title: Crystal Structure of the CTP1L Endolysin Reveals How Its Activity Is Regulated by a Secondary Translation Product.
abstract: Bacteriophages produce endolysins, which lyse the bacterial host cell to release newly produced virions. The timing of lysis is regulated and is thought to involve the activation of a molecular switch. We present a crystal structure of the activated endolysin CTP1L that targets Clostridium tyrobutyricum, consisting of a complex between the full-length protein and an N-terminally truncated C-terminal cell wall binding domain (CBD). The truncated CBD is produced through an internal translation start site within the endolysin gene. Mutants affecting the internal translation site change the oligomeric state of the endolysin and reduce lytic activity. The activity can be modulated by reconstitution of the full-length endolysin-CBD complex with free CBD. The same oligomerization mechanism applies to the CD27L endolysin that targets Clostridium difficile and the CS74L endolysin that targets Clostridium sporogenes. When the CTP1L endolysin gene is introduced into the commensal bacterium Lactococcus lactis, the truncated CBD is also produced, showing that the alternative start codon can be used in other bacterial species. The identification of a translational switch affecting oligomerization presented here has implications for the design of effective endolysins for the treatment of bacterial infections.
authors.affiliation: From the European Molecular Biology Laboratory, Notkestrasse 85, 22607 Hamburg, Germany.
authors.forename: Matthew
authors.initials: M
authors.lastname: Dunne
authors.affiliation: the European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Leicht
authors.affiliation: the Heinrich Pette Institute, Leibniz Institute for Experimental Virology, Martinistrasse 52, 20251 Hamburg, Germany.
authors.forename: Boris
authors.initials: B
authors.lastname: Krichel
authors.affiliation: From the European Molecular Biology Laboratory, Notkestrasse 85, 22607 Hamburg, Germany.
authors.forename: Haydyn D T
authors.identifier: http://orcid.org/0000-0002-5664-744X
authors.initials: HD
authors.lastname: Mertens
authors.affiliation: the Synchrotron Soleil, L'Orme des Merisiers, BP 48, Saint Aubin, 91192 Gif sur Yvette, France.
authors.forename: Andrew
authors.initials: A
authors.lastname: Thompson
authors.affiliation: the European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany.
authors.forename: Jeroen
authors.identifier: http://orcid.org/0000-0001-7549-9326
authors.initials: J
authors.lastname: Krijgsveld
authors.affiliation: From the European Molecular Biology Laboratory, Notkestrasse 85, 22607 Hamburg, Germany.
authors.forename: Dmitri I
authors.identifier: http://orcid.org/0000-0003-0830-5696
authors.initials: DI
authors.lastname: Svergun
authors.affiliation: the Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Departamento de Tecnología de Alimentos, Carretera de La Coruña km 7, 28040 Madrid, Spain.
authors.forename: Natalia
authors.initials: N
authors.lastname: Gómez-Torres
authors.affiliation: the Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Departamento de Tecnología de Alimentos, Carretera de La Coruña km 7, 28040 Madrid, Spain.
authors.forename: Sonia
authors.initials: S
authors.lastname: Garde
authors.affiliation: the Heinrich Pette Institute, Leibniz Institute for Experimental Virology, Martinistrasse 52, 20251 Hamburg, Germany, the European XFEL GmbH, Notkestrasse 85, 22607 Hamburg, Germany, and.
authors.forename: Charlotte
authors.identifier: http://orcid.org/0000-0002-1991-7922
authors.initials: C
authors.lastname: Uetrecht
authors.affiliation: the Institute of Food Research, Colney, Norwich NR4 7UA, United Kingdom.
authors.forename: Arjan
authors.initials: A
authors.lastname: Narbad
authors.affiliation: the Institute of Food Research, Colney, Norwich NR4 7UA, United Kingdom melinda.mayer@ifr.ac.uk.
authors.forename: Melinda J
authors.initials: MJ
authors.lastname: Mayer
authors.affiliation: From the European Molecular Biology Laboratory, Notkestrasse 85, 22607 Hamburg, Germany, r.meijers@embl-hamburg.de.
authors.forename: Rob
authors.identifier: http://orcid.org/0000-0003-2872-6279
authors.initials: R
authors.lastname: Meijers
mesh_terms: D000595:Amino Acid Sequence; D001435:Bacteriophages / Q000201:enzymology / Q000235:genetics; D048014:Clostridium tyrobutyricum / Q000187:drug effects; D018387:Codon, Initiator; D010450:Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000633:toxicity; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D055503:Protein Multimerization
chemical_list: D018387:Codon, Initiator; D010450:Endopeptidases; C001143:endolysin
keywords: Clostridia; antimicrobial; bacteriophage; endolysin; enzyme catalysis; mass spectrometry (MS); native mass spectrometry; oligomerization; protein structure; secondary translation