¹H, ¹⁵N and ¹³C resonance assignments, secondary structure, and the conformation of substrate in the binary folate complex of Escherichia coli dihydrofolate reductase

Summary

By using fully ¹⁵N- and ¹⁵N/¹³C-labeled Escherichia coli dihydrofolate reductase, the sequence-specific ¹H and ¹⁵N NMR assignments were achieved for 95% of the backbone resonances and for 90% of the ¹³C^α resonances in the binary folate complex. These assignments were made through a variety of three-dimensional proton-detected ¹⁵N and ¹³C experiments. A smaller but significant subset of side-chain ¹H and ¹³C assignments were also determined. In this complex, only one ¹⁵N or ¹³C resonance was detected per ¹⁵N or ¹³C protein nucleus, which indicated a single conformation. Proton-detected ¹³C experiments were also performed with unlabeled DHFR, complexed with ¹³C-7/¹³C-9 folate to probe for multiple conformations of the substrate in its binary complex. As was found for the protein resonances, only a single bound resonance corresponding to a productive conformation could be detected for C-7. These results are consistent with an earlier report based on ¹H NMR data [Falzone, C.J. et al. (1990) Biochemistry, 29, 9667–9677] and suggest that the E. coli enzyme is not involved in any catalytically unproductive binding modes in the binary complex. This feature of the E. coli enzyme seems to be unique among the bacterial forms of DHFR that have been studied to date.