Molecular insights into ZER1 recognition of N-terminal residue mutations.

Abstract

N-terminal glycine (Gly/N-degron), as a degradation signal, can be recognized by specific E3 ubiquitin ligases and plays a crucial role in protein degradation and cellular homeostasis. As a substrate receptor in the Cullin 2-RING E3 ligase complex, ZER1 mediates protein degradation via the Gly/N-degron pathway by recognizing N-terminal glycine and other small residues. This study employed all-atom molecular dynamics (MD) simulations and binding free energy calculations to explore ZER1's recognition of the wild-type peptide GFLHVGQD (WT) and its N-terminal mutants (G1S, G1A, G1T, and G1C). The results show that van der Waals and electrostatic interactions are the primary driving forces stabilizing the ZER1-peptide complex. While N-terminal mutations moderately enhanced binding affinity, their impact on the overall structural stability of ZER1 was minimal. Per-residue energy decomposition revealed that the N-terminal residue is vital for subsequent recognition and degradation processes, whereas the second (F2) and third (L3) residues play dominant roles at the binding interface, contributing most significantly to binding free energy. Hydrogen bond analysis further highlighted the critical roles of key residues, F2 and H4, in anchoring the peptide within the ZER1 binding pocket. This study provides molecular-level insights into the Gly/N-degron pathway, emphasizing the role of the N-terminal residue and the critical contributions of adjacent residues. The findings offer a theoretical foundation for further exploration of protein degradation mechanisms and the development of therapeutic strategies targeting ZER1-mediated pathways.