Crystal structure of the Ate1 arginyl-tRNA-protein transferase and arginylation of N-degron substrates

Abstract

N-degron pathways are proteolytic systems that target proteins bearing N-terminal (Nt) degradation signals (degrons) called N-degrons. Nt-Arg of a protein is among Nt-residues that can be recognized as destabilizing ones by the Arg/N-degron pathway. A proteolytic cleavage of a protein can generate Arg at the N terminus of a resulting C-terminal (Ct) fragment either directly or after Nt-arginylation of that Ct-fragment by the Ate1 arginyl-tRNA-protein transferase (R-transferase), which uses Arg-tRNA Arg as a cosubstrate. Ate1 can Nt-arginylate Nt-Asp, Nt-Glu, and oxidized Nt-Cys* (Cys-sulfinate or Cys-sulfonate) of proteins or short peptides. Ate1 genes of fungi, animals, and plants have been cloned decades ago, but a three-dimensional structure of Ate1 remained unknown. A detailed mechanism of arginylation is unknown as well. We describe here the crystal structure of the Ate1 R-transferase from the budding yeast Kluyveromyces lactis . The 58-kDa R-transferase comprises two domains that recognize, together, an acidic Nt-residue of an acceptor substrate, the Arg residue of Arg-tRNA Arg , and a 3′-proximal segment of the tRNA Arg moiety. The enzyme’s active site is located, at least in part, between the two domains. In vitro and in vivo arginylation assays with site-directed Ate1 mutants that were suggested by structural results yielded inferences about specific binding sites of Ate1. We also analyzed the inhibition of Nt-arginylation activity of Ate1 by hemin (Fe 3+ -heme), and found that hemin induced the previously undescribed disulfide-mediated oligomerization of Ate1. Together, these results advance the understanding of R-transferase and the Arg/N-degron pathway.

Functionally annotating cysteine disulfides and metal binding sites in the plant kingdom using AlphaFold2 predicted structures

“…To showcase the potential relevance of these metal binding site predictions, we turned to a recently discovered metal ligand binding site in the arginyl-tRNA-protein transferase (ATE), which is conserved in eukaryotes. Whereas binding studies using recombinant Saccharomyces cerevisiae Ate1 indicate the presence of Fe-S clusters [45], the crystal structure of recombinant Kluyveromyces lactis Ate1 (Figure 2B -Left) identified instead a Cys4 Zn 2+ binding site in the corresponding binding pocket [44]. In the AlphaFold2 predicted structures of both yeast Ate1 as well as their plant orthologs, we identified consistently a Cys4 Zn 2+ binding site, like shown in Figure 2B for Chlamydomonas, Selaginella and Arabidopsis, and thus alike those reported for Kluyveromyces lactis.…”

“…Similarly, metal binding protein domains such as Cys2His2 zinc finger domains are more prevalent in land plants and, oppositely, lower proportions of Fe-S cluster containing domains, such as the [4Fe-4S] cluster in the radical SAM domain [43], are more abundant in green algae (Figure S4). [44] and AlphaFold2 predicted structures for Chlamydomonas (UniProtKB A0A2K3D0B7), Selaginella (D8RCG9), and Arabidopsis (Q9ZT48) orthologs. Zn 2+ ligands were modeled by the metal ligand searching algorithm [40].…”

“…For an overview per metal ligand see Figure S3. (B) Cys4 Zn 2+ binding site in Kluyveromyces lactis Arg-tRNA-protein transferase 1 (Ate1) (PDB 7WG4, green)[44] and AlphaFold2 predicted structures for Chlamydomonas (UniProtKB A0A2K3D0B7), Selaginella (D8RCG9), and Arabidopsis (Q9ZT48) orthologs. Zn 2+ ligands were modeled by the metal ligand searching algorithm[40].…”

Functionally annotating cysteine disulfides and metal binding sites in the plant kingdom using AlphaFold2 predicted structures

“…For an overview per metal ligand see Figure S3. ( B ) Cys 4 Zn 2+ binding site in Kluyveromyces lactis Arg-tRNA-protein transferase 1 (Ate1) (PDB 7WG4, green) [44] and AlphaFold2 predicted structures for Chlamydomonas (UniProtKB A0A2K3D0B7), Selaginella (D8RCG9), and Arabidopsis (Q9ZT48) orthologs. Zn 2+ ligands were modeled by the metal ligand searching algorithm [40].…”

“…To showcase the potential relevance of these metal binding site predictions, we turned to a recently discovered metal ligand binding site in the arginyl-tRNA-protein transferase (ATE), which is conserved in eukaryotes. Whereas binding studies using recombinant Saccharomyces cerevisiae Ate1 indicate the presence of Fe-S clusters [45], the crystal structure of recombinant Kluyveromyces lactis Ate1 (Figure 2B – Left ) identified instead a Cys 4 Zn 2+ binding site in the corresponding binding pocket [44]. In the AlphaFold2 predicted structures of both yeast Ate1 as well as their plant orthologs, we identified consistently a Cys 4 Zn 2+ binding site, like shown in Figure 2B for Chlamydomonas, Selaginella and Arabidopsis , and thus alike those reported for Kluyveromyces lactis .…”