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


ABSTRACT: 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.

SUBMITTER: Kuiper EG 

PROVIDER: S-EPMC6937583 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Substrate recognition by the <i>Pseudomonas aeruginosa</i> EF-Tu-modifying methyltransferase EftM.

Kuiper Emily G EG   Dey Debayan D   LaMore Paige A PA   Owings Joshua P JP   Prezioso Samantha M SM   Goldberg Joanna B JB   Conn Graeme L GL  

The Journal of biological chemistry 20191121 52


The opportunistic bacterial pathogen <i>Pseudomonas aeruginosa</i> is a leading cause of serious infections in individuals with cystic fibrosis, compromised immune systems, or severe burns. <i>P. aeruginosa</i> 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 <i>P. aeruginosa</i> infections in vulne  ...[more]

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