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- PDB-4oeb: Structure of membrane binding protein pleurotolysin A from Pleuro... -

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Basic information

Entry
Database: PDB / ID: 4oeb
TitleStructure of membrane binding protein pleurotolysin A from Pleurotus ostreatus
ComponentsPleurotolysin A
KeywordsMEMBRANE BINDING PROTEIN / beta-sandwich fold / pleurotolysin B
Function / homologyMutm (Fpg) Protein; Chain: A, domain 2 - #50 / Hemolysin, aegerolysin type / Aegerolysin / Mutm (Fpg) Protein; Chain: A, domain 2 / hemolysis by symbiont of host erythrocytes / Sandwich / Mainly Beta / Pleurotolysin A
Function and homology information
Biological speciesPleurotus ostreatus (oyster mushroom)
MethodX-RAY DIFFRACTION / SYNCHROTRON / SAD / Resolution: 1.85 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 12, 2014Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 18, 2015Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Pleurotolysin A
B: Pleurotolysin A
C: Pleurotolysin A
D: Pleurotolysin A
hetero molecules


Theoretical massNumber of molelcules
Total (without water)61,3798
Polymers60,9954
Non-polymers3844
Water8,971498
1
A: Pleurotolysin A
hetero molecules


Theoretical massNumber of molelcules
Total (without water)15,3452
Polymers15,2491
Non-polymers961
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
B: Pleurotolysin A
hetero molecules


Theoretical massNumber of molelcules
Total (without water)15,3452
Polymers15,2491
Non-polymers961
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
3
C: Pleurotolysin A
hetero molecules


Theoretical massNumber of molelcules
Total (without water)15,3452
Polymers15,2491
Non-polymers961
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
4
D: Pleurotolysin A
hetero molecules


Theoretical massNumber of molelcules
Total (without water)15,3452
Polymers15,2491
Non-polymers961
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)131.700, 131.700, 65.213
Angle α, β, γ (deg.)90.00, 90.00, 120.00
Int Tables number171
Space group name H-MP62

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Components

#1: Protein
Pleurotolysin A / PriA


Mass: 15248.686 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pleurotus ostreatus (oyster mushroom) / Gene: PlyA, plyA / Production host: Escherichia coli (E. coli) / References: UniProt: Q8X1M9
#2: Chemical
ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: SO4
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 498 / 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: 2.68 Å3/Da / Density % sol: 54.05 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 6
Details: 20 mM sodium cacodylate, pH 6.0, 14% PEG3350, 0.2 M magnesium sulfate, VAPOR DIFFUSION, HANGING DROP, temperature 293K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: Australian Synchrotron / Beamline: MX1 / Wavelength: 0.95364 Å
DetectorType: ADSC QUANTUM 210r / Detector: CCD / Date: Apr 16, 2008
RadiationMonochromator: double crystal Si(111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.95364 Å / Relative weight: 1
ReflectionResolution: 1.85→43.109 Å / Num. all: 54873 / Num. obs: 54873 / % possible obs: 100 % / Observed criterion σ(F): 2 / Observed criterion σ(I): 2 / Redundancy: 88.1 % / Biso Wilson estimate: 27.25 Å2 / Rmerge(I) obs: 0.113 / Net I/σ(I): 49.9
Reflection shellResolution: 1.85→2.11 Å / Redundancy: 87.5 % / Rmerge(I) obs: 0.561 / Mean I/σ(I) obs: 11.5 / Num. unique all: 6421 / % possible all: 100

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Processing

Software
NameVersionClassification
ADSCQuantumdata collection
SHARPphasing
BUSTER2.10.0refinement
MOSFLMdata reduction
SCALAdata scaling
RefinementMethod to determine structure: SAD / Resolution: 1.85→43.109 Å / Cor.coef. Fo:Fc: 0.9396 / Cor.coef. Fo:Fc free: 0.9291 / SU R Cruickshank DPI: 0.115 / Cross valid method: THROUGHOUT / σ(F): 0 / SU R Blow DPI: 0.119 / SU Rfree Blow DPI: 0.109 / SU Rfree Cruickshank DPI: 0.107
RfactorNum. reflection% reflectionSelection details
Rfree0.1982 2783 5.07 %RANDOM
Rwork0.1724 ---
obs0.1737 54873 99.47 %-
Displacement parametersBiso mean: 35.26 Å2
Baniso -1Baniso -2Baniso -3
1--5.7899 Å20 Å20 Å2
2---5.7899 Å20 Å2
3---11.5798 Å2
Refine analyzeLuzzati coordinate error obs: 0.259 Å
Refinement stepCycle: LAST / Resolution: 1.85→43.109 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4166 0 20 498 4684
Refine LS restraints
Refine-IDTypeNumberRestraint functionWeightDev ideal
X-RAY DIFFRACTIONt_dihedral_angle_d1438SINUSOIDAL2
X-RAY DIFFRACTIONt_trig_c_planes112HARMONIC2
X-RAY DIFFRACTIONt_gen_planes604HARMONIC5
X-RAY DIFFRACTIONt_it4278HARMONIC20
X-RAY DIFFRACTIONt_nbd
X-RAY DIFFRACTIONt_improper_torsion
X-RAY DIFFRACTIONt_pseud_angle
X-RAY DIFFRACTIONt_chiral_improper_torsion572SEMIHARMONIC5
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_contact5374SEMIHARMONIC4
X-RAY DIFFRACTIONt_bond_d4278HARMONIC20.01
X-RAY DIFFRACTIONt_angle_deg5814HARMONIC21.15
X-RAY DIFFRACTIONt_omega_torsion3.77
X-RAY DIFFRACTIONt_other_torsion16.47
LS refinement shellResolution: 1.85→1.9 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.211 205 5.26 %
Rwork0.2154 3695 -
all0.2152 3900 -
obs--99.47 %
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)
10.9589-0.21790.19560.6281-0.13954.09980.07430.1213-0.0151-0.0191-0.1373-0.0231-0.00980.77210.0631-0.14290.03390.00070.08630.0116-0.08118.0773-42.5508-8.1404
21.2618-0.25160.55150.6089-0.33354.42530.0963-0.1051-0.22830.0663-0.00340.11280.3258-0.7597-0.0929-0.1347-0.05310.00090.03340.017-0.0624-2.7085-46.94494.5235
30.86470.00330.66151.71561.21815.51860.0979-0.0227-0.07860.21140.00230.00350.49450.6324-0.1002-0.0680.0409-0.0051-0.01470.0313-0.095918.2263-48.165421.0359
41.1275-0.79160.74371.5932-0.81713.90760.2850.2732-0.235-0.3007-0.08460.11930.5927-0.1149-0.2004-0.05480.063-0.0640.0022-0.0684-0.0615-0.7983-50.4157-25.0302
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1{ A|* }A2 - 136
2X-RAY DIFFRACTION2{ B|* }B2 - 136
3X-RAY DIFFRACTION3{ C|* }C2 - 136
4X-RAY DIFFRACTION4{ D|* }D2 - 136

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