Essential protein-protein interactions between Plasmodium falciparum thymidylate synthase and dihydrofolate reductase domains

In Plasmodium falciparum, dihydrofolate reductase and thymidylate synthase activities are conferred by a single 70-kDa bifunctional polypeptide (DHFR-TS, dihydrofolate reductase-thymidylate synthase) which assembles into a functional 140-kDa homodimer, In mammals, the two enzymes are smaller distinct molecules encoded on different genes, A 27-kDa amino domain of malarial DHFR-TS is sufficient to provide DHFR activity, but the structural requirements for TS function have not been established, Although the 3'-end of DHFR-TS has high homology to TS sequences from other species, expression of this protein fragment failed to yield active TS enzyme, and it failed to complement TS- Escherichia coli, Unexpectedly, even partial 5'-deletion of full-length DHFR-TS gene abolished TS function:on the 3'-end, Thus, it was hypothesized that the amino end of the bifunctional parasite protein plays an important role in TS function. When the 27-kDa amino domain (DHFR) was provided in trans, a previously inactive 40-kDa carboxyl domain from malarial DHFR-TS regained its TS function. Physical characterization of the "split enzymes" revealed that the 27- and the 40-kDa fragments of DHFR-TS had reassembled into a 140-kDa hybrid complex, Thus, in malarial DHFR-TS, there are physical interactions between the DHFR domain and the TS domain, and these interactions are necessary:to obtain a catalytically active TS. Interference with these essential protein-protein interactions could lead to new selective strategies to treat malaria resistant to traditional DHFR-TS inhibitors.