Journal: 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.
Mass: 18.015 Da / Num. of mol.: 87 / Source method: isolated from a natural source / Formula: H2O
Sequence details
VAL195 HAS BEEN MUTATED TO A PROLINE TO REDUCE AUTOCLEAVAGE
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Experimental details
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Experiment
Experiment
Method: X-RAY DIFFRACTION / Number of used crystals: 1
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Sample preparation
Crystal
Density 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 grow
pH: 8 / Details: 20MM TRIS PH8.0 AND 6 % PEG 8000
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Data collection
Diffraction
Mean temperature: 100 K
Diffraction source
Source: SYNCHROTRON / Site: PETRA III, EMBL c/o DESY / Beamline: P14 (MX2) / Wavelength: 1.223
Detector
Type: MARMOSAIC 225 mm CCD / Detector: CCD / Details: DIRECT BEAM (NO MIRRORS)
Radiation
Monochromator: SI 1 1 1 / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelength
Wavelength: 1.223 Å / Relative weight: 1
Reflection
Resolution: 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 shell
Highest resolution: 2.1 Å
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Processing
Software
Name
Version
Classification
REFMAC
5.7.0029
refinement
DENZO
datareduction
SCALEPACK
datascaling
MOLREP
phasing
Refinement
Method 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.
Rfactor
Num. reflection
% reflection
Selection details
Rfree
0.26382
221
4.7 %
RANDOM
Rwork
0.17217
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obs
0.17617
4489
91.03 %
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Solvent computation
Ion probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: BABINET MODEL WITH MASK