Structural investigations of arginyltransferases

Abstract

Arginyltransferases (ATE1s) are a class of essential eukaryotic enzymes that catalyze arginylation, the post‐translational transfer of Arg from an aminoacylated tRNA to a range of protein targets. This modification typically occurs at N‐terminal acidic residues, though ATE1 can also covalently attach Arg to mid‐chain residues. Arginylation may have either degradative or non‐degradative effects. For example, some arginylated proteins are processed through the Arg N‐degron pathway, which marks these post‐translationally modified proteins for degradation by the ubiquitin‐proteasome system. In contrast, arginylation may also manifest non‐degradative effects such as thermodynamic stability, subcellular relegalization, or functional changes. The diversity of ATE1’s targets confers its role as a global regulator, influencing functions such as cardiovascular development, neurological processing, and even the stress response. However, a lack of ATE1 structural knowledge has limited the determination of its three‐dimensional fold, how it is regulated, and how it recognizes its substrates. Using a combination of X‐ray crystallography, cryo‐EM, and size‐exclusion chromatography‐coupled small angle X‐ray scattering (SEC‐SAXS), our lab has successfully solved the structure of Saccharomyces cerevisiae ATE1 (ScATE1). The three‐dimensional structure of ScATE1 reveals a bilobed protein containing a canonical GCN5‐related N‐acetyltransferase (GNAT) fold. Structural superpositions and electrostatic analyses indicate this domain as the location of catalytic activity and tRNA binding. Furthermore, the structure reveals the spatial connectivity of the N‐terminal domain, which we previously showed binds an [Fe‐S] cluster, to the enzymatic active site, hinting at the cluster’s regulatory influence. As the first atomic‐level structure of any ATE1, this achievement brings us closer to answering pressing questions regarding the molecular‐level mechanism of eukaryotic post‐translational arginylation.