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- PDB-6z05: Campylobacter jejuni serine protease HtrA -

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Basic information

Entry
Database: PDB / ID: 6z05
TitleCampylobacter jejuni serine protease HtrA
ComponentsDegQ family serine endoprotease
KeywordsLYASE / Campylobacter jejuni / serine protease / HtrA / dodecamer
Function / homology
Function and homology information


peptidase Do / periplasmic space / serine-type endopeptidase activity
Similarity search - Function
Peptidase S1C, Do / PDZ domain / Peptidase S1C / Domain present in PSD-95, Dlg, and ZO-1/2. / PDZ domain / PDZ superfamily / Peptidase S1, PA clan
Similarity search - Domain/homology
Periplasmic serine endoprotease DegP-like
Similarity search - Component
Biological speciesCampylobacter jejuni (Campylobacter)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 5.8 Å
AuthorsGrinzato, A. / Kandiah, E. / Zanotti, G.
CitationJournal: Gut Microbes / Year: 2020
Title: Functional analysis and cryo-electron microscopy of serine protease HtrA.
Authors: Urszula Zarzecka / Alessandro Grinzato / Eaazhisai Kandiah / Dominik Cysewski / Paola Berto / Joanna Skorko-Glonek / Giuseppe Zanotti / Steffen Backert /
Abstract: is a predominant zoonotic pathogen causing gastroenteritis and other diseases in humans. An important bacterial virulence factor is the secreted serine protease HtrA (HtrA ), which targets tight ... is a predominant zoonotic pathogen causing gastroenteritis and other diseases in humans. An important bacterial virulence factor is the secreted serine protease HtrA (HtrA ), which targets tight and adherens junctional proteins in the gut epithelium. Here we have investigated the function and structure of HtrA using biochemical assays and cryo-electron microscopy. Mass spectrometry analysis identified differences and similarities in the cleavage site specificity for HtrA by comparison to the HtrA counterparts from and . We defined the architecture of HtrA at 5.8 Å resolution as a dodecamer, built of four trimers. The contacts between the trimers are quite loose, a fact that explains the flexibility and mobility of the dodecameric assembly. This flexibility has also been studied through molecular dynamics simulation, which revealed opening of the dodecamer to expose the proteolytically active site of the protease. Moreover, we examined the rearrangements at the level of oligomerization in the presence or absence of substrate using size exclusion chromatography, which revealed hexamers, dodecamers and larger oligomeric forms, as well as remarkable stability of higher oligomeric forms (> 12-mers) compared to previously tested homologs from other bacteria. Extremely dynamic decay of the higher oligomeric forms into lower forms was observed after full cleavage of the substrate by the proteolytically active variant of HtrA . Together, this is the first report on the in-depth functional and structural analysis of HtrA , which may allow the construction of therapeutically relevant HtrA inhibitors in the near future.
History
DepositionMay 7, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 30, 2020Provider: repository / Type: Initial release

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Structure visualization

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Assembly

Deposited unit
A: DegQ family serine endoprotease
B: DegQ family serine endoprotease
C: DegQ family serine endoprotease
D: DegQ family serine endoprotease
E: DegQ family serine endoprotease
F: DegQ family serine endoprotease
G: DegQ family serine endoprotease
H: DegQ family serine endoprotease
I: DegQ family serine endoprotease
J: DegQ family serine endoprotease
K: DegQ family serine endoprotease
L: DegQ family serine endoprotease


Theoretical massNumber of molelcules
Total (without water)566,18912
Polymers566,18912
Non-polymers00
Water0
1


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

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Components

#1: Protein
DegQ family serine endoprotease / Serine protease HtrA


Mass: 47182.379 Da / Num. of mol.: 12
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Campylobacter jejuni (Campylobacter)
Gene: htrA, AY595_01940, B7T14_05270, BB943_00930, BB980_02965, BOI95_01210, CA962_01090, CKH46_05385, CW563_00090, DK813_04915, DQX79_02990, DUH84_04555, DW530_01050, F0N90_05545, F1O60_00260, F3L27_ ...Gene: htrA, AY595_01940, B7T14_05270, BB943_00930, BB980_02965, BOI95_01210, CA962_01090, CKH46_05385, CW563_00090, DK813_04915, DQX79_02990, DUH84_04555, DW530_01050, F0N90_05545, F1O60_00260, F3L27_00375, F6319_03730, F7858_01545, FR416_00510, FRM19_03410, FRQ83_01860, FZ422_01615, FZU65_03575
Production host: Escherichia coli (E. coli) / References: UniProt: A0A620LS18

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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: HtrA dodecamer / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Campylobacter jejuni (Campylobacter)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 44 e/Å2 / Film or detector model: FEI FALCON III (4k x 4k)

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Processing

CTF correctionType: NONE
SymmetryPoint symmetry: T (tetrahedral)
3D reconstructionResolution: 5.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 113843 / Symmetry type: POINT

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