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- PDB-6xos: CryoEM structure of human presequence protease in partial open state 1 -

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Basic information

Entry
Database: PDB / ID: 6xos
TitleCryoEM structure of human presequence protease in partial open state 1
ComponentsPresequence protease, mitochondrial
KeywordsHYDROLASE / Partial open state
Function / homology
Function and homology information


Mitochondrial protein import / Hydrolases; Acting on peptide bonds (peptidases); Metalloendopeptidases / protein targeting to mitochondrion / enzyme activator activity / protein processing / metalloendopeptidase activity / metallopeptidase activity / mitochondrial matrix / mitochondrion / proteolysis / zinc ion binding
Similarity search - Function
: / Peptidase M16C associated / Peptidase M16C associated / PreP C-terminal domain / Peptidase M16C associated / Cytochrome Bc1 Complex; Chain A, domain 1 / Metalloenzyme, LuxS/M16 peptidase-like / Peptidase M16, C-terminal / Peptidase M16 inactive domain / Peptidase M16, N-terminal ...: / Peptidase M16C associated / Peptidase M16C associated / PreP C-terminal domain / Peptidase M16C associated / Cytochrome Bc1 Complex; Chain A, domain 1 / Metalloenzyme, LuxS/M16 peptidase-like / Peptidase M16, C-terminal / Peptidase M16 inactive domain / Peptidase M16, N-terminal / Insulinase (Peptidase family M16) / Metalloenzyme, LuxS/M16 peptidase-like / 2-Layer Sandwich / Alpha Beta
Similarity search - Domain/homology
Presequence protease, mitochondrial
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsLiang, W.G. / Zhao, M. / Tang, W.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute on Aging (NIH/NIA)R01 GM121964 United States
CitationJournal: Nat Commun / Year: 2022
Title: Structural basis for the mechanisms of human presequence protease conformational switch and substrate recognition.
Authors: Wenguang G Liang / Juwina Wijaya / Hui Wei / Alex J Noble / Jordan M Mancl / Swansea Mo / David Lee / John V Lin King / Man Pan / Chang Liu / Carla M Koehler / Minglei Zhao / Clinton S ...Authors: Wenguang G Liang / Juwina Wijaya / Hui Wei / Alex J Noble / Jordan M Mancl / Swansea Mo / David Lee / John V Lin King / Man Pan / Chang Liu / Carla M Koehler / Minglei Zhao / Clinton S Potter / Bridget Carragher / Sheng Li / Wei-Jen Tang /
Abstract: Presequence protease (PreP), a 117 kDa mitochondrial M16C metalloprotease vital for mitochondrial proteostasis, degrades presequence peptides cleaved off from nuclear-encoded proteins and other ...Presequence protease (PreP), a 117 kDa mitochondrial M16C metalloprotease vital for mitochondrial proteostasis, degrades presequence peptides cleaved off from nuclear-encoded proteins and other aggregation-prone peptides, such as amyloid β (Aβ). PreP structures have only been determined in a closed conformation; thus, the mechanisms of substrate binding and selectivity remain elusive. Here, we leverage advanced vitrification techniques to overcome the preferential denaturation of one of two ~55 kDa homologous domains of PreP caused by air-water interface adsorption. Thereby, we elucidate cryoEM structures of three apo-PreP open states along with Aβ- and citrate synthase presequence-bound PreP at 3.3-4.6 Å resolution. Together with integrative biophysical and pharmacological approaches, these structures reveal the key stages of the PreP catalytic cycle and how the binding of substrates or PreP inhibitor drives a rigid body motion of the protein for substrate binding and catalysis. Together, our studies provide key mechanistic insights into M16C metalloproteases for future therapeutic innovations.
History
DepositionJul 7, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 7, 2021Provider: repository / Type: Initial release
Revision 1.1Apr 13, 2022Group: Database references / Category: citation / citation_author / database_2
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _database_2.pdbx_DOI / _database_2.pdbx_database_accession
Revision 1.2Apr 20, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.title / _citation_author.name
Revision 1.3May 15, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Assembly

Deposited unit
A: Presequence protease, mitochondrial


Theoretical massNumber of molelcules
Total (without water)114,9011
Polymers114,9011
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: SAXS
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area0 Å2
ΔGint0 kcal/mol
Surface area40430 Å2

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Components

#1: Protein Presequence protease, mitochondrial / hPreP / Pitrilysin metalloproteinase 1 / hMP1


Mass: 114901.461 Da / Num. of mol.: 1 / Fragment: UNP residues 33-1037
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PITRM1, KIAA1104, MP1, PREP
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: Q5JRX3, Hydrolases; Acting on peptide bonds (peptidases); Metalloendopeptidases

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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: CryoEM map of Human Presequence Protease in partial open state 1
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Buffer solutionpH: 7.7
Details: 20 mM Tris, pH 7.7, 150 mM NaCl, 10mM KCl, 20 mM EDTA and 1 mM 2-mercaptoethanol
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: unspecified
VitrificationInstrument: SPOTITON / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 308 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 defocus max: 2500 nm / Nominal defocus min: 1500 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm
Image recordingAverage exposure time: 6 sec. / Electron dose: 62 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.17_3644: / Classification: refinement
EM software
IDNameCategory
1RELIONparticle selection
4CTFFINDCTF correction
10RELIONinitial Euler assignment
11RELIONfinal Euler assignment
12RELIONclassification
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 130572 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0097896
ELECTRON MICROSCOPYf_angle_d1.0810708
ELECTRON MICROSCOPYf_dihedral_angle_d7.0591037
ELECTRON MICROSCOPYf_chiral_restr0.0651174
ELECTRON MICROSCOPYf_plane_restr0.0071398

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