Effects of Distal Mutations on Ligand-Binding Affinity in <i>E. coli</i> Dihydrofolate Reductase
Chen‐Hua Huang, Yun‐Wen Chen, Tsun-Tsao Huang, Ya‐Ting Kao
Abstract
DHFR and each mutant. We propose that the effects of these distal mutations on ligand-binding affinity stem from the spatial steric hindrance, the disturbance on the hydrogen network, or the modification of the protein flexibility. The modified N-terminus tag in DHFR acts as a cap on the entrance of the substrate-binding cavity, squeezes the adenosine binding subdomain, and influences the binding of NADPH in some mutants. If the mutation positions are away from the N-terminus tag and the adenosine binding subdomain, the additive effects due to the N-terminus tag were not observed. In the double-mutant-cycle analysis, double mutations show nonadditive properties upon either cofactor or substrate binding. Also, in general, the first point mutation strongly affects the ligand binding compared to the second one.