Substrate recognition by the Pseudomonas aeruginosa EF-Tu?modifying methyltransferase EftM

• Type of Material: Article
• Authors: Emily G. Kuiper, Emory University; Debayan Dey, Emory University; Paige A. LaMore, Emory University; Joshua P. Owings, Emory University; Samantha M. Prezioso, Emory University; Joanna Goldberg, Emory University; Graeme Conn, Emory University
• Language: English
• Date: 2019-12-27
• Publisher: AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
• Publication Version: Final Published Version
• Copyright Statement: © 2019 Kuiper et al.
• License: Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International
• Final Published Version (URL): http://dx.doi.org/10.1074/jbc.RA119.011213
• Title of Journal or Parent Work: JOURNAL OF BIOLOGICAL CHEMISTRY
• Volume: 294
• Issue: 52
• Start Page: 20109
• End Page: 20121
• Grant/Funding Information: This work was supported by Cystic Fibrosis Foundation Grant GOLDBE17P0 (to J. B. G. and G. L. C.), Postdoctoral Fellowship DEY18F0 (to D. D.), and in part by NIAID Training Grant T32-AI106699 from the National Institutes of Health to the Antimicrobial Resistance and Therapeutic Discovery Training Program of Emory University (awarded to S. M. P.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
• Keywords: translation elongation factor; Pseudomonas aeruginosa (P; aeruginosa); SERVER; GENERATION; OMPB; EF-Tu; post-translational modification (PTM); EftM; Science & Technology; enzyme mechanism; PROTEIN; Life Sciences & Biomedicine; Biochemistry & Molecular Biology; CHROMATIN; MODULATION; protein methylation; PHOSPHORYLCHOLINE; substrate specificity; S-adenosylmethionine (SAM); POSTTRANSLATIONAL MODIFICATIONS
• Research Categories: Biology, Molecular; Chemistry, Biochemistry

Abstract

The opportunistic bacterial pathogen Pseudomonas aeruginosa is a leading cause of serious infections in individuals with cystic fibrosis, compromisedimmunesystems, or severe burns. P. aeruginosa adhesion to host epithelial cells is enhanced by surface-exposedtranslationelongationfactorEF-TucarryingaLys-5trimethylation, incorporated by the methyltransferase EftM. Thus, the EF-Tu modification by EftM may represent a target to prevent P. aeruginosa infections in vulnerable individuals. Here, we extend our understanding of EftM activity by defining the molecular mechanism by which it recognizes EF-Tu. Acting on the observation that EftM can bind to EF-Tu lacking its N-terminal peptide (encompassing the Lys-5 target site), we generated an EftM homology model and used it in protein/protein docking studies to predict EftM/EF-Tu interactions. Using site-directed mutagenesis of residues in both proteins, coupled with binding and methyltransferase activity assays, we experimentally validated the predicted protein/protein interface.Wealso show that EftM cannot methylate the isolated N-terminal EF-Tu peptide and that binding-induced conformational changes in EftM are likely needed to enable placement of the first 5-6 amino acids of EF-Tu into a conserved peptide-binding channel in EftM. In this channel, a group of residues that are highly conserved in EftM proteins position the N-terminalsequenceto facilitate Lys-5 modification.Ourfindings reveal that EftM employs molecular strategies for substrate recognition common among both class I (Rossmann fold) and class II (SET domain) methyltransferases and pave the way for studies seeking a deeper understanding of EftM's mechanism of action on EF-Tu.