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Published August 2, 2022 | Supplemental Material + Published
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Crystal structure of the Ate1 arginyl-tRNA-protein transferase and arginylation of N-degron substrates


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-tRNAᴬʳᵍ 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-tRNAᴬʳᵍ, and a 3′-proximal segment of the tRNAᴬʳᵍ 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 (Fe³⁺-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.

Additional Information

© 2022 the Author(s). Published by PNAS. This article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND). Contributed by Alexander Varshavsky; received June 3, 2022; accepted June 14, 2022; reviewed by Ulrich Hartl and William Tansey. We thank colleagues at the beamlines 5C and 11C, Pohang Accelerator Laboratory, South Korea, the beamline BL17A at the Photon Factory, Japan, and at the beamline BL44XU, Spring-8, Japan, for assistance with collecting X-ray data. This work was supported by National Research Foundation of Korea Grants 2020R1A2C3008285, 2020R1A5A1019023, 2021M3A9G8024747, and 2021M3A9I4030068 (to H.K.S.); Grants 2020R1A3B2078127 and 2017R1A5A1015366 (to C.-S.H); and NIH Grant GM031530 (to A.V.). Author contributions: B.H.K., M.K.K., and H.K.S. designed research; B.H.K., M.K.K., S.J.O., K.T.N., J.H.K., and C.-S.H. performed research; B.H.K., M.K.K., S.J.O., K.T.N., J.H.K., A.V., C.-S.H., and H.K.S. analyzed data; and A.V. and H.K.S. wrote the paper. Data Availability Statement: All relevant data in the paper are available in the main text, in SI Appendix, or in the Protein Data Bank under the following accession codes: 7WFX (EV_Ortho) (109), 7WG1 (DV_Ortho) (110), 7WG2 (EV_Tetra) (111), and 7WG4 (DV_Tetra) (112).

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August 22, 2023
December 22, 2023