title: The CD27L and CTP1L endolysins targeting Clostridia contain a built-in trigger and release factor.
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.
authors: European Molecular Biology Laboratory (EMBL), Hamburg, Germany.
authors: Matthew
authors: M
authors: Dunne
authors: European Molecular Biology Laboratory (EMBL), Hamburg, Germany.
authors: Haydyn D T
authors: HD
authors: Mertens
authors: European Molecular Biology Laboratory (EMBL), Hamburg, Germany.
authors: Vasiliki
authors: V
authors: Garefalaki
authors: European Molecular Biology Laboratory (EMBL), Hamburg, Germany.
authors: Cy M
authors: CM
authors: Jeffries
authors: Synchrotron Soleil, L'Orme des Merisiers, Saint Aubin, Gif sur Yvette, France.
authors: Andrew
authors: A
authors: Thompson
authors: European Molecular Biology Laboratory (EMBL), Heidelberg, Germany.
authors: Edward A
authors: EA
authors: Lemke
authors: European Molecular Biology Laboratory (EMBL), Hamburg, Germany.
authors: Dmitri I
authors: DI
authors: Svergun
authors: Institute of Food Research, Colney, Norwich, United Kingdom.
authors: Melinda J
authors: MJ
authors: Mayer
authors: Institute of Food Research, Colney, Norwich, United Kingdom.
authors: Arjan
authors: A
authors: Narbad
authors: European Molecular Biology Laboratory (EMBL), Hamburg, Germany.
authors: Rob
authors: R
authors: Meijers
mesh_terms: D001435:Bacteriophages / Q000201:enzymology / Q000235:genetics; D016360:Clostridioides difficile / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000821:virology; D018360:Crystallography, X-Ray; D010450:Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008954:Models, Biological; D017434:Protein Structure, Tertiary; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D014764:Viral Proteins; D010450:Endopeptidases; C001143:endolysin