Abstract 2347 Characterization of the metal-binding properties of arginyltransferase 1 (ATE1)

Arginylation is a critically important eukaryotic post-translational modification that modulates protein stability and regulates protein half-life thereby affecting global cellular homeostasis. Protein arginylation is catalyzed by arginyltransferase 1 (ATE1), an enzyme nearly conserved across the eukaryotic domain that transfers an Arg moiety from an aminoacylated Arg-tRNAArg to an acceptor protein in a non-ribosomal, ATP-independent process. We have recently determined the X-ray crystal structure of Saccharomyces cerevisiae ATE1 (ScATE1), which revealed the presence of a bilobed structure and the GCN5-related N-acetyltransferase fold (GNAT) enzymatic active site. A cryo-EM structure of ScATE1 in complex with uncharged tRNA confirmed these results and revealed that the 3' hydroxyl end of the tRNA molecule buries itself inside this polar GNAT fold. Adjacent to this region in three-dimensional space is the presence of the conserved N-terminal regulatory domain of ATE1 that contains four critical Cys residues. We have shown that this regulatory domain binds an O2-sensitive [Fe-S] cluster that regulates arginylation both in vitro as well as in vivo. However, the cryo-EM structure as well as a homologous ATE1 structure suggests the presence of a Zn2+ ion in this location when ATE1 is purified from recombinant sources. To date, no evidence has shown the relevance of this Zn2+ ion to the function of ATE1, and we hypothesize that the metal-binding domain of ATE1 may be promiscuous. To test this hypothesis, I have performed in vitro metal-binding experiments in which several thiophilic metal ions (Co2+, Zn2+, and Cd2+) were titrated into purified apo ScATE1. The presence of any of these metals generally disrupts the fold of ScATE1, with Cd2+ showing the most notable destabilization. For the protein that remains stable, the presence of Zn2+ or Cd2+ appears to results in a compaction of structure based on changes in size-exclusion elution profiles of metal-bound ScATE1. Enzymatic assays and structural studies are currently underway to understand the effects (if any) that these non-native metals illicit on the function and the structure of ScATE1. The results of these studies will help define the metal-binding properties of ATE1 and will determine how the presence of these metals regulates the structure and the function of this enzyme that is critical to eukaryotic homeostasis. The research was funded by NIGMS grant R35GM133497.