Mutation-induced remodeling of the binding mechanism between GID4 and the PGLW peptide revealed by molecular dynamics simulations.

GID4 functions as a substrate receptor in the Pro/N-degron pathway by recognizing peptides with an N-terminal proline. While the overall binding mode has been characterized, the residue-specific mechanism underlying peptide recognition remains unclear. In this study, molecular dynamics simulations combined with MM-GB/SA calculations, per-residue energy decomposition, and hydrogen-bond analysis were performed to investigate the interaction between GID4 and the PGLW peptide and the effects of key residue mutations. The results show that peptide binding stabilizes the GID4 binding pocket and restrains the motions of surrounding hairpin loops. The relative binding free energy differences of Q132A, E237A, and Y258A compared with the WT system were 18.91, 18.94, and 20.62 kcal/mol, respectively. Per-residue decomposition identified E237 (-5.63 kcal/mol) and K135 (-4.54 kcal/mol) on GID4 and P1 (-7.83 kcal/mol), L3 (-10.01 kcal/mol), and W4 (-5.85 kcal/mol) on the peptide as major favorable contributors in the WT complex. Mutations of key residues, especially Q132, E237, and Y258, were associated with weakened calculated binding affinity, likely through disruption of electrostatic complementarity, local packing, and the extended hydrogen-bond network. Overall, the results suggest that the mutation-induced decrease in calculated binding affinity is associated with reorganization of the hydrogen-bond network and disruption of cooperative interactions at the binding interface.