The CD27L and CTP1L Endolysins Targeting Clostridia Contain a Built-in Trigger and Release Factor

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. © 2014 dunne et al.