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- PDB-4r8f: Crystal structure of yeast aminopeptidase 1 (Ape1) -

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Basic information

Entry
Database: PDB / ID: 4r8f
TitleCrystal structure of yeast aminopeptidase 1 (Ape1)
ComponentsVacuolar aminopeptidase 1
KeywordsHYDROLASE / peptidase / Cvt pathway / Autophagy
Function / homology
Function and homology information


aminopeptidase I / Cvt complex / cytoplasm to vacuole targeting by the Cvt pathway / fungal-type vacuole / metalloaminopeptidase activity / proteolysis / zinc ion binding / identical protein binding / cytoplasm
Similarity search - Function
Aminopeptidase i, Domain 2 / Aminopeptidase i, Domain 2 / Peptidase M18 / Peptidase M18, domain 2 / Aminopeptidase I zinc metalloprotease (M18) / Zn peptidases / Aminopeptidase / Roll / 3-Layer(aba) Sandwich / Mainly Beta / Alpha Beta
Similarity search - Domain/homology
Vacuolar aminopeptidase 1
Similarity search - Component
Biological speciesSaccharomyces cerevisiae S288c (yeast)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.5 Å
AuthorsSu, M.-Y. / Chang, C.-I.
CitationJournal: Autophagy / Year: 2015
Title: Structure of yeast Ape1 and its role in autophagic vesicle formation.
Authors: Ming-Yuan Su / Wen-Hsin Peng / Meng-Ru Ho / Shih-Chieh Su / Yuan-Chih Chang / Guang-Chao Chen / Chung-I Chang /
Abstract: In Saccharomyces cerevisiae, a constitutive biosynthetic transport pathway, termed the cytoplasm-to-vacuole targeting (Cvt) pathway, sequesters precursor aminopeptidase I (prApe1) dodecamers in the ...In Saccharomyces cerevisiae, a constitutive biosynthetic transport pathway, termed the cytoplasm-to-vacuole targeting (Cvt) pathway, sequesters precursor aminopeptidase I (prApe1) dodecamers in the form of a large complex into a Cvt vesicle using autophagic machinery, targeting it into the vacuole (the yeast lysosome) where it is proteolytically processed into its mature form, Ape1, by removal of an amino-terminal 45-amino acid propeptide. prApe1 is thought to serve as a scaffolding cargo critical for the assembly of the Cvt vesicle by presenting the propeptide to mediate higher-ordered complex formation and autophagic receptor recognition. Here we report the X-ray crystal structure of Ape1 at 2.5 Å resolution and reveal its dodecameric architecture consisting of dimeric and trimeric units, which associate to form a large tetrahedron. The propeptide of prApe1 exhibits concentration-dependent oligomerization and forms a stable tetramer. Structure-based mutagenesis demonstrates that disruption of the inter-subunit interface prevents dodecameric assembly and vacuolar targeting in vivo despite the presence of the propeptide. Furthermore, by examining the vacuolar import of propeptide-fused exogenous protein assemblies with different quaternary structures, we found that 3-dimensional spatial distribution of propeptides presented by a scaffolding cargo is essential for the assembly of the Cvt vesicle for vacuolar delivery. This study describes a molecular framework for understanding the mechanism of Cvt or autophagosomal biogenesis in selective macroautophagy.
History
DepositionSep 2, 2014Deposition site: RCSB / Processing site: PDBJ
Revision 1.0Aug 12, 2015Provider: repository / Type: Initial release
Revision 1.1Nov 22, 2017Group: Refinement description / Category: software
Revision 1.2Aug 24, 2022Group: Database references
Category: citation / citation_author ...citation / citation_author / database_2 / struct_ref_seq_dif
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.title / _citation_author.name / _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_ref_seq_dif.details
Revision 1.3Nov 8, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Vacuolar aminopeptidase 1
B: Vacuolar aminopeptidase 1
C: Vacuolar aminopeptidase 1
D: Vacuolar aminopeptidase 1


Theoretical massNumber of molelcules
Total (without water)207,5464
Polymers207,5464
Non-polymers00
Water1,63991
1
A: Vacuolar aminopeptidase 1
B: Vacuolar aminopeptidase 1
C: Vacuolar aminopeptidase 1
D: Vacuolar aminopeptidase 1

A: Vacuolar aminopeptidase 1
B: Vacuolar aminopeptidase 1
C: Vacuolar aminopeptidase 1
D: Vacuolar aminopeptidase 1

A: Vacuolar aminopeptidase 1
B: Vacuolar aminopeptidase 1
C: Vacuolar aminopeptidase 1
D: Vacuolar aminopeptidase 1


Theoretical massNumber of molelcules
Total (without water)622,63812
Polymers622,63812
Non-polymers00
Water21612
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_445-y-1,x-y-1,z1
crystal symmetry operation3_545-x+y,-x-1,z1
Buried area71010 Å2
ΔGint-239 kcal/mol
Surface area160200 Å2
MethodPISA
Unit cell
Length a, b, c (Å)140.171, 140.171, 348.677
Angle α, β, γ (deg.)90.00, 90.00, 120.00
Int Tables number146
Space group name H-MH3

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Components

#1: Protein
Vacuolar aminopeptidase 1 / Aminopeptidase III / Aminopeptidase yscI / Leucine aminopeptidase IV / LAPIV / Polypeptidase / ...Aminopeptidase III / Aminopeptidase yscI / Leucine aminopeptidase IV / LAPIV / Polypeptidase / Vacuolar aminopeptidase I


Mass: 51886.516 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288c (yeast) / Gene: LAP4, APE1, API, YSC1, YKL103C, YKL455 / Plasmid: pET28a / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P14904, aminopeptidase I
#2: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 91 / Source method: isolated from a natural source / Formula: H2O

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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: 3.18 Å3/Da / Density % sol: 61.27 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 7.2
Details: 0.1M Tris-HCl pH7.2, 1.1M NaCl, 42.5% PEG 400, 0.1M MgCl2, VAPOR DIFFUSION, SITTING DROP, temperature 293.0K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: NSRRC / Beamline: BL15A / Wavelength: 1.2 Å
DetectorType: RAYONIX MX300HE / Detector: CCD / Date: Jun 19, 2014
RadiationMonochromator: LN2-Cooled, Fixed-Exit Double Crystal Monochromator
Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.2 Å / Relative weight: 1
ReflectionResolution: 2.5→116.23 Å / Num. all: 87912 / Num. obs: 84889 / % possible obs: 96.56 % / Observed criterion σ(F): 2 / Observed criterion σ(I): 0
Reflection shellResolution: 2.5→2.59 Å / Rmerge(I) obs: 0.554 / Mean I/σ(I) obs: 2.04 / % possible all: 98

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Processing

Software
NameVersionClassification
HKL-2000data collection
REFMAC5.8.0073refinement
HKL-2000data reduction
HKL-2000data scaling
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 4DYO

4dyo


Resolution: 2.5→116.23 Å / Cor.coef. Fo:Fc: 0.927 / Cor.coef. Fo:Fc free: 0.9 / SU B: 9.891 / SU ML: 0.216 / Cross valid method: THROUGHOUT / ESU R: 0.402 / ESU R Free: 0.261 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.24637 4222 5 %RANDOM
Rwork0.21532 ---
obs0.2169 80667 95.69 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso mean: 42.309 Å2
Baniso -1Baniso -2Baniso -3
1-0.09 Å20.05 Å2-0 Å2
2--0.09 Å2-0 Å2
3----0.3 Å2
Refinement stepCycle: LAST / Resolution: 2.5→116.23 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms13544 0 0 91 13635
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0050.01913839
X-RAY DIFFRACTIONr_bond_other_d0.0010.0213214
X-RAY DIFFRACTIONr_angle_refined_deg1.0921.95718723
X-RAY DIFFRACTIONr_angle_other_deg0.726330428
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.24551714
X-RAY DIFFRACTIONr_dihedral_angle_2_deg35.78524.295617
X-RAY DIFFRACTIONr_dihedral_angle_3_deg19.056152329
X-RAY DIFFRACTIONr_dihedral_angle_4_deg19.9961564
X-RAY DIFFRACTIONr_chiral_restr0.0630.22092
X-RAY DIFFRACTIONr_gen_planes_refined0.0040.02115566
X-RAY DIFFRACTIONr_gen_planes_other0.0010.023170
X-RAY DIFFRACTIONr_nbd_refined
X-RAY DIFFRACTIONr_nbd_other
X-RAY DIFFRACTIONr_nbtor_refined
X-RAY DIFFRACTIONr_nbtor_other
X-RAY DIFFRACTIONr_xyhbond_nbd_refined
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_metal_ion_refined
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined
X-RAY DIFFRACTIONr_symmetry_vdw_other
X-RAY DIFFRACTIONr_symmetry_hbond_refined
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_symmetry_metal_ion_refined
X-RAY DIFFRACTIONr_symmetry_metal_ion_other
X-RAY DIFFRACTIONr_mcbond_it1.2954.1656919
X-RAY DIFFRACTIONr_mcbond_other1.2954.1656918
X-RAY DIFFRACTIONr_mcangle_it2.3596.2288612
X-RAY DIFFRACTIONr_mcangle_other2.3596.2288613
X-RAY DIFFRACTIONr_scbond_it1.0184.3826920
X-RAY DIFFRACTIONr_scbond_other1.0184.3826921
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other1.8866.51510112
X-RAY DIFFRACTIONr_long_range_B_refined4.19933.00315203
X-RAY DIFFRACTIONr_long_range_B_other4.19933.00415204
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 2.497→2.562 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.315 189 -
Rwork0.285 4007 -
obs--63.93 %

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