THE SOLUTION STRUCTURE OF THE COMPLEX OF LACTOBACILLUS-CASEI DIHYDROFOLATE-REDUCTASE WITH METHOTREXATE

We have determined the three-dimensional solution structure of the com plex of Lactobacillus casei dihydrofolate reductase (18.3 kDa, 162 ami no acid residues) formed with the anticancer drug methotrexate using 2 531 distance, 361 dihedral angle and 48 hydrogen bond restraints obtai ned from analysis of multidimensional NMR spectra. Simulated annealing calculations produced a family of 21 structures fully consistent with the constraints. The structure has four a-helices and eight beta-stra nds with two other regions, comprising residues 11 to 14 and 126 to 12 7, also interacting with each other in a beta-sheet manner. The methot rexate binding site is very well defined and the structure around its glutamate moiety was improved by including restraints reflecting the p reviously determined specific interactions between the glutamate alpha -carboxylate group with Arg57 and the gamma-carboxylate group with His 28. The overall fold of the binary complex in solution is very similar to that observed in the X-ray studies of the ternary complex of L. ca sei dihydrofolate reductase formed with methotrexate and NADPH (the st ructures of the binary and ternary complexes have a root-mean-square d ifference over the backbone atoms of 0.97 Angstrom). Thus no major con formational change takes place when NADPH binds to the binary complex. In the binary complex, the loop comprising residues 9 to 23 which for ms part of the active site has been shown to be in the ''closed'' conf ormation as defined by M. R. Sawaya & J. Kraut, who considered the cor responding loops in crystal structures of complexes of dihydrofolate r eductases from several organisms. Thus the absence of the NADPH does n ot result in the ''occluded'' form of the loop as seen in crystal stud ies of some other dihydrofolate reductases in the absence of coenzyme. Some regions of the structure in the binary complex which form intera ction sites for NADPH are less well defined than other regions. Howeve r, in general terms, the NADPH binding site appears to be essentially pre-formed in the binary complex. This may contribute to the tighter b inding of coenzyme in the presence of methotrexate. (C) 1998 Academic Press Limited.