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- PDB-4mkq: Crystal structure of the Pore-Forming Toxin Monalysin mutant dele... -

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Basic information

Entry
Database: PDB / ID: 4mkq
TitleCrystal structure of the Pore-Forming Toxin Monalysin mutant deleted of the membrane-spanning domain
Components
  • MONALYSIN
  • Monalysin
KeywordsTOXIN / Pore-Forming Toxin
Function / homology
Function and homology information


hemolysis in another organism / porin activity / pore complex / monoatomic ion transport / protein homooligomerization / toxin activity / host cell plasma membrane / extracellular region
Similarity search - Function
Monalysin, beta barrel Pore-forming domain / Beta barrel Pore-forming domain
Similarity search - Domain/homology
Biological speciesPseudomonas entomophila (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.65 Å
AuthorsLeone, P. / Roussel, A.
CitationJournal: J Biol Chem / Year: 2015
Title: X-ray and Cryo-electron Microscopy Structures of Monalysin Pore-forming Toxin Reveal Multimerization of the Pro-form.
Authors: Philippe Leone / Cecilia Bebeacua / Onya Opota / Christine Kellenberger / Bruno Klaholz / Igor Orlov / Christian Cambillau / Bruno Lemaitre / Alain Roussel /
Abstract: β-Barrel pore-forming toxins (β-PFT), a large family of bacterial toxins, are generally secreted as water-soluble monomers and can form oligomeric pores in membranes following proteolytic cleavage ...β-Barrel pore-forming toxins (β-PFT), a large family of bacterial toxins, are generally secreted as water-soluble monomers and can form oligomeric pores in membranes following proteolytic cleavage and interaction with cell surface receptors. Monalysin has been recently identified as a β-PFT that contributes to the virulence of Pseudomonas entomophila against Drosophila. It is secreted as a pro-protein that becomes active upon cleavage. Here we report the crystal and cryo-electron microscopy structure of the pro-form of Monalysin as well as the crystal structures of the cleaved form and of an inactive mutant lacking the membrane-spanning region. The overall structure of Monalysin displays an elongated shape, which resembles those of β-pore-forming toxins, such as Aerolysin, but is devoid of a receptor-binding domain. X-ray crystallography, cryo-electron microscopy, and light-scattering studies show that pro-Monalysin forms a stable doughnut-like 18-mer complex composed of two disk-shaped nonamers held together by N-terminal swapping of the pro-peptides. This observation is in contrast with the monomeric pro-form of the other β-PFTs that are receptor-dependent for membrane interaction. The membrane-spanning region of pro-Monalysin is fully buried in the center of the doughnut, suggesting that upon cleavage of pro-peptides, the two disk-shaped nonamers can, and have to, dissociate to leave the transmembrane segments free to deploy and lead to pore formation. In contrast with other toxins, the delivery of 18 subunits at once, nearby the cell surface, may be used to bypass the requirement of receptor-dependent concentration to reach the threshold for oligomerization into the pore-forming complex.
History
DepositionSep 5, 2013Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 11, 2015Provider: repository / Type: Initial release
Revision 1.1Apr 15, 2015Group: Database references
Revision 1.2Apr 22, 2015Group: Database references
Revision 1.3Jun 10, 2015Group: Database references
Revision 1.4Aug 2, 2017Group: Refinement description / Source and taxonomy / Category: entity_src_gen / software
Revision 1.5Sep 20, 2023Group: Data collection / Database references / 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
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_ref_seq_dif.details

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: MONALYSIN
B: MONALYSIN
C: Monalysin
D: Monalysin


Theoretical massNumber of molelcules
Total (without water)43,9884
Polymers43,9884
Non-polymers00
Water5,188288
1
A: MONALYSIN
B: MONALYSIN
C: Monalysin
D: Monalysin

A: MONALYSIN
B: MONALYSIN
C: Monalysin
D: Monalysin


Theoretical massNumber of molelcules
Total (without water)87,9778
Polymers87,9778
Non-polymers00
Water1448
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation4_655-x+1,-y,z1
Buried area11290 Å2
ΔGint-42 kcal/mol
Surface area30510 Å2
MethodPISA
Unit cell
Length a, b, c (Å)129.458, 129.458, 78.888
Angle α, β, γ (deg.)90.00, 90.00, 120.00
Int Tables number172
Space group name H-MP64

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Components

#1: Protein MONALYSIN


Mass: 19108.105 Da / Num. of mol.: 2 / Fragment: see remark 999
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas entomophila (bacteria) / Strain: L48 / Gene: PSEEN3174 / Production host: Escherichia coli (E. coli) / References: UniProt: Q1I8U1
#2: Protein/peptide Monalysin


Mass: 2886.110 Da / Num. of mol.: 2 / Fragment: see remark 999
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas entomophila (bacteria) / Strain: L48 / Gene: PSEEN3174 / Production host: Escherichia coli (E. coli) / References: UniProt: Q1I8U1
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 288 / Source method: isolated from a natural source / Formula: H2O
Compound detailsAUTHORS STATE THAT PEPTIDE CHAINS (C AND D) AND PROTEIN CHAINS (A AND B) BELONG TO A SAME SINGLE ...AUTHORS STATE THAT PEPTIDE CHAINS (C AND D) AND PROTEIN CHAINS (A AND B) BELONG TO A SAME SINGLE CHAIN WITHOUT BEEN CLEAVED BETWEEN RESIDUES 35 AND 36. AUTHORS HAVE STRONG EVIDENCE OF THE DOMAIN SWAPPING BUT NO EXPERIMENTAL DATA COULD PERMIT TO ASSIGN THE CHAINS UNAMBIGUOUSLY. THEREFORE, THEY WERE ASSIGNED DISTINCT CHAIN IDS.
Sequence detailsTHE SEQUENCE OF CHAIN C AND D MATCHES UNIPROT ENTRY Q1I8U1 BETWEEN RESIDUES 9 AND 35. THE SEQUENCE ...THE SEQUENCE OF CHAIN C AND D MATCHES UNIPROT ENTRY Q1I8U1 BETWEEN RESIDUES 9 AND 35. THE SEQUENCE OF CHAIN A AND B MATCHES UNIPROT ENTRY Q1I8U1 BETWEEN RESIDUES 36 AND 271, WITH A DELETION OF MEMBRANE SPANNING DOMAIN (RESIDUES 102-170), THAT IS REPLACED BY A GLY501-SER502 DIPEPTIDE LINKER. SEE REMARK 400 FOR CHAIN ID ASSIGNMENT DETAILS.

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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: 4.34 Å3/Da / Density % sol: 71.65 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 6.5
Details: 0.1M sodium cacodylate, 0.2M calcium acetate, 18% PEG8000, pH 6.5, VAPOR DIFFUSION, SITTING DROP, temperature 293K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: ID29 / Wavelength: 0.97887 Å
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97887 Å / Relative weight: 1
ReflectionResolution: 2.65→30 Å / Num. all: 21936 / Num. obs: 21936 / % possible obs: 100 % / Observed criterion σ(F): 0 / Observed criterion σ(I): 0 / Redundancy: 3.4 % / Biso Wilson estimate: 53.13 Å2 / Rmerge(I) obs: 0.132 / Net I/σ(I): 7.8
Reflection shellResolution: 2.65→2.79 Å / Redundancy: 3.4 % / Rmerge(I) obs: 0.534 / Mean I/σ(I) obs: 2 / Num. unique all: 3186 / % possible all: 100

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Processing

Software
NameVersionClassification
ADSCQuantumdata collection
MOLREPphasing
BUSTER2.11.4refinement
XDSdata reduction
SCALAdata scaling
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB entry 4MJT

4mjt


Resolution: 2.65→29.94 Å / Cor.coef. Fo:Fc: 0.8983 / Cor.coef. Fo:Fc free: 0.8748 / SU R Cruickshank DPI: 0.288 / Cross valid method: THROUGHOUT / σ(F): 0
RfactorNum. reflection% reflectionSelection details
Rfree0.2157 1121 5.11 %RANDOM
Rwork0.1874 ---
obs0.1888 21920 99.66 %-
all-21936 --
Displacement parametersBiso mean: 38.36 Å2
Baniso -1Baniso -2Baniso -3
1--7.497 Å20 Å20 Å2
2---7.497 Å20 Å2
3---14.994 Å2
Refine analyzeLuzzati coordinate error obs: 0.296 Å
Refinement stepCycle: LAST / Resolution: 2.65→29.94 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2852 0 0 288 3140
Refine LS restraints
Refine-IDTypeDev idealNumberRestraint functionWeight
X-RAY DIFFRACTIONt_bond_d0.0072934HARMONIC2
X-RAY DIFFRACTIONt_angle_deg0.933998HARMONIC2
X-RAY DIFFRACTIONt_dihedral_angle_d948SINUSOIDAL2
X-RAY DIFFRACTIONt_trig_c_planes70HARMONIC2
X-RAY DIFFRACTIONt_gen_planes432HARMONIC5
X-RAY DIFFRACTIONt_it2934HARMONIC20
X-RAY DIFFRACTIONt_omega_torsion2.54
X-RAY DIFFRACTIONt_other_torsion19.45
X-RAY DIFFRACTIONt_chiral_improper_torsion368SEMIHARMONIC5
X-RAY DIFFRACTIONt_ideal_dist_contact3270SEMIHARMONIC4
LS refinement shellResolution: 2.65→2.78 Å / Total num. of bins used: 11
RfactorNum. reflection% reflection
Rfree0.2348 138 4.81 %
Rwork0.2299 2732 -
all0.2302 2870 -
obs--99.66 %

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