Non-polymers4,83354
Water1086
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area38440 Å2
ΔGint-746 kcal/mol
Surface area99670 Å2
MethodPISA
2
G: M17 leucyl aminopeptidase
H: M17 leucyl aminopeptidase
I: M17 leucyl aminopeptidase
J: M17 leucyl aminopeptidase
K: M17 leucyl aminopeptidase
L: M17 leucyl aminopeptidase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)352,12436
Polymers349,6216
Non-polymers2,50230
Water1086
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area29290 Å2
ΔGint-751 kcal/mol
Surface area94910 Å2
MethodPISA
Unit cell
Length a, b, c (Å)117.035, 201.549, 166.224
Angle α, β, γ (deg.)90.000, 106.013, 90.000
Int Tables number4
Space group name H-MP1211
Space group name HallP2yb
Symmetry operation#1: x,y,z
#2: -x,y+1/2,-z

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Components

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Protein , 1 types, 12 molecules ABCDEFGHIJKL

#1: Protein
M17 leucyl aminopeptidase


Mass: 58270.199 Da / Num. of mol.: 12
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(DE3) (bacteria)
References: UniProt: A0A1G4HHP8, UniProt: A5K3U9*PLUS, leucyl aminopeptidase

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Non-polymers , 5 types, 1525 molecules

#2: Chemical...
ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 30 / Source method: obtained synthetically / Formula: C3H8O3
#3: Chemical
ChemComp-PEG / DI(HYDROXYETHYL)ETHER


Mass: 106.120 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: C4H10O3
#4: Chemical...
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 24 / Source method: obtained synthetically / Formula: Zn
#5: Chemical
ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 18 / Source method: obtained synthetically / Formula: SO4
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 1441 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestN

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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: 2.69 Å3/Da / Density % sol: 54.36 %
Crystal growTemperature: 298 K / Method: vapor diffusion, hanging drop / pH: 6.5 / Details: 17% PEG 3350, 0.1 M HEPES pH 6.5, 0.15 M (NH4)2SO4

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Australian Synchrotron / Beamline: MX2 / Wavelength: 0.9537 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Mar 28, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9537 Å / Relative weight: 1
ReflectionResolution: 2.29→48.95 Å / Num. obs: 327356 / % possible obs: 99.7 % / Redundancy: 4.5 % / Biso Wilson estimate: 35.02 Å2 / CC1/2: 0.992 / Rmerge(I) obs: 0.154 / Rpim(I) all: 0.081 / Rrim(I) all: 0.174 / Χ2: 0.54 / Net I/σ(I): 5.9
Reflection shellResolution: 2.29→2.33 Å / Redundancy: 4 % / Num. unique obs: 15478 / CC1/2: 0.408 / % possible all: 95.3

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Processing

Software
NameVersionClassification
PHENIX1.17.1_3660refinement
PDB_EXTRACTdata extraction
Aimlessdata scaling
XDSdata reduction
Cootmodel building
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 3KQZ

3kqz


Resolution: 2.33→44.32 Å / SU ML: 0.2867 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 24.4933
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2435 15699 4.99 %
Rwork0.2054 298718 -
obs0.2073 314417 99.93 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 42.35 Å2
Refinement stepCycle: LAST / Resolution: 2.33→44.32 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms43042 0 378 1441 44861
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.002544121
X-RAY DIFFRACTIONf_angle_d0.497559786
X-RAY DIFFRACTIONf_chiral_restr0.0446928
X-RAY DIFFRACTIONf_plane_restr0.00247576
X-RAY DIFFRACTIONf_dihedral_angle_d8.90576200
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.33-2.360.31475350.27889890X-RAY DIFFRACTION99.94
2.36-2.380.29324980.25849942X-RAY DIFFRACTION99.94
2.38-2.410.3144980.26489963X-RAY DIFFRACTION99.94
2.41-2.440.30995270.26149946X-RAY DIFFRACTION99.9
2.44-2.480.28775020.25659978X-RAY DIFFRACTION99.91
2.48-2.510.28585330.25399889X-RAY DIFFRACTION99.88
2.51-2.550.3094840.25549985X-RAY DIFFRACTION99.98
2.55-2.580.29955330.24659875X-RAY DIFFRACTION99.89
2.58-2.620.29525400.23869921X-RAY DIFFRACTION99.88
2.62-2.670.29555150.23719967X-RAY DIFFRACTION99.85
2.67-2.710.28545380.2379936X-RAY DIFFRACTION99.78
2.71-2.760.28615170.23479848X-RAY DIFFRACTION99.45
2.76-2.820.28225450.22159938X-RAY DIFFRACTION99.93
2.82-2.870.27635240.22169960X-RAY DIFFRACTION100
2.87-2.940.26415630.21049910X-RAY DIFFRACTION100
2.94-30.26075320.20689956X-RAY DIFFRACTION100
3-3.080.25134940.21259975X-RAY DIFFRACTION99.98
3.08-3.160.26265050.21489965X-RAY DIFFRACTION100
3.16-3.260.26145030.20979996X-RAY DIFFRACTION100
3.26-3.360.24435820.19619866X-RAY DIFFRACTION99.98
3.36-3.480.23655450.1929945X-RAY DIFFRACTION100
3.48-3.620.24515000.20059973X-RAY DIFFRACTION100
3.62-3.780.23015080.18610003X-RAY DIFFRACTION100
3.78-3.980.21275350.18189952X-RAY DIFFRACTION99.99
3.98-4.230.20195560.16999955X-RAY DIFFRACTION100
4.23-4.560.17985200.16399982X-RAY DIFFRACTION99.99
4.56-5.020.20145500.16749966X-RAY DIFFRACTION99.99
5.02-5.740.23094810.20310068X-RAY DIFFRACTION99.99
5.74-7.230.24035240.216910049X-RAY DIFFRACTION100
7.23-44.320.22385120.203610119X-RAY DIFFRACTION99.65

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