SASDAL5: CD27L (Clostridium difficile bacteriophage 27 endolysin)

Basic information

• Entry: Database: SASBDB / ID: SASDAL5

Sample

CD27L

• Clostridium difficile bacteriophage 27 endolysin (protein), CD27L, Clostridioides difficile

• Biological species: Clostridioides difficile (bacteria)
• Citation: 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, 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.

Contact author

• Haydyn Mertens (EMBL-Hamburg, European Molecular Biology Laboratory (EMBL) - Hamburg outstation, Notkestraße 85, Geb. 25A, 22607 Hamburg, Deutschland, Germany)

Models

• Model #269: ; Type: atomic / Radius of dummy atoms: 1.90 A / Comment: OLIGOMER component 1: head-head CD27L dimer / Chi-square value: 2.4649; Search similar-shape structures of this assembly by Omokage search (details)
• Model #270: ; Type: atomic / Radius of dummy atoms: 1.90 A / Comment: OLIGOMER component 2: side-side CD27L dimer / Chi-square value: 2.4649; Search similar-shape structures of this assembly by Omokage search (details)
• Model #271: ; Type: atomic / Radius of dummy atoms: 1.90 A / Comment: OLIGOMER component 3a / Chi-square value: 2.4649; Search similar-shape structures of this assembly by Omokage search (details)
• Model #272: ; Type: atomic / Radius of dummy atoms: 1.90 A / Comment: OLIGOMER component 3b / Chi-square value: 2.4649; Search similar-shape structures of this assembly by Omokage search (details)
• Model #273: ; Type: atomic / Radius of dummy atoms: 1.90 A / Chi-square value: 2.4649; Search similar-shape structures of this assembly by Omokage search (details)
• Model #274: ; Type: atomic / Radius of dummy atoms: 1.90 A / Comment: OLIGOMER component 3c / Chi-square value: 2.4649; Search similar-shape structures of this assembly by Omokage search (details)
• Model #275: ; Type: atomic / Radius of dummy atoms: 1.90 A / Comment: OLIGOMER component 3d / Chi-square value: 2.4649; Search similar-shape structures of this assembly by Omokage search (details)
• Model #276: ; Type: atomic / Radius of dummy atoms: 1.90 A / Comment: OLIGOMER component 3e / Chi-square value: 2.4649; Search similar-shape structures of this assembly by Omokage search (details)

Sample

• Sample: Name: CD27L / Sample MW: 64.2 kDa / Specimen concentration: 0.90-4.00 / Concentration method: A280
• Buffer: Name: 20 mM HEPES 150 mM NaCl / pH: 7.5 / Composition: 150 mM NaCl

Entity #94

Name: CD27L / Type: protein
Description: Clostridium difficile bacteriophage 27 endolysin
Formula weight: 32.1 / Num. of mol.: 2 / Source: Clostridioides difficile
Sequence:

MGSSHHHHHH SSGLVPRGSH MKICITVGHS ILKSGACTSA DGVVNEYQYN KSLAPVLADT FRKEGHKVDV IIsPEKQFKT KNEEKSYKIP RVNSGGYDLL IELHLNASNG QGKGSEVLYY SNKGLEYATR ICDKLGTVFK NRGAKLDKRL YILNSSKPTA VLIESFFCDN KEDYDKAKKL GHEGIAKLIV EGVLNKNINN EGVKQMYKHT IVYDGEVDKI SATVVGWGYN DGKILICDIK DYVPGQTQNL YVVGGGACEK ISSITKEKFI MIKGNDRFDT LYKALDFIN

Experimental information

• Beam: Instrument name: DORIS III X33 / City: Hamburg / 国: Germany / Shape: 0.6 / Type of source: X-ray synchrotronSynchrotron / Wavelength: 0.15 Å / Dist. spec. to detc.: 2.7 mm
• Detector: Name: Pilatus 1M-W / Pixsize x: 0.172 mm

Scan

Title: CD27L wild-type / Measurement date: Mar 17, 2011 / Storage temperature: 10 °C / Cell temperature: 10 °C / Exposure time: 15 sec. / Number of frames: 8 / Unit: 1/nm /

	Min	Max
Q	0.1292	6.3452

Distance distribution function P(R)

Sofotware P(R): GNOM 4.5a / Number of points: 427 /

	Min	Max
Q	0.1332	2.421
P(R) point	24	450
R	0	10.57

Result

Type of curve: merged / Standard: BSA

	Experimental	Standard	Porod
MW	42.1 kDa	42.1 kDa	45.1 kDa
Volume	-	-	72.15 nm3

	P(R)	Guinier
Forward scattering, I0	44	45.6
Radius of gyration, Rg	3.3 nm	3.3 nm

	Min	Max
D	-	10.6
Guinier point	1	100