Abstract
We have determined the crystal structure of dihydrofolate reductase-thymidylate synthase (DHFR-TS) from Cryptosporidium hominis, revealing a unique linker domain containing an 11-residue alpha-helix that has extensive interactions with the opposite DHFR-TS monomer of the homodimeric enzyme. Analysis of the structure of DHFR-TS from C. hominis and of previously solved structures of DHFR-TS from Plasmodium falciparum and Leishmania major reveals that the linker domain primarily controls the relative orientation of the DHFR and TS domains. Using the tertiary structure of the linker domains, we have been able to place a number of protozoa in two distinct and dissimilar structural families corresponding to two evolutionary families and provide the first structural evidence validating the use of DHFR-TS as a tool of phylogenetic classification. Furthermore, the structure of C. hominis DHFR-TS calls into question surface electrostatic channeling as the universal means of dihydrofolate transport between TS and DHFR in the bifunctional enzyme.
Highlights
Thymidylate synthase (TS)1 and dihydrofolate reductase (DHFR) are essential enzymes in the cell cycle of all organisms, since they catalyze the production of dTMP, required for DNA replication
To better understand whether bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) is a good tool for the phylogenetic classification of some families of protozoa, we have solved the crystal structure of DHFR-TS from Cryptosporidium hominis (ChDHFR-TS), previously called Cryptosporidium parvum type 1 [7], to 2.8 Å and performed a structure-function analysis with previously solved structures of DHFR-TS from Plasmodium falciparum (PfDHFR-TS) [8] and Leishmania major (LmDHFR-TS) [9]
The structural differences we discovered between the linker domains of the Apicomplexan and kinetoplastid DHFR-TS fam
Summary
The protein was purified on a methotrexate affinity column and eluted with 2 mM dihydrofolate. Fractions containing pure protein were concentrated to 7 mg/ml and incubated with 2 mM ligands (methotrexate, NADPH, CB3717, and dUMP) for approximately 1 h on ice. Using hanging drop vapor diffusion, a promising crystallization condition was refined to 10% polyethylene glycol 6000, 50 mM ammonium sulfate, 150 mM lithium sulfate, and 100 mM Tris, pH 8.0. The crystals were soaked in 15% ethylene glycol for 5 min and in 25% ethylene glycol for another 5 min before being plunged into liquid nitrogen for cryogenic data collection. Structure Determination—The structure was determined by molecular replacement using a model of thymidylate synthase from P. carinii (Protein Data Bank 1F28) [14], from which all ligands were removed, as
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