Molecular mechanism of ZER1-mediated recognition of N-terminal glycine in protein degradation

The N-terminal glycine (Gly/N-degron) serves as a degradation signal recognized by specific E3 ligases, playing a crucial role in protein quality control and maintaining intracellular protein homeostasis. Zinc finger E3 ubiquitin protein ligase 1 (ZER1), a substrate receptor of the Cullin 2-RING E3 ubiquitin ligase, recognizes N-terminal glycine and other small N-terminal residues (such as serine, alanine, and cysteine), mediating protein degradation through the Gly/N-degron pathway. In this study, we employed all-atom molecular dynamics (MD) simulations and binding free energy calculations to study the binding mode of ZER1 with N-terminal glycine and the effects of key residue mutations on this recognition process. The results show that the binding of glycine-containing peptides and key residue mutations have minimal impact on ZER1 structural stability. During ZER1-peptide binding, van der Waals and electrostatic interactions are the primary driving forces. Residues W552, N553, D556, N597, I678, N579, R681, and K716 play significant roles in the recognition process, with mutations in these residues affecting the electrostatic distribution and hydrophobic properties of the ZER1 binding site, thereby influencing peptide binding. Additionally, ZER1 forms a complex hydrogen-bond network with the peptide, which stabilizes the peptide like an anchor. D556A and N597A disrupt the hydrogen bond between the N-terminal residue (G1) and ZER1. These findings provide a molecular-level understanding of the N-terminal degron pathway and lay a foundation for future related research.