Multifaceted N-Degron Recognition and Ubiquitylation by GID/CTLH E3 Ligases

• Published in: Journal of molecular biology Vol. 434; no. 2; p. 167347
• Main Authors: Chrustowicz, Jakub, Sherpa, Dawafuti, Teyra, Joan, Loke, Mun Siong, Popowicz, Grzegorz M., Basquin, Jerome, Sattler, Michael, Prabu, J. Rajan, Sidhu, Sachdev S., Schulman, Brenda A.
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 30.01.2022
• Subjects: bacteriophages; complement; Humans; molecular biology; N-degron pathway; Phage display; Protein Binding; Protein Domains; Protein Interaction Domains and Motifs; Protein–protein interaction; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins - metabolism; Structural biology; Ubiquitin; Ubiquitin - metabolism; ubiquitin-protein ligase; Ubiquitin-Protein Ligases - chemistry; Ubiquitin-Protein Ligases - metabolism; Ubiquitination; yeasts; Ubiquitin; Phage display; N-degron pathway; Protein–protein interaction; Structural biology
• ISSN: 0022-2836; 1089-8638; 1089-8638
• DOI: 10.1016/j.jmb.2021.167347

[Display omitted] •Phage display identifies peptide motifs optimally binding GID/CTLH E3 ligases.•Substrate receptor loopsstructurally conform to diverse interacting peptides.•Naturally-occurring N-degrons bind GID/CTLH E3s suboptimally.•Degron, ubiquitylated domain, and E3 assembly combinatorially direct degradation. N-degron E3 ubiquitin ligases recognize specific residues at the N-termini of substrates. Although molecular details of N-degron recognition are known for several E3 ligases, the range of N-terminal motifs that can bind a given E3 substrate binding domain remains unclear. Here, we discovered capacity of Gid4 and Gid10 substrate receptor subunits of yeast “GID”/human “CTLH” multiprotein E3 ligases to tightly bind a wide range of N-terminal residues whose recognition is determined in part by the downstream sequence context. Screening of phage displaying peptide libraries with exposed N-termini identified novel consensus motifs with non-Pro N-terminal residues binding Gid4 or Gid10 with high affinity. Structural data reveal that conformations of flexible loops in Gid4 and Gid10 complement sequences and folds of interacting peptides. Together with analysis of endogenous substrate degrons, the data show that degron identity, substrate domains harboring targeted lysines, and varying E3 ligase higher-order assemblies combinatorially determine efficiency of ubiquitylation and degradation.