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- PDB-4ov8: Crystal Structure of the TMH1-lock mutant of the mature form of p... -

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Basic information

Entry
Database: PDB / ID: 4ov8
TitleCrystal Structure of the TMH1-lock mutant of the mature form of pleurotolysin B
ComponentsPleurotolysin B
KeywordsTOXIN / TMH1-lock / MACPF domain / pore-forming protein / Pleurtolysin A component
Function / homologyPleurotolysin B, C-terminal / Pleurotolysin B C-terminal domain / Membrane attack complex/perforin (MACPF) domain profile. / MAC/Perforin domain / Membrane attack complex component/perforin (MACPF) domain / Pleurotolysin B
Function and homology information
Biological speciesPleurotus ostreatus (oyster mushroom)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.15 Å
AuthorsKondos, S.C. / Law, R.H.P. / Whisstock, J.C. / Dunstone, M.A.
CitationJournal: PLoS Biol / Year: 2015
Title: Conformational changes during pore formation by the perforin-related protein pleurotolysin.
Authors: Natalya Lukoyanova / Stephanie C Kondos / Irene Farabella / Ruby H P Law / Cyril F Reboul / Tom T Caradoc-Davies / Bradley A Spicer / Oded Kleifeld / Daouda A K Traore / Susan M Ekkel / Ilia ...Authors: Natalya Lukoyanova / Stephanie C Kondos / Irene Farabella / Ruby H P Law / Cyril F Reboul / Tom T Caradoc-Davies / Bradley A Spicer / Oded Kleifeld / Daouda A K Traore / Susan M Ekkel / Ilia Voskoboinik / Joseph A Trapani / Tamas Hatfaludi / Katherine Oliver / Eileen M Hotze / Rodney K Tweten / James C Whisstock / Maya Topf / Helen R Saibil / Michelle A Dunstone /
Abstract: Membrane attack complex/perforin-like (MACPF) proteins comprise the largest superfamily of pore-forming proteins, playing crucial roles in immunity and pathogenesis. Soluble monomers assemble into ...Membrane attack complex/perforin-like (MACPF) proteins comprise the largest superfamily of pore-forming proteins, playing crucial roles in immunity and pathogenesis. Soluble monomers assemble into large transmembrane pores via conformational transitions that remain to be structurally and mechanistically characterised. Here we present an 11 Å resolution cryo-electron microscopy (cryo-EM) structure of the two-part, fungal toxin Pleurotolysin (Ply), together with crystal structures of both components (the lipid binding PlyA protein and the pore-forming MACPF component PlyB). These data reveal a 13-fold pore 80 Å in diameter and 100 Å in height, with each subunit comprised of a PlyB molecule atop a membrane bound dimer of PlyA. The resolution of the EM map, together with biophysical and computational experiments, allowed confident assignment of subdomains in a MACPF pore assembly. The major conformational changes in PlyB are a ∼70° opening of the bent and distorted central β-sheet of the MACPF domain, accompanied by extrusion and refolding of two α-helical regions into transmembrane β-hairpins (TMH1 and TMH2). We determined the structures of three different disulphide bond-trapped prepore intermediates. Analysis of these data by molecular modelling and flexible fitting allows us to generate a potential trajectory of β-sheet unbending. The results suggest that MACPF conformational change is triggered through disruption of the interface between a conserved helix-turn-helix motif and the top of TMH2. Following their release we propose that the transmembrane regions assemble into β-hairpins via top down zippering of backbone hydrogen bonds to form the membrane-inserted β-barrel. The intermediate structures of the MACPF domain during refolding into the β-barrel pore establish a structural paradigm for the transition from soluble monomer to pore, which may be conserved across the whole superfamily. The TMH2 region is critical for the release of both TMH clusters, suggesting why this region is targeted by endogenous inhibitors of MACPF function.
History
DepositionFeb 20, 2014Deposition site: RCSB / Processing site: PDBJ
Revision 1.0Feb 18, 2015Provider: repository / Type: Initial release
Revision 1.1Nov 8, 2023Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / struct_ref_seq_dif / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_ref_seq_dif.details / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Pleurotolysin B
hetero molecules


Theoretical massNumber of molelcules
Total (without water)52,65610
Polymers52,1591
Non-polymers4979
Water3,135174
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)71.181, 71.181, 174.675
Angle α, β, γ (deg.)90.00, 90.00, 120.00
Int Tables number152
Space group name H-MP3121

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Components

#1: Protein Pleurotolysin B


Mass: 52159.473 Da / Num. of mol.: 1 / Fragment: TMH1-lock, UNP residues 49-523 / Mutation: Y166C, G266C, C487A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pleurotus ostreatus (oyster mushroom) / Gene: mPlyB, plyB / Plasmid: pET3a / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3) pLysS codon plus / References: UniProt: Q5W9E8
#2: Chemical ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: SO4
#3: Chemical
ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Cl
#4: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C3H8O3
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 174 / 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: 2.45 Å3/Da / Density % sol: 49.78 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 5.5
Details: 25% PEG 8000, 0.1M sodium cacodylate, 0.2M ammonium sullfate, pH 5.5, VAPOR DIFFUSION, HANGING DROP, temperature 293K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: Australian Synchrotron / Beamline: MX2 / Wavelength: 0.98 Å
DetectorType: ADSC QUANTUM 315r / Detector: CCD / Date: Feb 18, 2012
RadiationMonochromator: Silicon Double Crystal / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.98 Å / Relative weight: 1
ReflectionResolution: 2.15→174.68 Å / Num. all: 28792 / Num. obs: 28792 / % possible obs: 100 % / Observed criterion σ(F): 1.2 / Observed criterion σ(I): 1 / Rmerge(I) obs: 0.091
Reflection shellResolution: 2.15→2.27 Å

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Processing

Software
NameVersionClassification
Blu-IceGUIdata collection
PHENIX(phenix.phaser)model building
PHENIX(phenix.refine: 1.8.1_1168)refinement
XDSdata reduction
SCALAdata scaling
PHENIXphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 4OEJ

4oej


Resolution: 2.15→61.644 Å / SU ML: 0.25 / σ(F): 1.34 / Phase error: 25.13 / Stereochemistry target values: ML
RfactorNum. reflection% reflectionSelection details
Rfree0.2426 1456 5.07 %random
Rwork0.2072 ---
all0.2091 28729 --
obs0.2091 28729 99.97 %-
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 2.15→61.644 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3530 0 22 174 3726
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0043621
X-RAY DIFFRACTIONf_angle_d0.8214928
X-RAY DIFFRACTIONf_dihedral_angle_d12.2191322
X-RAY DIFFRACTIONf_chiral_restr0.052562
X-RAY DIFFRACTIONf_plane_restr0.004640
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Total num. of bins used: 10 / % reflection obs: 100 %

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection Rwork
2.1501-2.22690.33781380.27082689
2.2269-2.31610.29441370.28142683
2.3161-2.42150.2991430.24232683
2.4215-2.54910.29781600.24072679
2.5491-2.70890.29271400.23742673
2.7089-2.9180.27041470.22122707
2.918-3.21160.29731240.21352746
3.2116-3.67630.22091670.18812709
3.6763-4.63160.19981650.1672757
4.6316-61.66970.20571350.20052947
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
11.08640.67670.36153.0611.60590.8378-0.6064-0.04360.27370.3206-0.0317-0.257-1.21540.49180.3610.8358-0.2775-0.46940.4020.03140.605323.82541.835320.0438
22.44170.33870.8125.5103-0.15373.2335-0.42620.16840.18950.02070.1354-0.0495-0.6524-0.2390.29970.33440.0549-0.13260.2654-0.06880.28269.624730.56813.2207
31.4877-0.18391.54123.14040.35044.1416-0.283-0.1260.22280.17980.1297-0.3966-0.19680.17090.13020.24460.0159-0.03260.1935-0.0090.245515.427924.488414.9739
42.6115-0.87950.78252.08691.36873.59960.1214-0.2718-0.1902-0.060.0590.06831.3337-0.345-0.17150.6114-0.1047-0.05010.21820.02160.291912.22522.846711.3658
52.3545-1.60431.47592.24150.58882.94890.2221-0.4972-0.1803-0.075-0.24180.06291.1485-0.2788-0.03480.6079-0.11470.00430.314-0.00370.302613.77174.34148.6634
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection details
1X-RAY DIFFRACTION1(chain A and resid 63:100)
2X-RAY DIFFRACTION2(chain A and resid 101:181)
3X-RAY DIFFRACTION3(chain A and resid 184:410)
4X-RAY DIFFRACTION4(chain A and resid 411:487)
5X-RAY DIFFRACTION5(chain A and resid 488:519)

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