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- PDB-4cu2: C-terminal domain of CTP1L endolysin mutant V195P that reduces au... -

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Basic information

Entry
Database: PDB / ID: 4cu2
TitleC-terminal domain of CTP1L endolysin mutant V195P that reduces autoproteolysis
ComponentsENDOLYSINLysin
KeywordsHYDROLASE / BACTERIAL LYSIS / AUTOPROTEOLYSIS
Function / homology
Function and homology information


peptidoglycan catabolic process / cell wall macromolecule catabolic process / lysozyme / lysozyme activity
Similarity search - Function
Rossmann fold - #12090 / Glycosyl hydrolase family 25 (GH25) domain profile. / Glycoside hydrolase, family 25 / Glycosyl hydrolases family 25 / Glycoside hydrolase superfamily / Rossmann fold / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
Biological speciesClostridium phage phiCTP1 (virus)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.11 Å
AuthorsDunne, M. / Mertens, H.D.T. / Garefalaki, V. / Jeffries, C.M. / Thompson, A. / Lemke, E.A. / Svergun, D.I. / Mayer, M.J. / Narbad, A. / Meijers, R.
CitationJournal: PLoS Pathog / Year: 2014
Title: The CD27L and CTP1L endolysins targeting Clostridia contain a built-in trigger and release factor.
Authors: Matthew Dunne / Haydyn D T Mertens / Vasiliki Garefalaki / Cy M Jeffries / Andrew Thompson / Edward A Lemke / Dmitri I Svergun / Melinda J Mayer / Arjan Narbad / Rob Meijers /
Abstract: The bacteriophage ΦCD27 is capable of lysing Clostridium difficile, a pathogenic bacterium that is a major cause for nosocomial infection. A recombinant CD27L endolysin lyses C. difficile in vitro, ...The bacteriophage ΦCD27 is capable of lysing Clostridium difficile, a pathogenic bacterium that is a major cause for nosocomial infection. A recombinant CD27L endolysin lyses C. difficile in vitro, and represents a promising alternative as a bactericide. To better understand the lysis mechanism, we have determined the crystal structure of an autoproteolytic fragment of the CD27L endolysin. The structure covers the C-terminal domain of the endolysin, and represents a novel fold that is identified in a number of lysins that target Clostridia bacteria. The structure indicates endolysin cleavage occurs at the stem of the linker connecting the catalytic domain with the C-terminal domain. We also solved the crystal structure of the C-terminal domain of a slow cleaving mutant of the CTP1L endolysin that targets C. tyrobutyricum. Two distinct dimerization modes are observed in the crystal structures for both endolysins, despite a sequence identity of only 22% between the domains. The dimers are validated to be present for the full length protein in solution by right angle light scattering, small angle X-ray scattering and cross-linking experiments using the cross-linking amino acid p-benzoyl-L-phenylalanine (pBpa). Mutagenesis on residues contributing to the dimer interfaces indicates that there is a link between the dimerization modes and the autocleavage mechanism. We show that for the CTP1L endolysin, there is a reduction in lysis efficiency that is proportional to the cleavage efficiency. We propose a model for endolysin triggering, where the extended dimer presents the inactive state, and a switch to the side-by-side dimer triggers the cleavage of the C-terminal domain. This leads to the release of the catalytic portion of the endolysin, enabling the efficient digestion of the bacterial cell wall.
History
DepositionMar 16, 2014Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 6, 2014Provider: repository / Type: Initial release
Revision 1.1Dec 17, 2014Group: Data collection
Revision 1.2Mar 7, 2018Group: Data collection / Source and taxonomy / Category: diffrn_source / entity_src_gen
Item: _diffrn_source.pdbx_synchrotron_site / _entity_src_gen.pdbx_gene_src_scientific_name ..._diffrn_source.pdbx_synchrotron_site / _entity_src_gen.pdbx_gene_src_scientific_name / _entity_src_gen.pdbx_host_org_scientific_name / _entity_src_gen.pdbx_host_org_strain
Revision 1.3May 1, 2024Group: Data collection / Database references ...Data collection / Database references / Other / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_database_status / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_database_status.status_code_sf

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: ENDOLYSIN


Theoretical massNumber of molelcules
Total (without water)8,9971
Polymers8,9971
Non-polymers00
Water1,56787
1
A: ENDOLYSIN

A: ENDOLYSIN

A: ENDOLYSIN

A: ENDOLYSIN


Theoretical massNumber of molelcules
Total (without water)35,9884
Polymers35,9884
Non-polymers00
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation3_655-x+1,y,-z1
crystal symmetry operation2_655-x+1,-y,z1
crystal symmetry operation4_555x,-y,-z1
Buried area5410 Å2
ΔGint-11.9 kcal/mol
Surface area14860 Å2
MethodPISA
Unit cell
Length a, b, c (Å)44.932, 48.821, 77.226
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number23
Space group name H-MI222
Components on special symmetry positions
IDModelComponents
11A-2007-

HOH

21A-2008-

HOH

31A-2077-

HOH

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Components

#1: Protein ENDOLYSIN / Lysin


Mass: 8997.040 Da / Num. of mol.: 1 / Fragment: C-TERMINAL DOMAIN, RESIDUES 195-274 / Mutation: YES
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Clostridium phage phiCTP1 (virus) / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: D9ZNF3
#2: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 87 / Source method: isolated from a natural source / Formula: H2O
Sequence detailsVAL195 HAS BEEN MUTATED TO A PROLINE TO REDUCE AUTOCLEAVAGE

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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: 2.4 Å3/Da / Density % sol: 48 %
Description: THERE ARE ICE RINGS, AND THE DETECTOR GEOMETRY LIMITED HIGH RESOLUTION. BEAMLINE WAS UNDER COMMISSIONING.
Crystal growpH: 8 / Details: 20MM TRIS PH8.0 AND 6 % PEG 8000

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: PETRA III, EMBL c/o DESY / Beamline: P14 (MX2) / Wavelength: 1.223
DetectorType: MARMOSAIC 225 mm CCD / Detector: CCD / Details: DIRECT BEAM (NO MIRRORS)
RadiationMonochromator: SI 1 1 1 / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.223 Å / Relative weight: 1
ReflectionResolution: 2.1→20 Å / Num. obs: 4489 / % possible obs: 92 % / Observed criterion σ(I): 0 / Redundancy: 5.7 % / Rmerge(I) obs: 0.02 / Net I/σ(I): 48.9
Reflection shellHighest resolution: 2.1 Å

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Processing

Software
NameVersionClassification
REFMAC5.7.0029refinement
DENZOdata reduction
SCALEPACKdata scaling
MOLREPphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: CD27L C-TERMINAL DOMAIN

Resolution: 2.11→16.54 Å / Cor.coef. Fo:Fc: 0.952 / Cor.coef. Fo:Fc free: 0.9 / SU B: 5.669 / SU ML: 0.143 / Cross valid method: THROUGHOUT / ESU R: 0.262 / ESU R Free: 0.236 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS.
RfactorNum. reflection% reflectionSelection details
Rfree0.26382 221 4.7 %RANDOM
Rwork0.17217 ---
obs0.17617 4489 91.03 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: BABINET MODEL WITH MASK
Displacement parametersBiso mean: 22.701 Å2
Baniso -1Baniso -2Baniso -3
1-3.18 Å20 Å20 Å2
2---1.06 Å20 Å2
3----2.12 Å2
Refinement stepCycle: LAST / Resolution: 2.11→16.54 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms633 0 0 87 720
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0060.02657
X-RAY DIFFRACTIONr_bond_other_d0.0010.02623
X-RAY DIFFRACTIONr_angle_refined_deg1.0341.959896
X-RAY DIFFRACTIONr_angle_other_deg0.70231424
X-RAY DIFFRACTIONr_dihedral_angle_1_deg5.26583
X-RAY DIFFRACTIONr_dihedral_angle_2_deg40.65324.68832
X-RAY DIFFRACTIONr_dihedral_angle_3_deg12.56715109
X-RAY DIFFRACTIONr_dihedral_angle_4_deg22.338154
X-RAY DIFFRACTIONr_chiral_restr0.0610.2103
X-RAY DIFFRACTIONr_gen_planes_refined0.0030.02768
X-RAY DIFFRACTIONr_gen_planes_other0.0010.02156
X-RAY DIFFRACTIONr_nbd_refined
X-RAY DIFFRACTIONr_nbd_other
X-RAY DIFFRACTIONr_nbtor_refined
X-RAY DIFFRACTIONr_nbtor_other
X-RAY DIFFRACTIONr_xyhbond_nbd_refined
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_metal_ion_refined
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined
X-RAY DIFFRACTIONr_symmetry_vdw_other
X-RAY DIFFRACTIONr_symmetry_hbond_refined
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_symmetry_metal_ion_refined
X-RAY DIFFRACTIONr_symmetry_metal_ion_other
X-RAY DIFFRACTIONr_mcbond_it
X-RAY DIFFRACTIONr_mcbond_other
X-RAY DIFFRACTIONr_mcangle_it
X-RAY DIFFRACTIONr_mcangle_other
X-RAY DIFFRACTIONr_scbond_it
X-RAY DIFFRACTIONr_scbond_other
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other
X-RAY DIFFRACTIONr_long_range_B_refined
X-RAY DIFFRACTIONr_long_range_B_other
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 2.107→2.162 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.221 14 -
Rwork0.202 340 -
obs--96.99 %

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