Abstract 1180 Structural investigations of Mus musculus ATE1

Post-translational modifications (PTMs) are chemical alterations of the polypeptide that affect protein function through changes in oligomerization, activity, cellular localization, and other important molecular properties. Some PTMs involve the addition and/or removal of an amino acid. One such eukaryotic modification is arginylation, a PTM in which the enzyme arginyltransferase 1 (ATE1) catalyzes the non-ribosomal addition of the amino acid Arg to a polypeptide. While understudied, arginylation and ATE1 are critically essential to eukaryotic cellular homeostasis. We have recently reported the first structure of Saccharomyces cerevisiae (baker's yeast) ATE1 (ScATE1), but the structure of a mammalian ATE1 remains unknown. In this work, we have used AlphaFold to predict the structure of (Mus musculus) ATE1 isoform 1 (MmATE1-1). This calculated model suggests the presence of conserved structural features seen in ScATE1; however, the presence of an intrinsically disordered region (IDR) is also predicted. To explore the validity of this model, we have cloned, overproduced, and purified mouse MmATE1-1 for structural and biophysical studies. Purified mouse ATE1-1 produced high yields of soluble, monomeric protein that is calculated to have a slightly open conformation based on gel filtration and multi-angle light scattering (MALS) studies. While crystallization experiments are currently ongoing, we have analyzed MmATE1-1 using small-angle X-ray scattering (SAXS) and compared its SAXS profile to that of ScATE1. Additionally, we have begun to employ hydrogen-deuterium-exchange mass spectrometry (HDX-MS) to characterize further the solution state structure of MmATE1-1. When combined, these data illustrate that mammalian ATE1 is structurally more complex and more dynamic than its yeast paralog. Thank you to my funding sources as wells as the SIBYLS beamline. GRISE- T32-GM144876 CBI- T32 GM066706 R35 GM133497 SSRL Beamline 7 - 3.