¹⁵N NMR studies of the conformation of E. coli dihydrofolate reductase in complex with folate or methotrexate

We have employed ¹⁵N NMR to characterize the conformations of Escherichia coli dihydrofolate reductase (ECDHFR) in complex with [5-¹⁵N]folate or [5-¹⁵N]methotrexate (MTX). Two ¹⁵N resonances were observed for DHFR/MTX binary complex. The relative population of these two conformations is pH dependent. Addition of NADP⁺ or NADPH results in the disappearance of the low field resonance. In contrast, only one conformation was observed for both the DHFR/folate and DHFR/folate/NADP⁺ complexes. However, the ¹⁵N chemical shift of [5-¹⁵N]folate in the binary DHFR/folate complex is 7.28 ppm upfield from that of the ternary complex, suggesting the possible loss of a hydrogen bonding to N5 of folate in the ternary complex.