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- EMDB-18389: P.aeruginosa clone C construct PaFtsH2-H1-link32 in negative stain -

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Basic information

Entry
Database: EMDB / ID: EMD-18389
TitleP.aeruginosa clone C construct PaFtsH2-H1-link32 in negative stain
Map dataConstruct PaFtsH2H1-link-32
Sample
  • Organelle or cellular component: Construct PFtsH2-H1-link32
Keywordsprotease / MEMBRANE PROTEIN
Biological speciesPseudomonas aeruginosa SG17M (bacteria)
Methodsingle particle reconstruction / negative staining / Resolution: 22.8 Å
AuthorsMawla GD / Mansour Kamal S / Cao L-Y / Purhonen P / Hebert H / Sauer RT / Baker TA / Romling U
Funding support Sweden, 1 items
OrganizationGrant numberCountry
Swedish Research Council Sweden
CitationJournal: J Biol Chem / Year: 2024
Title: The membrane-cytoplasmic linker defines activity of FtsH proteases in Pseudomonas aeruginosa clone C.
Authors: Gina D Mawla / Shady M Kamal / Lian-Ying Cao / Pasi Purhonen / Hans Hebert / Robert T Sauer / Tania A Baker / Ute Römling /
Abstract: Pandemic Pseudomonas aeruginosa clone C strains encode two inner-membrane associated ATP-dependent FtsH proteases. PaftsH1 is located on the core genome and supports cell growth and intrinsic ...Pandemic Pseudomonas aeruginosa clone C strains encode two inner-membrane associated ATP-dependent FtsH proteases. PaftsH1 is located on the core genome and supports cell growth and intrinsic antibiotic resistance, whereas PaftsH2, a xenolog acquired through horizontal gene transfer from a distantly related species, is unable to functionally replace PaftsH1. We show that purified PaFtsH2 degrades fewer substrates than PaFtsH1. Replacing the 31-amino acid-extended linker region of PaFtsH2 spanning from the C-terminal end of the transmembrane helix-2 to the first seven highly divergent residues of the cytosolic AAA+ ATPase module with the corresponding region of PaFtsH1 improves hybrid-enzyme substrate processing in vitro and enables PaFtsH2 to substitute for PaFtsH1 in vivo. Electron microscopy indicates that the identity of this linker sequence influences FtsH flexibility. We find membrane-cytoplasmic (MC) linker regions of PaFtsH1 characteristically glycine-rich compared to those from FtsH2. Consequently, introducing three glycines into the membrane-proximal end of PaFtsH2's MC linker is sufficient to elevate its activity in vitro and in vivo. Our findings establish that the efficiency of substrate processing by the two PaFtsH isoforms depends on MC linker identity and suggest that greater linker flexibility and/or length allows FtsH to degrade a wider spectrum of substrates. As PaFtsH2 homologs occur across bacterial phyla, we hypothesize that FtsH2 is a latent enzyme but may recognize specific substrates or is activated in specific contexts or biological niches. The identity of such linkers might thus play a more determinative role in the functionality of and physiological impact by FtsH proteases than previously thought.
History
DepositionSep 5, 2023-
Header (metadata) releaseJan 17, 2024-
Map releaseJan 17, 2024-
UpdateFeb 14, 2024-
Current statusFeb 14, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_18389.png

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Map

FileDownload / File: emd_18389.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationConstruct PaFtsH2H1-link-32
Voxel sizeX=Y=Z: 2.19 Å
Density
Contour LevelBy AUTHOR: 0.9
Minimum - Maximum-0.107883416 - 2.6597548
Average (Standard dev.)0.07747284 (±0.28375638)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-64-64-64
Dimensions128128128
Spacing128128128
CellA=B=C: 280.32 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_18389_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_18389_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Construct PFtsH2-H1-link32

EntireName: Construct PFtsH2-H1-link32
Components
  • Organelle or cellular component: Construct PFtsH2-H1-link32

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Supramolecule #1: Construct PFtsH2-H1-link32

SupramoleculeName: Construct PFtsH2-H1-link32 / type: organelle_or_cellular_component / ID: 1 / Parent: 0
Details: A construct containing 32-residue composed of the membrane-cytoplasmic linker (25 amino acids long) and first seven resides of the AAA+ ATPase module from PaFtsH1 with all other enzyme ...Details: A construct containing 32-residue composed of the membrane-cytoplasmic linker (25 amino acids long) and first seven resides of the AAA+ ATPase module from PaFtsH1 with all other enzyme sequences derived from PaFtsH2
Source (natural)Organism: Pseudomonas aeruginosa SG17M (bacteria)

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 8
Details: 50mM Tris-HCl,10mM KCl,5mM MgSO4,10uM ZnCl2,60mM imidazole,10% glycerol,0.1% Igepal,2mM 2-mercaptoethanol
StainingType: NEGATIVE / Material: uranyl acetate

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Electron microscopy

MicroscopeJEOL 2100F
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.7000000000000001 µm
Sample stageSpecimen holder model: JEOL
Image recordingFilm or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Average electron dose: 20.0 e/Å2

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Image processing

Startup modelType of model: OTHER
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: EMAN2
Final angle assignmentType: PROJECTION MATCHING / Software - Name: EMAN2
Final reconstructionApplied symmetry - Point group: C6 (6 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 22.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: EMAN2 / Number images used: 3510
FSC plot (resolution estimation)

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