The SMD module of the CSFp strategy was used to identify potential undesirable collisions of ligands entering and exiting the binding pocket and to alter the structural pre-organization in the unbound

The CSFp strategy's SMD module was employed to identify potential unfavorable collisions of ligands during entry and exit from the binding pocket, and to modify the structural pre-organization in the unbound protein state. D-PL, which possesses two hydrophobic methyl groups, exhibited a 23.5-fold lower activity against D-Lac compared to 2-hydroxybutyrolactone. This disparity may be attributed to the unfavorable collisions of D-PL's methyl group with the hydrophobic residues of the binding channel during entry and exit from the pocket. To investigate this hypothesis, SMD simulations were conducted to monitor the frequency of unfavorable collisions of D-PL with hydrophobic residues during the pull-off process. Asn96, Phe308, and Gln364 were found to form longer non-polar interactions with D-PL during this process, leading to increased difficulty in its movement. Subsequently, these residues were mutated to alanine, and the results revealed that N96A had no effect on activity, Q364A caused D-Lac inactivation, and F308A had 1.4-fold higher activity than WT. This suggests that the aromatic side chain of F308 impedes the entry and exit of D-Lac. To remove the spatial barrier of F308, F308 was mutated to a smaller glycine, and deletion of the F308 locus was considered. The results showed that Δ308 led to the complete inactivation of D-Lac, while F308G exhibited an 8-fold increase in activity compared to WT. MD simulations were utilized to investigate the inactivation of Δ308, which resulted in an overall decrease in loop flexibility near the binding pocket, leading to the inability of the loop to complete substrate recognition and binding