Coupling effects of distal loops on structural stability and enzymatic activity of Escherichia coli dihydrofolate reductase revealed by deletion mutants.

Residues distal from the active site in dihydrofolate reductase (DHFR) have regulatory roles in catalytic reaction and also folding stability. The couplings of the distal residues to the ones in the active site have been analyzed using site-directed mutants. To expand our understanding of the structural and functional influences of distal residue mutation, we explored the structural stability and enzymatic activity of deletion mutants. Deletion has greater structural and dynamical impacts on the corresponding part than site-directed mutation does. Thus, deletion amplifies the effects caused by distal mutations, which should make the mutual couplings among the distant residues more apparent. We focused on residues 52, 67, 121, and 145 in the four distinct loops of DHFR. All the single-residue deletion mutants showed marked reduction in stability, except for Delta52 in an alphaC-betaC loop. Double deletion mutants showed that the loop alphaC-betaC has nonadditive couplings with the betaF-betaG and betaG-betaH loops regarding stability. Single deletion to the loops alphaC-betaC or betaC-betaD resulted in considerable activity reduction, demonstrating that the loops couple to the residues near the active site. The four loops were shown to be functionally interdependent from the double deletion experiments.