Structural comparisons of complexes of methotrexate analogs with Lactobacillus casei dihydrofolate reductase by two dimensional proton NMR at 500 MHz

Abstract

We have used two-dimensional (2D) NMR methods to examine complexes of Lactobacillus casei dihydrofolate reductase and methotrexate (MTX) analogues having structural modifications of the benzoyl ring [the 3'',5''-dichloro and 3'',5''-dichloro analogues (II and III)] and also the glutamic acid moiety [the .alpha.- and .gamma.-monoamides (IV and V)]. Assignments of the 1H signals in the spectra of the various complexes were made by comparison of their 2D spectra with those of complexes containing methotrexate where we have previously assigned resonances from 32 of the 162 amino acid residues. In the complexes formed with the dihalomethotrexate analogues, the glutamic acid and pteridine ring moieties were shown to bind to the enzyme in a manner similar to that found in the methotrexate-enzyme complex. Perturbations in 1H chemical shifts of protons in Phe-49, Leu-54, and Leu-27 and the methotrexate H7 and NMe protons were observed in the different complexes and were accounted for by changes in orientation of the benzoyl ring in the various complexes (15.degree. and 25.degree. in the difluoro- and dichloromethotrexate complexes, respectively). Binding of oxidized or reduced coenzyme (NADP+ or NADPH) to the binary complexes did not result in different shifts for Leu-27, Leu-54, or Leu-19 protons, and thus, the orientation of the benzoyl ring of the methotrexate analogues is not perturbed greatly by the presence of either oxidized or reduced coenzyme. In the complex with the .gamma.-monoamide analogue, the 1H signals of assigned residues in the protein had almost identical shifts with the corresponding protons in the methotrexate-enzyme complex for all residues except His-28 and, to a lesser extent, Leu-27. This indicates that while the His-28 interaction with the MTX .gamma.-CO2- is no longer present in this complex with the .gamma.-amide, there has not been a major change in the overall structure of the two complexes. This behavior contrasts with that seen for the .alpha.-amide complex where 1H signals from protons in several amino acid residues are different compared with their values in the complex formed with methotrexate. The residues most affected are all near to the benzoyl ring of methotrexate: this indicates that the disruption of the .alpha.-CO2- interaction with Arg-57 is accompanied by a change in orientation of the benzoyl ring.