Lysine residues are key to the formation of spatial structures of proteins and the regulation protein functions. Lysine is one of the three ribosomally coded amino acid residues with positively charged side chains at physiological pH. Its side chain can be involved in manifold noncovalent interactions in a protein, including van der Waals interactions, hydrogen bonds and electrostatic interactions with negatively charged residues. For example, salt bridge formation between lysine residues and acidic residues is thought to be important in forming the leucine zipper structure18
. The lysine residue plays a key role in general acid-base catalyzed enzymatic reactions in which proton transfer is required19
. Neutralization of the basic side chain of lysine could lead to significant effects on protein function, as has been described for lysine acetylation, ubiquitination and methylation13,20
. Given the importance of lysine in protein folding and function, it is expected that lysine succinylation is likely to have a significant impact on the substrate proteins.
Lysine succinylation induces more substantial changes to a protein's chemical properties than do lysine methylation and acetylation, two PTMs known to have important cellular roles. At physiological pH (7.4), succinylation, acetylation and monomethylation at a lysine residue will change the charge status from +1 to −1, from +1 to 0 and not at all, respectively. Thus, the change of charge status at the lysine residue caused by succinylation is comparable to that caused by protein phosphorylation at serine, threonine or tyrosine residues (from 0 to −2 charges). In addition, succinylation adds a bigger structural moiety than acetylation or methylation. Accordingly, the more dramatic structural alteration resulting from lysine succinylation, as demonstrated in the mutagenesis experiment, is likely to lead to more significant changes in protein structure and function.
Enzymatic reactions of short-chain lysine acylations, such as acetylation, propionylation and butyrylation, use their corresponding high-energy CoAs to perform reactions. By analogy to these reactions, and in combination with our isotopic succinate labeling experiments and observations of differential succinylation among cancer cell lines (Supplementary Fig. 12
), we therefore propose that succinyl-CoA is the cofactor of enzyme-mediated lysine succinylation. Succinyl-CoA is an important metabolic intermediate in a variety of metabolic pathways, including TCA cycle, porphyrin synthesis and catabolism of odd-chain fatty acids and some branched-chain amino acids. Homeostasis of succinyl-CoA is critical to normal cellular physiology. Mutations in the genes that are involved in succinyl-CoA metabolism, such as ketoglutarate dehydrogenase, succinyl-CoA-3-ketoacid-coenzyme A transferase and succinyl-CoA synthetase21–23
, could lead to diseases.
In summary, we present evidence to conclusively establish lysine succinylation as a new PTM. Given the high abundance of lysine succinylation and its induced chemical changes, it is highly likely that lysine succinylation could have important cellular functions. This study provides a stepping stone for dissection of this PTM pathway and studies of its biological significance.