The structure-function relationship of ATE1 R-transferase of the autophagic Arg/N-degron pathway

ATE1 (arginyltransferase 1; EC 2.3.2) transfers the amino acid arginine (Arg) from Arg-tRNA^Arg to the N-terminal (Nt) residues of proteins, such as aspartate (Asp), glutamate (Glu), and oxidized cysteine (Cys). The resulting Nt-Arg acts as an N-degron that regulates the degradation of various biomaterials via the ubiquitin/Ub-proteasome system (UPS) or the autophagy-lysosome system (ALS). In the UPS, Arg/N-degrons are recognized by cognate N-recognins, leading to substrate ubiquitination and proteasomal degradation. In the ALS, the same degrons bind the macroautophagy/autophagy receptor SQSTM1/p62 (sequestosome 1) to facilitate self-polymerization of SQSTM1 associated with cargoes and SQSTM1 interaction with LC3-II on phagophores. A key unresolved question is why only a small subset of proteins acquires Arg/N-degrons, given the rather weak binding affinity of ATE1 for Nt-substrates. In this study, we determined the cryo-EM structures of human ATE1 in complex with Arg-tRNA^Arg and an Nt-Asp peptide. ATE1 harbors two adjacent pockets that each bind an Nt-substrate or Arg-tRNA^Arg, the latter being wrapped by a long, unstructured loop. In the apo state, two ATE1 monomers form a homodimer. ATE1 achieves the selectivity for its peptidyl-ligands through these multivalent interactions, with K_d values in the micro-molar range. These results reveal the structural principle of Nt-arginylation at the crossroads of the UPS and ALS.<b>Abbreviations</b>: ALS: autophagy-lysosome system; Arg: arginine; Asp: aspartate; ATE1: arginyltransferase 1; Cys: cysteine; CysO₂(H): Cys sulfinic acid; Glu: glutamate; Nt: N-terminal; UBR: ubiquitin protein ligase E3 component n-recognin; UPS: ubiquitin-proteasome system; ZZ: ZZ-type zinc finger.