Two- and three-dimensional (2D and 3D) NMR techniques have been used to assign the signals from nearly all of the protons in Lactobacillus casei dihydrofolate reductase (DHFR) (M(r) 18,300) in its 11 complex with the antibacterial drug trimethoprim. A sample of uniformly 15N-labeled protein was examined using 3D 15N/1H experiments [nuclear Overhauser, heteronuclear multiple quantum coherence (NOESY-HMQC) and total correlation, heteronuclear multiple quantum coherence (TOCSY-HMQC) experiments]. Twenty-two intermolecular NOEs between trimethoprim and protein protons and four intramolecular NOEs in the ligand have been detected. Some were obtained by using heteronuclear editing and 2D HMQC-NOESY experiments on complexes formed with 15N-and 13C-labeled trimethoprim molecules ([1,3-15N2,2-amino-15N]-and [7-13C,4'-methoxy-13C]trimethoprim) bound to unlabeled protein. The ligand-protein NOEs were used as distance constraints in conjunction with minimum energy and simulated annealing calculations (carried out with X-PLOR) to dock the trimethoprim ligand into dihydrofolate reductase, using as a starting structure the crystal coordinates from a related complex with a similar overall protein structure. The restrained minimum energy calculations and the simulated annealing calculations gave 83 calculated structures with distance violations of < 0.1 A. In all of these, the two aromatic rings of trimethoprim occupied essentially the same region of conformational space in the binding site (RMSD = 0.63 A). The protein residues nearest to the bound trimethoprim were found to be very similar in all of the structures and agreed well with corresponding contact residues observed in the X-ray crystal studies on trimethoprim complexes formed with Escherichia coli and chicken liver DHFRs.