Recognition of nonproline N-terminal residues by the Pro/N-degron pathway

Dong, Cheng; Chen, Shun-Jia; Melnykov, Artem; Weirich, Sara; Sun, Kelly; Jeltsch, Albert; Varshavsky, Alexander; Min, Jinrong

doi:10.1073/pnas.2007085117

Title: Recognition of nonproline N-terminal residues by the Pro/N-degron pathway

Journal Article · 08 June 2020 · Proceedings of the National Academy of Sciences of the United States of America

DOI: https://doi.org/10.1073/pnas.2007085117 · OSTI ID:1642239

^[1];

^[2]; Melnykov, Artem ^[3]; Weirich, Sara ^[4]; Sun, Kelly ^[5]; Jeltsch, Albert ^[4]; Varshavsky, Alexander ^[2]; Min, Jinrong ^[6]

Tianjin Medical Univ. (China)
California Inst. of Technology (CalTech), Pasadena, CA (United States)
Auragent Bioscience, St. Louis, MO (United States)
Univ. Stuttgart (Germany)
Univ. of Toronto, ON (Canada)
Univ. of Toronto, ON (Canada); Central China Normal Univ., Wuhan (China)

Eukaryotic N-degron pathways are proteolytic systems whose unifying feature is their ability to recognize proteins containing N-terminal (Nt) degradation signals called N-degrons, and to target these proteins for degradation by the 26S proteasome or autophagy. GID4, a subunit of the GID ubiquitin ligase, is the main recognition component of the proline (Pro)/N-degron pathway. GID4 targets proteins through their Nt-Pro residue or a Pro at position 2, in the presence of specific downstream sequence motifs. Here we show that human GID4 can also recognize hydrophobic Nt-residues other than Pro. One example is the sequence Nt-IGLW, bearing Nt-Ile. Nt-IGLW binds to wild-type human GID4 with a $$K_d$$ of 16 μM, whereas the otherwise identical Nt-Pro–bearing sequence PGLW binds to GID4 more tightly, with a $$K_d$$ of 1.9 μM. Despite this difference in affinities of GID4 for Nt-IGLW vs. Nt-PGLW, we found that the GID4-mediated Pro/N-degron pathway of the yeast Saccharomyces cerevisiae can target an Nt-IGLW–bearing protein for rapid degradation. We solved crystal structures of human GID4 bound to a peptide bearing Nt-Ile or Nt-Val. We also altered specific residues of human GID4 and measured the affinities of resulting mutant GID4s for Nt-IGLW and Nt-PGLW, thereby determining relative contributions of specific GID4 residues to the GID4-mediated recognition of Nt-Pro vs. Nt-residues other than Pro. These and related results advance the understanding of targeting by the Pro/N-degron pathway and greatly expand the substrate recognition range of the GID ubiquitin ligase in both human and yeast cells.

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Research Organization:: Argonne National Laboratory (ANL), Argonne, IL (United States). Advanced Photon Source (APS)

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER); Natural Sciences and Engineering Research Council of Canada (NSERC); AbbVie; Bayer Pharma AG; Boehringer Ingelheim; Canada Foundation for Innovation (CFI); Eshelman Institute for Innovation; Ontario Genomics Institute (OGI); European Union (EU); European Federation of Pharmaceutical Industries and Associations; Janssen; Merck; Novartis Pharma AG; Ontario Ministry of Research, Innovation, and Science; Pfizer; São Paulo Research Foundation (FAPESP); Takeda; Wellcome; National Natural Science Foundation of China (NSFC); National Institutes of Health (NIH)

Grant/Contract Number:: AC02-06CH11357; RGPIN-2016-06300; 115766; 31900865; 1R01DK039520; 1R01GM031530

OSTI ID:: 1642239

Journal Information:: Proceedings of the National Academy of Sciences of the United States of America, Vol. 117, Issue 25; ISSN 0027-8424

Publisher:: National Academy of SciencesCopyright Statement

Country of Publication:: United States

Language:: ENGLISH

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