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.

中文翻译：

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GID/CTLH E3 连接酶对 N-degron 的多方面识别和泛素化

N-degron E3 泛素连接酶识别底物 N 端的特定残基。尽管 N-degron 识别的分子细节对于几种 E3 连接酶是已知的，但可以结合给定 E3 底物结合域的 N 末端基序的范围仍不清楚。在这里，研究酵母“GID”/人“CTLH”多蛋白 E3 连接酶的 Gid4 和 Gid10 底物受体亚基，其已知底物带有 N 端脯氨酸，我们发现了与不同 N 端序列高亲和力结合的能力，部分确定按上下文。筛选具有暴露的 N 末端的噬菌体展示肽库，鉴定了具有非 Pro N 末端残基的新型共有基序，以高亲和力明显结合 Gid4 或 Gid10。结构数据显示 Gid4 和 Gid10 中的灵活环符合不同相互作用肽序列的互补折叠。连同对内源性底物降解的分析，数据显示去核身份、含有靶向赖氨酸的底物域和不同的 E3 连接酶高阶组装组合决定了泛素化和降解的效率。