GID4 recognition of Pro/N-degron peptides: Conformational selection and induced fit

The N-degron pathway is essential for protein quality control and cellular homeostasis. GID4, a subunit of the GID ubiquitin ligase, is the main recognition component of the Pro/N-degron pathway. It binds protein substrates through their N-terminal proline, but its binding model and recognition of nonproline residues remain unclear. In this study, we performed molecular dynamics simulations and binding energy calculations to explore GID4's binding with Pro/N-degron peptides and nonproline residues. Our analysis revealed that in its apo state, GID4's hairpin loops (L1, L2, L3, and L4) facilitate alternating open-closed states in the binding pocket. This conformational change allows selective recognition of proteins with N-terminal degradation signals, followed by loop adaptation that secures the substrate. The binding process follows a combined mechanism of conformational selection and induced fit. Mutation of the N-terminal proline reduces its binding contribution but has minimal impact on interactions with other residues. These findings provide new insights into GID4's substrate recognition mechanisms, potentially aiding the regulation of protein degradation pathways.