Structural Basis for Recognizing Phosphoarginine and Evolving Residue-Specific Protein Phosphatases in Gram-Positive Bacteria

• A pArg-selective antibody was used with a phage-display approach
• A phospho-enzyme/arginine complex reveals the molecular details of targeting pArg
• A size-and-polarity filter defines phospho-residue specificity of protein phosphatases
• Identification of a eukaryotic arginine phosphatase points to broader impact of pArg

SummaryMany cellular pathways are regulated by the competing activity of protein kinases and phosphatases. The recent identification of arginine phosphorylation as a protein modification in bacteria prompted us to analyze the molecular basis of targeting phospho-arginine. In this work, we characterize an annotated tyrosine phosphatase, YwlE, that counteracts the protein arginine kinase McsB. Strikingly, structural studies of YwlE reaction intermediates provide a direct view on a captured arginine residue. Together with biochemical data, the crystal structures depict the evolution of a highly specific phospho-arginine phosphatase, with the use of a size-and-polarity filter for distinguishing phosphorylated arginine from other phosphorylated side chains. To confirm the proposed mechanism, we performed bioinformatic searches for phosphatases, employing a similar selectivity filter, and identified a protein in Drosophila melanogaster exhibiting robust arginine phosphatase activity. In sum, our findings uncover the molecular framework for specific targeting of phospho-arginine and suggest that protein arginine (de)phosphorylation may be relevant in eukaryotes.

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