H-1/N-15 HSQC NMR studies of ligand carboxylate group interactions with arginine residues in complexes of brodimoprim analogues and Lactobacillus casei dihydrofolate reductase

H-1 and N-15 NMR studies have been undertaken on complexes of Lactobacillus casei dihydrofolate reductase (DHFR)-formed with analogues of the antibact erial drug brodimoprim (2,4-diamino-5-(3',5'-dimethoxy-4'-bromobenzyl)pyrim idine) in order to monitor interactions between carboxylate groups on the l igands and basic residues in the protein. These analogues had been designed by computer modeling with carboxylated alkyl chains introduced at the 3'-O position in order to improve their binding properties by making additional interactions with basic groups in the protein. Specific interactions betwe en ligand carboxylate groups and the conserved Arg57 residue have been dete cted in studies of H-1/N-15 HSQC spectra of complexes of DHFR with both the 4-carboxylate and the 4,6-dicarboxylate brodimoprim analogues. The spectra from both complexes showed four resolved signals for the four NHeta proton s of the guanidino group of Arg57, and this is consistent with hindered rot ation in the guanidino group resulting from interactions with the 4-carboxy late group in each analogue. In the spectra of each complex, one of the pro tons from each of the two NH2 groups and both nitrogens are considerably de shielded compared to the shielding values normally observed for such nuclei . This pattern of deshielding is that expected for a symmetrical end-on int eraction of the carboxylate oxygens with the NHeta 12 and NHeta 22 guanidin o protons. The differences in the degree of deshielding between the complex es of the two structurally similar brodimoprim analogues and the methotrexa te indicates that the shielding is very sensitive to geometry, most probabl y to hydrogen bond lengths. The H-1/N-15 HSQC spectrum of the DHFR complex with the brodimoprim-6-carboxylate analogue does not feature any deshielded Arg NHeta protons and this argues against a similar interaction with the A rg57 in this case, It has not proved possible to determine whether the 6-ca rboxylate in this analogue is interacting directly with any residue in the protein. H-1/N-15 HSQC spectra have been fully assigned for the complexes w ith the three brodimoprim analogues and chemical shift mapping used to expl ore interactions in the binding site. The H-1 signals of the bound ligands for all three brodimoprim analogues have been assigned. Their H-1 chemical shifts were found to be fairly similar in;the different complexes indicatin g that the 2,4-diaminopyrimidine and the benzyl ring are binding in essenti ally the same binding sites and with the same overall conformation in the d ifferent complexes. The rotation rate about the (NCzeta)-C-epsilon bond in the brodimoprim-4,6-dicarboxylate complex with DHFR has been determined fro m a zz-HSQC exchange experiment, and its value is quite similar to that obs erved in the DHFR methotrexate complex (24 +/- 10 s(-1) at 8 degrees C and 50 +/- 10 s(-1) at 15 degrees C, respectively). The H-1 and N-15 chemical s hift differences of selected amide and guanidino NH groups, measured betwee n the DHFR complexes, provided further evidence about the interactions invo lving Arg57 with the 4-carboxylate and 4,6-dicarboxylate brodimoprim analog ues.