13C NMR studies of complexes of Escherichia coli dihydrofolate reductase formed with methotrexate and with folic acid

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Abstract
13C NMR studies of 13C‐labelled ligands bound to dihydrofolate reductase provide (DHFR) a powerful means of detecting and characterizing multiple bound conformations. Such studies of complexes of Escherichia coli DHFR with [4,7,8a,9‐13C]‐ and [2,4a,6‐13C]methotrexate (MTX) and [4,6.8a‐13C]‐ and [2,4a,7,9‐13C]folic acid confirm that in the binary complexes, MTX binds in two conformational forms and folate binds as a single conformation. Earlier studies on the corresponding complexes with Lactobacillus casei DHFR indicated that, in this case, MTX binds as a single conformation whereas folate binds in multiple conformational forms (both in its binary complex and ternary complex with NADP+); two of the bound conformational states for the folate complexes are very different from each other in that there is a 180° difference in their pteridine ring orientation. In contrast, the two different conformational states observed for MTX bound to E. coli DHFR do not show such a major difference in ring orientation and bind with N1 protonated in both forms. The major difference appears to involve the manner in which the 4‐NH2 group of MTX binds to the enzyme (although the same protein residues are probably involved in both interactions). Addition of either NADP+ or NADPH to the E. coli DHFR‐MTX complex results in a single set of 13C signals for bound methotrexate consistent with only one conformational form in the ternary complexes.