The Impact of Second-Shell Residues on Substrate Binding at the Active Site of Thymidylate Synthase from...

Employing classical alchemical free-energy simulations, we quantitatively evaluated how mutations in second-shell residues (Y44, Q106, Y108, and T181) affect the energetics of substrate (dUMP) binding to the active site of MtbThyX, an important drug target for Mycobacterium tuberculosis. The substrate binding affinity of nine mutants was compared with the wild-type MtbThyX. We found that specific mutations (viz., T181F or T181L) in the second-shell residue can decrease substrate binding by more than 8 kcal/mol. The decrease in substrate binding affinity is clearly demonstrated by the disruption of the active site architecture observed in molecular dynamics structures. These structures indicate increased exposure of the active site to the solvent, disruption of essential electrostatic interactions, and a change in the relative positioning of the substrate (dUMP) and the cofactor (FAD). The calculated free energy changes (ΔΔG) provide a plausible explanation for previous observations, showing that some mutations have mild or no impact on bacterial fitness (e.g., Y108F, T181S, and T181A), while others (e.g., T181F, Y44F, Y108S, Y44A, Q106A, and T181L) are detrimental. Good convergence of the estimated free energy differences (uncertainty below 1 kcal/mol) was achieved through about 30 μs of total sampling across several independent simulations. This study explores the relationships between thermodynamics and structural characteristics (including wild-type and mutant forms) to clarify the mechanism of substrate recognition in the active site of MtbThyX. This approach may offer important insights into how protein mutations affect substrate binding in other ThyX homologues.