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- PDB-8vjl: SpoIVFB:pro-sigmaK complex -

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Basic information

Entry
Database: PDB / ID: 8vjl
TitleSpoIVFB:pro-sigmaK complex
Components
  • RNA polymerase sigma factor
  • Stage IV sporulation protein FB
KeywordsMEMBRANE PROTEIN / spoIVFB / sigmaK / S2P / site-2-protease / protease
Function / homology
Function and homology information


Hydrolases; Acting on peptide bonds (peptidases); Metalloendopeptidases / sporulation resulting in formation of a cellular spore / sigma factor activity / DNA-templated transcription initiation / metallopeptidase activity / proteolysis / DNA binding / metal ion binding / membrane
Similarity search - Function
RNA polymerase sigma-K type / : / Peptidase M50 / Peptidase family M50 / Cro/C1-type HTH domain profile. / Cro/C1-type helix-turn-helix domain / Sigma-70 factors family signature 1. / Sigma-70 factors family signature 2. / RNA polymerase sigma-70 / RNA polymerase sigma-70 region 4 ...RNA polymerase sigma-K type / : / Peptidase M50 / Peptidase family M50 / Cro/C1-type HTH domain profile. / Cro/C1-type helix-turn-helix domain / Sigma-70 factors family signature 1. / Sigma-70 factors family signature 2. / RNA polymerase sigma-70 / RNA polymerase sigma-70 region 4 / Sigma-70, region 4 / RNA polymerase sigma-70 region 2 / RNA polymerase sigma-70 like domain / Sigma-70 region 2 / RNA polymerase sigma factor, region 2 / RNA polymerase sigma factor, region 3/4-like / Winged helix-like DNA-binding domain superfamily
Similarity search - Domain/homology
DI-PALMITOYL-3-SN-PHOSPHATIDYLETHANOLAMINE / RNA polymerase sigma factor / Stage IV sporulation protein FB
Similarity search - Component
Biological speciesBacillus subtilis subsp. subtilis str. 168 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsOrlando, M.A. / Pouillon, H.J.T. / Mandal, S. / Kroos, L. / Orlando, B.J.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM146721 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM043585 United States
CitationJournal: Nat Commun / Year: 2024
Title: Substrate engagement by the intramembrane metalloprotease SpoIVFB.
Authors: Melanie A Orlando / Hunter J T Pouillon / Saikat Mandal / Lee Kroos / Benjamin J Orlando /
Abstract: S2P intramembrane metalloproteases regulate diverse signaling pathways across all three domains of life. However, the mechanism by which S2P metalloproteases engage substrates and catalyze peptide ...S2P intramembrane metalloproteases regulate diverse signaling pathways across all three domains of life. However, the mechanism by which S2P metalloproteases engage substrates and catalyze peptide hydrolysis within lipid membranes has remained elusive. Here we determine the cryo-EM structure of the S2P family intramembrane metalloprotease SpoIVFB from Bacillus subtilis bound to its native substrate Pro-σ. The structure and accompanying biochemical data demonstrate that SpoIVFB positions Pro-σ at the enzyme active site through a β-sheet augmentation mechanism, and reveal key interactions between Pro-σ and the interdomain linker connecting SpoIVFB transmembrane and CBS domains. The cryo-EM structure and molecular dynamics simulation reveal a plausible path for water to access the membrane-buried active site of SpoIVFB, and suggest a possible role of membrane lipids in facilitating substrate capture. These results provide key insight into how S2P intramembrane metalloproteases capture and position substrates for hydrolytic proteolysis within the hydrophobic interior of a lipid membrane.
History
DepositionJan 7, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 2, 2024Provider: repository / Type: Initial release
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Revision 1.1Oct 30, 2024Group: Data collection / Database references / Structure summary
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Stage IV sporulation protein FB
B: RNA polymerase sigma factor
C: Stage IV sporulation protein FB
D: RNA polymerase sigma factor
E: Stage IV sporulation protein FB
F: RNA polymerase sigma factor
G: Stage IV sporulation protein FB
H: RNA polymerase sigma factor
hetero molecules


Theoretical massNumber of molelcules
Total (without water)213,72914
Polymers208,3248
Non-polymers5,4056
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein
Stage IV sporulation protein FB


Mass: 36991.512 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus subtilis subsp. subtilis str. 168 (bacteria)
Gene: spoIVFB, bofB, BSU27970 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: P26937, Hydrolases; Acting on peptide bonds (peptidases); Metalloendopeptidases
#2: Protein
RNA polymerase sigma factor


Mass: 15089.531 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus subtilis subsp. subtilis str. 168 (bacteria)
Gene: sigK / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A9E3K6
#3: Chemical
ChemComp-LMN / Lauryl Maltose Neopentyl Glycol / 2,2-didecylpropane-1,3-bis-b-D-maltopyranoside


Mass: 1005.188 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C47H88O22 / Comment: detergent*YM
#4: Chemical ChemComp-PEF / DI-PALMITOYL-3-SN-PHOSPHATIDYLETHANOLAMINE / 3-[AMINOETHYLPHOSPHORYL]-[1,2-DI-PALMITOYL]-SN-GLYCEROL


Mass: 691.959 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C37H74NO8P / Comment: phospholipid*YM
Has ligand of interestN
Has protein modificationN

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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: Complex of SpoIVFB and pro-sigmaK / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.052 MDa / Experimental value: NO
Source (natural)Organism: Bacillus subtilis subsp. subtilis str. 168 (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 8
Details: 25mM Tris, 150mM NaCl, 5mM betamercaptoethanol, 2mM MgCl2, 2mM ATP and 0.005% LMNG
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMsodium chlorideNaCl1
225 mMTris-baseC4H11NO31
35 mMbeta-mercaptoethanolC2H6OS1
42 mMmagnesium chlorideMgCl21
52 mMadenosine triphosphateC10H16N5O13P31
60.005 %lauryl maltose neopentyl glycyolC47H88O221
SpecimenConc.: 8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Detergent solubilized complex of spoIVFB and pro-sigmaK(1-127)
Specimen supportDetails: Glow discharge in a Pelco EasyGlow at 15mA for 45s / Grid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 700 nm / Cs: 2.7 mm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 60.5 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
2Leginon3.5image acquisition
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 36371 / Symmetry type: POINT
Atomic model buildingSpace: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00313684
ELECTRON MICROSCOPYf_angle_d0.55318526
ELECTRON MICROSCOPYf_dihedral_angle_d9.491924
ELECTRON MICROSCOPYf_chiral_restr0.0382142
ELECTRON MICROSCOPYf_plane_restr0.0042224

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