TY  - JOUR
A1  - Mezhyrova, Julija
A1  - Martin, Janosch
A1  - Peetz, Oliver
A1  - Dötsch, Volker
A1  - Morgner, Nina
A1  - Ma, Yi
A1  - Bernhard, Frank
T1  - Membrane insertion mechanism and molecular assembly of the bacteriophage lysis toxin ΦX174-E
T2  - The FEBS journal
N2  - The bacteriophage ΦX174 causes large pore formation in Escherichia coli and related bacteria. Lysis is mediated by the small membrane-bound toxin ΦX174-E, which is composed of a transmembrane domain and a soluble domain. The toxin requires activation by the bacterial chaperone SlyD and inhibits the cell wall precursor forming enzyme MraY. Bacterial cell wall biosynthesis is an important target for antibiotics; therefore, knowledge of molecular details in the ΦX174-E lysis pathway could help to identify new mechanisms and sites of action. In this study, cell-free expression and nanoparticle technology were combined to avoid toxic effects upon ΦX174-E synthesis, resulting in the efficient production of a functional full-length toxin and engineered derivatives. Pre-assembled nanodiscs were used to study ΦX174-E function in defined lipid environments and to analyze its membrane insertion mechanisms. The conformation of the soluble domain of ΦX174-E was identified as a central trigger for membrane insertion, as well as for the oligomeric assembly of the toxin. Stable complex formation of the soluble domain with SlyD is essential to keep nascent ΦX174-E in a conformation competent for membrane insertion. Once inserted into the membrane, ΦX174-E assembles into high-order complexes via its transmembrane domain and oligomerization depends on the presence of an essential proline residue at position 21. The data presented here support a model where an initial contact of the nascent ΦX174-E transmembrane domain with the peptidyl-prolyl isomerase domain of SlyD is essential to allow a subsequent stable interaction of SlyD with the ΦX174-E soluble domain for the generation of a membrane insertion competent toxin.
KW  - cell-free expression
KW  - molecular switch
KW  - nanodiscs
KW  - peptide antibiotics
KW  - phage lysis proteins
Y1  - 2020
UR  - http://publikationen.ub.uni-frankfurt.de/frontdoor/index/index/docId/63868
UR  - https://nbn-resolving.org/urn:nbn:de:hebis:30:3-638686
SN  - 1742-4658
N1  - Yi Ma was financially supported by Guangdong Natural Science Foundation (2019A1515011251), the Fundamental Research Funds for the Central Universities (2018MS54), and the opening fund of State Key Laboratory of Fermentation and Enzyme Engineering and China Scholarship Council (201706155087). Nina Morgner was funded by the Heisenberg Professor ERC starting grant. The work was further funded by the Collaborative Research Centre (SFB) 807 of the German Research Foundation (DFG), the state of Hessen (Center for Biomolecular Magnetic Resonance), and by the research training group Complex Light Control (CLiC) at the Goethe University Frankfurt.
N1  - The peer review history for this article is available at https://publons.com/publon/10.1111/febs.15642.
VL  - 288
IS  - 10
SP  - 3300
EP  - 3316
PB  - Wiley-Blackwell
CY  - Oxford [u.a.]
ER  -