Document Type
Article
Publication Date
2-21-2024
Abstract
Bacteria express lytic enzymes such as glycosidases, which have potentially self-destructive peptidoglycan (PG)-degrading activity and, therefore, require careful regulation in bacteria. The PG glycosidase EtgA is regulated by localization to the assembling type III secretion system (T3SS), generating a hole in the PG layer for the T3SS to reach the outer membrane. The EtgA localization was found to be mediated via EtgA interacting with the T3SS inner rod protein EscI. To gain structural insights into the EtgA recognition of EscI, we determined the 2.01 Å resolution structure of an EscI (51–87)-linker-EtgA fusion protein designed based on AlphaFold2 predictions. The structure revealed EscI residues 72–87 forming an α-helix interacting with the backside of EtgA, distant from the active site. EscI residues 56–71 also were found to interact with EtgA, with these residues stretching across the EtgA surface. The ability of the EscI to interact with EtgA was also probed using an EscI peptide. The EscI peptide comprising residues 66–87, slightly larger than the observed EscI α-helix, was shown to bind to EtgA using microscale thermophoresis and thermal shift differential scanning fluorimetry. The EscI peptide also had a two-fold activity-enhancing effect on EtgA, whereas the EscI-EtgA fusion protein enhanced activity over four-fold compared to EtgA. Our studies suggest that EtgA regulation by EscI could be trifold involving protein localization, protein activation, and protein stabilization components. Analysis of the sequence conservation of the EscI EtgA interface residues suggested a possible conservation of such regulation for related proteins from different bacteria.
Keywords
escI inner rod protein, lytic transglycosylase, muramidase, peptidoglycan glycosidases EtgA, protein crystallography, type III secretion system
Publication Title
Protein Science
Grant
1R21AI148875
Rights
© 2024 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.
Creative Commons License
This work is licensed under a Creative Commons Attribution-NonCommercial-No Derivative Works 4.0 International License.
Recommended Citation
Boorman J, Zeng X, Lin J, van den Akker F. Structural insights into peptidoglycan glycosidase EtgA binding to the inner rod protein EscI of the type III secretion system via a designed EscI-EtgA fusion protein. Protein Science. 2024; 33(3):e4930. https://doi.org/10.1002/pro.4930
Manuscript Version
Final Publisher Version