J. Am. Chem. Soc., published online 23 September 2011, doi: 10.1021/ja205137d

AMPylation is a recently discovered post-translational modification (PTM) reaction that involves transfer of an adenylyl 5′-monophosphate (AMP) group to the side chain of hydroxylated amino acids in proteins. The PTM is installed by AMPylator enzymes that use ATP as an AMP donor and contain either a filamentation induced by cAMP (fic) or adenylyl transferase (AT) catalytic domain. Many AMPylators, such as VopS, are bacterial virulence factors that appear to target eukaryotic small GTPases such as Cdc42, but AMPylators have recently been identified in humans (HYPE) and in Drosophila melanogaster (dFic). Though AMPylation appears to be widespread, the role of these PTMs and the substrate scope of AMPylators remains unclear. Grammel et al. now report a method for the selective tagging of AMPylated proteins using Cu(I)-catalyzed alkyne-azide cycloaddition reactions, or 'click' chemistry. The authors synthesized N6-propargyl adenosine 5′-triphosphate (N6pATP) and demonstrated that it functions as an effective ATP analog for all known classes of AMPylators and that the resulting AMPylated proteins can be selectively tagged with fluorescent or biotinylated groups through click chemistry. This bioorthogonal labeling strategy was applied to the validation of known AMPylation substrates from mammalian cell lysates on a proteome level, suggesting that it may be useful for future investigations of AMPylator substrate specificity in cells.