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

Dong, Cheng and Chen, Shun-Jia and Melnykov, Artem and Weirich, Sara and Sun, Kelly and Jeltsch, Albert and Varshavsky, Alexander and Min, Jinrong (2020) Recognition of nonproline N-terminal residues by the Pro/N-degron pathway. Proceedings of the National Academy of Sciences of the United States of America, 117 (25). pp. 14158-14167. ISSN 0027-8424. PMCID PMC7322002. https://resolver.caltech.edu/CaltechAUTHORS:20200609-091521870

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Abstract

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.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1073/pnas.2007085117DOIArticle
https://www.pnas.org/content/suppl/2020/06/07/2007085117.DCSupplementalPublisherSupporting Information
http://www.ncbi.nlm.nih.gov/pmc/articles/pmc7322002/PubMed CentralArticle
ORCID:
AuthorORCID
Dong, Cheng0000-0002-2891-8759
Chen, Shun-Jia0000-0002-5489-4930
Melnykov, Artem0000-0002-4733-4249
Varshavsky, Alexander0000-0002-4011-258X
Additional Information:© 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.
Funders:
Funding AgencyGrant Number
Department of Energy (DOE)DE-AC02-06CH11357
Natural Sciences and Engineering Research Council of Canada (NSERC)RGPIN-2016-06300
Structural Genomics ConsortiumUNSPECIFIED
AbbVieUNSPECIFIED
Bayer Pharma AGUNSPECIFIED
Boehringer IngelheimUNSPECIFIED
Canada Foundation for InnovationUNSPECIFIED
Eshelman Institute for InnovationUNSPECIFIED
Genome CanadaUNSPECIFIED
Ontario Genomics InstituteOGI-055
Innovative Medicines InitiativeUNSPECIFIED
European UnionUNSPECIFIED
European Federation of Pharmaceutical Industries and Associations115766
JanssenUNSPECIFIED
MerckUNSPECIFIED
Novartis Pharma AGUNSPECIFIED
Ontario Ministry of Research, Innovation, and ScienceUNSPECIFIED
PfizerUNSPECIFIED
Fundaçao de Amparo à Pesquisa do Estado de Sao Paulo (FAPESP)UNSPECIFIED
TakedaUNSPECIFIED
Wellcome TrustUNSPECIFIED
National Natural Science Foundation of China31900865
NIH1R01DK039520
NIH1R01GM031530
Subject Keywords:degron; GID; GID4; ubiquitin; degradation
Issue or Number:25
PubMed Central ID:PMC7322002
Record Number:CaltechAUTHORS:20200609-091521870
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20200609-091521870
Official Citation:Recognition of nonproline N-terminal residues by the Pro/N-degron pathway. Cheng Dong, Shun-Jia Chen, Artem Melnykov, Sara Weirich, Kelly Sun, Albert Jeltsch, Alexander Varshavsky, Jinrong Min. Proceedings of the National Academy of Sciences Jun 2020, 117 (25) 14158-14167; DOI: 10.1073/pnas.2007085117
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:103790
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:09 Jun 2020 17:55
Last Modified:06 Aug 2020 18:36

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