期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 294, 期 52, 页码 20109-20121出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.RA119.011213
关键词
enzyme mechanism; post-translational modification (PTM); protein methylation; Pseudomonas aeruginosa (P; aeruginosa); S-adenosylmethionine (SAM); substrate specificity; translation elongation factor; EF-Tu; EftM
资金
- Cystic Fibrosis Foundation [GOLDBE17P0, DEY18F0]
- NIAID from the National Institutes of Health [T32-AI106699]
The opportunistic bacterial pathogen Pseudomonas aeruginosa is a leading cause of serious infections in individuals with cystic fibrosis, compromised immune systems, or severe burns. P. aeruginosa adhesion to host epithelial cells is enhanced by surface-exposed translation elongation factor EF-Tu carrying a Lys-5 trimethylation, 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. We also 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-terminal sequence to facilitate Lys-5 modification. Our findings 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.
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