Crystal structure of avian dihydrofolate reductase containing phenyltriazine and NADPH.

Abstract

The structure of chicken liver dihydrofolate reductase (5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase (EC 1.5.1.3)),in a ternary complex with NADPH and a phenyltriazine inhibitor, has been determined at a nominal resolution of 2.9 A. Overall backbone chain folding is very similar to that observed in Escherichia coli and Lactobacillus casei dihydrofolate reductases.About 70% of the additional residues present in the avian enzyme occur in three loops far removed from the substrate and cofactor binding sites.The most dramatic structural difference is at one edge of the central j3 sheet, where a stretch of six residues is inserted near the NHz-terminal end of strand BG.This insertion produces a looped out region of 11 residues that do not participate in classical antiparallel interchain hydrogen bonding.The triazine ring of the phenyltriazine inhibitor is in a position analogous to that occupied by the pyrimidine portion of methotrexate when the latter binds to bacterial dihydrofolate reductase.A hydrogen-bonded charge-charge interaction occurs between the carboxylate side chain of Glu-30 and the inhibitor's N1 and 2amino group.This interaction closely resembles a similar one between Asp-27 (E.coli) and the pteridine ring of methotrexate.The inhibitor's phenyl ring occupies a space analogous to that utilized for binding the pyrazine and C9-NlO portion of methotrexate.NADPH lies in a long shallow groove winding across one face of the enzyme molecule.As in L. casei dihydrofolate reductase, the cofactor is held in an extended conformation by numerous hydrophobic, hydrogenbonded, and ionic interactions.Changes in enzyme structure induced by phenyltriazine binding to the holoenzyme have been studied by difference Fourier methods.Large movements (greater than 3 A) are observed for the side chains of Glu-30 and Tyr-31.Other features in the difference map can be attributed to a variety of minor adjustments of secondary and tertiary structure, and to the displacement or rearrangement of bound solvent molecules.Two side chains, Cys-11 and Asn-13, which are 12 A from the active site, have also moved by more than 3 A. How-