4CU2

C-terminal domain of CTP1L endolysin mutant V195P that reduces autoproteolysis

Summary for 4CU2

• Entry DOI: 10.2210/pdb4cu2/pdb
• Related: 4CU5
• Descriptor: ENDOLYSIN (2 entities in total)
• Functional Keywords: hydrolase, bacterial lysis, autoproteolysis
• Biological source: Clostridium phage phiCTP1
• Total number of polymer chains: 1
• Total formula weight: 8997.04

Authors

Dunne, 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. (deposition date: 2014-03-16, release date: 2014-08-06, Last modification date: 2024-05-01)

Primary citation

Dunne, 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.
The Cd27L and Ctp1L Endolysins Targeting Clostridia Contain a Built-in Trigger and Release Factor.
Plos Pathog., 10:04228-, 2014

PubMed 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.

PubMed: 25058163
DOI: 10.1371/JOURNAL.PPAT.1004228

Experimental method

X-RAY DIFFRACTION (2.11 Å)