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Published June 23, 2020 | Supplemental Material + Published
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Recognition of nonproline N-terminal residues by the Pro/N-degron pathway


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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© 2020 National Academy of Sciences. Published under the PNAS license. Edited by F. Ulrich Hartl, Max Planck Institute of Chemistry, Martinsried, Germany, and approved May 13, 2020 (received for review April 14, 2020). PNAS first published June 8, 2020. We thank Wolfram Tempel for help with data collection and structure determination. Experimental data of this study were produced, in part, through experiments at the Argonne National Laboratory and Structural Biology Center at the Advanced Photon Source. The Structural Biology Center Collaborative Access Team is operated by the University of Chicago Argonne, LLC, for the US Department of Energy and the Office of Biological and Environmental Research under contract DE-AC02-06CH11357. This work is supported by Natural Sciences and Engineering Research Council Grant RGPIN-2016-06300 and by the Structural Genomics Consortium (to J.M.). The Structural Genomics Consortium is a registered charity (number 1097737) that receives funds from AbbVie, Bayer Pharma AG, Boehringer Ingelheim, Canada Foundation for Innovation, Eshelman Institute for Innovation, Genome Canada through the Ontario Genomics Institute (OGI-055), Innovative Medicines Initiative (European Union/European Federation of Pharmaceutical Industries and Associations) (ULTRA-DD Grant 115766), Janssen, Merck, Novartis Pharma AG, Ontario Ministry of Research, Innovation, and Science, Pfizer, São Paulo Research Foundation-Fundaçao de Amparo à Pesquisa do Estado de Sao Paulo, Takeda, and Wellcome. This work was also supported by the National Natural Science Foundation of China, Grant 31900865 (to C.D.) and by the NIH Grants 1R01DK039520 and 1R01GM031530 (to A.V.). Data Availability: The atomic coordinates and structure factors of GID4-IGLWKS and GID4-VGLWKS complexes have been deposited in the Protein Data Bank (https://www.rcsb.org/) under the accession codes 6WZX and 6WZZ, respectively. All other relevant data in the paper are entirely available through both text and figures in the main text and SI Appendix (122, 123). Author contributions: C.D., S.-J.C., A.M., A.J., A.V., and J.M. designed research; C.D., S.-J.C., A.M., S.W., and K.S. performed research; C.D., S.-J.C., A.M., S.W., K.S., A.J., A.V., and J.M. analyzed data; and C.D., S.-J.C., A.M., A.J., A.V., and J.M. wrote the paper. The authors declare no competing interest. This article is a PNAS Direct Submission. Data deposition: The atomic coordinates and structure factors of GID4-IGLWKS and GID4-VGLWKS complexes have been deposited in the Protein Data Bank (https://www.rcsb.org/) under the accession codes 6WZX and 6WZZ, respectively. This article contains supporting information online at https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.2007085117/-/DCSupplemental.

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August 22, 2023
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