Chemoproteomic identification of CO₂-dependent lysine carboxylation in proteins

Carbon dioxide is an omnipresent gas that drives adaptive responses within organisms from all domains of life. The molecular mechanisms by which proteins serve as sensors of CO₂ are, accordingly, of great interest. Because CO₂ is electrophilic, one way it can modulate protein biochemistry is by carboxylation of the amine group of lysine residues. However, the resulting CO₂-carboxylated lysines spontaneously decompose, giving off CO₂, which makes studying this modification difficult. Here we describe a method to stably mimic CO₂-carboxylated lysine residues in proteins. We leverage this method to develop a quantitative approach to identify CO₂-carboxylated lysines of proteins and explore the lysine 'carboxylome' of the CO₂-responsive cyanobacterium Synechocystis sp. We uncover one CO₂-carboxylated lysine within the effector binding pocket of the metabolic signaling protein PII. CO₂-carboxylatation of this lysine markedly lowers the affinity of PII for its regulatory effector ligand ATP, illuminating a negative molecular control mechanism mediated by CO₂.