4pic
YwlE arginine phosphatase from Geobacillus stearothermophilus
Structural highlights
FunctionPAP_GEOSE Catalyzes the specific dephosphorylation of phosphoarginine residues in proteins. Probably counteracts the protein arginine kinase McsB in vivo. Exhibits almost no activity against pTyr peptides. Protein arginine phosphorylation has a physiologically important role and is involved in the regulation of many critical cellular processes, such as protein homeostasis, motility, competence, and stringent and stress responses, by regulating gene expression and protein activity.[1] Publication Abstract from PubMedArginine phosphorylation is an emerging post-translational protein modification implicated in the bacterial stress response. Although early reports suggested that arginine phosphorylation also occurs in higher eukaryotes, its overall prevalence was never studied by modern mass spectrometry methods, owing to technical difficulties arising from the acid lability of phosphoarginine. As shown recently, the McsB and YwlE proteins from Bacillus subtilis function as a highly specific protein arginine kinase and phosphatase couple, shaping the phosphoarginine proteome. Using a B. subtilis DeltaywlE strain as source for arginine-phosphorylated proteins, we could adapt mass spectrometry (MS) protocols to the special chemical properties of the arginine modification. Despite this progress, the analysis of protein arginine phosphorylation in eukaryotes is still challenging, given the great abundance of serine/threonine phosphorylations that would compete with phosphoarginine during the phosphopeptide enrichment procedure as well as during data-dependent MS acquisition. We thus set out to establish a method to selectively enrich arginine-phosphorylated proteins as an initial step in the phosphoproteomic analysis. For this purpose, we developed a substrate-trapping mutant of the YwlE phosphatase that retains binding affinity towards arginine-phosphorylated proteins but cannot hydrolyze the captured substrates. By testing a number of active site substitutions, we identified a YwlE mutant (C9A) that stably binds to arginine-phosphorylated proteins. We further improved the substrate-trapping efficiency by impeding the oligomerization of the phosphatase mutant. The engineered YwlE trap efficiently captured arginine-phosphorylated proteins from complex B. subtilis DeltaywlE cell extracts, thus facilitating identification of phosphoarginine sites in the large pool of cellular protein modifications. In conclusion, we present a novel tool for the selective enrichment and subsequent MS analysis of arginine phosphorylation, which is a largely overlooked protein modification that may have an important impact for eukaryotic cell signaling. Chasing phosphoarginine proteins: development of a selective enrichment method using a phosphatase trap.,Trentini DB, Fuhrmann J, Mechtler K, Clausen T Mol Cell Proteomics. 2014 May 13. pii: mcp.O113.035790. PMID:24825175[2] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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