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- PDB-4oej: Structure of membrane binding protein pleurotolysin B from Pleuro... -

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Basic information

Entry
Database: PDB / ID: 4oej
TitleStructure of membrane binding protein pleurotolysin B from Pleurotus ostreatus
ComponentsPleurotolysin B
KeywordsMEMBRANE BINDING PROTEIN / MACPF domain / Pore formation / pleurotolysin A
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 / ACETATE ION / Pleurotolysin B
Function and homology information
Biological speciesPleurotus ostreatus (oyster mushroom)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MIRAS / Resolution: 2.2 Å
AuthorsDunstone, M.A. / Caradoc-Davies, T.T. / Whisstock, J.C. / Law, R.H.P.
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
DepositionJan 13, 2014Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 18, 2015Provider: repository / Type: Initial release
Revision 1.1Feb 28, 2024Group: Data collection / Database references / Derived calculations
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _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

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Assembly

Deposited unit
A: Pleurotolysin B
hetero molecules


Theoretical massNumber of molelcules
Total (without water)57,80110
Polymers57,1611
Non-polymers6409
Water2,882160
1
A: Pleurotolysin B
hetero molecules

A: Pleurotolysin B
hetero molecules


Theoretical massNumber of molelcules
Total (without water)115,60220
Polymers114,3222
Non-polymers1,28018
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation4_555y,x,-z1
Buried area7880 Å2
ΔGint-32 kcal/mol
Surface area37630 Å2
MethodPISA
Unit cell
Length a, b, c (Å)71.580, 71.580, 174.920
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: 57160.996 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pleurotus ostreatus (oyster mushroom) / Gene: plyB / Production host: Escherichia coli (E. coli) / References: UniProt: Q5W9E8
#2: Chemical
ChemComp-ACT / ACETATE ION


Mass: 59.044 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C2H3O2
#3: Chemical
ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C3H8O3
#4: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Cl
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 160 / 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: 6

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Sample preparation

CrystalDensity Matthews: 2.26 Å3/Da / Density % sol: 45.65 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 5.6
Details: 0.1 M trisodium citrate, pH 5.6, 20% w/v PEG4000, 0.2 M ammonium acetate, VAPOR DIFFUSION, HANGING DROP, temperature 293K

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Data collection

Diffraction
IDMean temperature (K)Crystal-ID
11001
21
Diffraction source
SourceSiteBeamlineIDWavelength (Å)
SYNCHROTRONAustralian Synchrotron MX110.9797
SYNCHROTRONAustralian Synchrotron MX120.95665
Detector
TypeIDDetectorDate
ADSC QUANTUM 210r1CCDOct 9, 2008
ADSC QUANTUM 210r2CCDNov 18, 2008
Radiation
IDMonochromatorProtocolMonochromatic (M) / Laue (L)Scattering typeWavelength-ID
1double crystal Si(111)SINGLE WAVELENGTHMx-ray1
2double crystal Si(111)SINGLE WAVELENGTHMx-ray1
Radiation wavelength
IDWavelength (Å)Relative weight
10.97971
20.956651
ReflectionResolution: 2.2→87.37 Å / Num. obs: 27236 / % possible obs: 100 % / Observed criterion σ(F): 2.2 / Observed criterion σ(I): 2.2 / Redundancy: 10.2 % / Biso Wilson estimate: 43.77 Å2 / Rmerge(I) obs: 0.077 / Net I/σ(I): 18.8
Reflection shell
Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. unique allDiffraction-ID% possible all
2.2-2.329.90.6693.939051,2100
6.96-87.379.20.03643.19741,299.2

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Processing

Software
NameVersionClassification
ADSCQuantumdata collection
SHARPphasing
BUSTER2.10.0refinement
MOSFLMdata reduction
SCALAdata scaling
RefinementMethod to determine structure: MIRAS / Resolution: 2.2→26.56 Å / Cor.coef. Fo:Fc: 0.9473 / Cor.coef. Fo:Fc free: 0.9304 / SU R Cruickshank DPI: 0.226 / Cross valid method: THROUGHOUT / σ(F): 0 / SU R Blow DPI: 0.233 / SU Rfree Blow DPI: 0.18 / SU Rfree Cruickshank DPI: 0.179
RfactorNum. reflection% reflectionSelection details
Rfree0.2213 1361 5.01 %RANDOM
Rwork0.1892 ---
obs0.1908 27161 99.95 %-
Displacement parametersBiso mean: 59.54 Å2
Baniso -1Baniso -2Baniso -3
1--0.8787 Å20 Å20 Å2
2---0.8787 Å20 Å2
3---1.7574 Å2
Refine analyzeLuzzati coordinate error obs: 0.396 Å
Refinement stepCycle: LAST / Resolution: 2.2→26.56 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3475 0 41 160 3676
Refine LS restraints
Refine-IDTypeNumberRestraint functionWeightDev ideal
X-RAY DIFFRACTIONt_dihedral_angle_d1180SINUSOIDAL2
X-RAY DIFFRACTIONt_trig_c_planes74HARMONIC2
X-RAY DIFFRACTIONt_gen_planes532HARMONIC5
X-RAY DIFFRACTIONt_it3586HARMONIC20
X-RAY DIFFRACTIONt_nbd
X-RAY DIFFRACTIONt_improper_torsion
X-RAY DIFFRACTIONt_pseud_angle
X-RAY DIFFRACTIONt_chiral_improper_torsion482SEMIHARMONIC5
X-RAY DIFFRACTIONt_sum_occupancies
X-RAY DIFFRACTIONt_utility_distance
X-RAY DIFFRACTIONt_utility_angle
X-RAY DIFFRACTIONt_utility_torsion
X-RAY DIFFRACTIONt_ideal_dist_contact4093SEMIHARMONIC4
X-RAY DIFFRACTIONt_bond_d3586HARMONIC20.008
X-RAY DIFFRACTIONt_angle_deg4876HARMONIC21.07
X-RAY DIFFRACTIONt_omega_torsion3
X-RAY DIFFRACTIONt_other_torsion17.4
LS refinement shellResolution: 2.2→2.28 Å / Total num. of bins used: 14
RfactorNum. reflection% reflection
Rfree0.2178 117 4.17 %
Rwork0.2016 2690 -
all0.2023 2807 -
obs--99.95 %
Refinement TLS params.Method: refined / Origin x: 14.4198 Å / Origin y: 22.2295 Å / Origin z: 13.7641 Å
111213212223313233
T-0.1934 Å20.0286 Å2-0.0029 Å2--0.2021 Å20.0089 Å2---0.1588 Å2
L0.8232 °2-0.3779 °21.013 °2-1.7325 °20.0881 °2--4.8932 °2
S-0.0984 Å °-0.0926 Å °0.0117 Å °0.0992 Å °0.0984 Å °-0.1017 Å °-0.0164 Å °-0.1146 Å °-0.0001 Å °
Refinement TLS groupSelection details: { A|* }

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