Abstract 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, studying the Gid4 and Gid10 substrate receptor subunits of yeast “GID”/human “CTLH” multiprotein E3 ligases, whose known substrates bear N-terminal prolines, we discovered capacity for high-affinity binding to diverse N-terminal sequences determined in part by context. Screening of phage displaying peptide libraries with exposed N-termini identified novel consensus motifs with non-Pro N-terminal residues distinctly binding Gid4 or Gid10 with high affinity. Structural data reveal that flexible loops in Gid4 and Gid10 conform to complementary folds of diverse interacting peptide sequences. 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.

Multifaceted N-degron recognition and ubiquitylation by GID/CTLH E3 ligases