Structural modeling and biophysical characterization of arginyl‐tRNA transferase (ATE1)

Abstract

Arginylation is an important post-translational modification (PTM) defined by the non-ribosomal transfer of the amino acid arginine from an aminoacylated tRNA to a diverse set of polypeptide substrates. This reaction is catalyzed by arginyl-tRNA transferase (ATE1), a eukaryotic enzyme that is essential to maintain normal cellular homeostasis. Importantly, research has linked dysfunction of cellular arginylation to neurodegenerative diseases as well as cancer metastasis; however, little is known about ATE1's structure, interaction with its substrates, and its mechanism of action. To guide our understanding of ATE1's structure, function, and mechanism, we have determined a structural model of Saccharomyces cerevisiae ATE1 (ScATE1) and characterized this protein biophysically. Our results have revealed interesting similarities to, and differences from, the bacterial leucyl/phenylalanyl-tRNA transferase (L/F-transferase). Based on our structural predictions, we hypothesize that target protein recognition in ScATE1 follows a similar mechanism as L/F-transferases. Through large-scale sequence alignments, we have found a highly conserved, positively charged His residue cradled within the postulated enzyme active site, which we believe makes electrostatic contacts with the target polypeptide that is arginylated. While we hypothesize that transferase interactions with their respective aminoacyl-tRNAs are also comparable, we use electrophoretic mobility shift assays to determine that ATE1 binds uncharged tRNA weakly, suggesting a more complex interaction process than previously believed. Overall, these data provide a promising guide for further study into the structure, function, and mechanism of ATE1s.