Ligand Binding Studies of a Plasmid Encoded Dihydrofolate Reductase by 19F NMR

Abstract

Plasmid encoded-R67 dihydrofolate reductase (DHFR) catalyzes the NADPH-dependent reduction of dihydrofolate (DHF) to tetrahydrofolate. R67 DHFR is a homotetramer with a single active site pore and two tryptophans per monomer (W38 and W45). W38 and its symmetry-related partners occur at the dimer-dimer interfaces while the W45 residues occur at the monomer-monomer interfaces. We have labeled these tryptophans using 19F-labeled indole with the 19F atoms at different positions (4-, 5-, 6- or 7-) of the indole ring. In vitro ligand binding studies of NADP+ to the apoprotein or a ternary complex with NADP+ and DHF showed characteristic spectra for each complex. The apoprotein gave rise to a sharp and a broad peak. Upon addition of NADP+, the sharp peak for W38 shows line broadening while the broad peak for W45 remains unchanged. In the ternary complex, the sharp peak in the apo and binary complex splits into three peaks while the broad peak remains unchanged. The appearance of three new resonances can be explained by how NADPH and DHF bind in the active site pore. Two symmetry related lysine 32s at the edge of one side of the pore constrain the position of NADPH by forming ionic interactions with the phosphate groups. However, on the other side of the pore, the glutamate tail of DHF is disordered and switches between ion pairs with the K32s on that side of the pore. This results in two different environments for the nearby W38 residue. Our NMR results are consistent with computational simulations of the glutamate-tail interacting with symmetry related lysine 32 residues at the edge of the pore. This approach will be valuable for determining the binding affinity of DHF towards R67 DHFR by in-cell NMR.