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.
Shallom
S
S
Department of Biology, The Catholic University of America, Washington, D.C. 20064, USA.
Zhang
K
K
Jiang
L
L
Rathod
P K
PK
D000818:Animals; D004789:Enzyme Activation; D005816:Genetic Complementation Test; D010963:Plasmodium falciparum / Q000201:enzymology*; D011485:Protein Binding; D017384:Sequence Deletion; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D013940:Thymidylate Synthase / Q000235:genetics / Q000378:metabolism*
D013762:Tetrahydrofolate Dehydrogenase; D013940:Thymidylate Synthase