Structure of human thymidylate synthase suggests advantages of chemotherapy with noncompetitive inhibitors.

Thymidylate synthase (TS) is a major target in the chemotherapy of colorectal cancer and some other neoplasms. The emergence of resistance to the treatment is often related to the increased levels of TS in cancer cells, which have been linked to the elimination of TS binding to its own mRNA upon drug binding, a feedback regulatory mechanism, and/or to the increased stability to intracellular degradation of TS.drug complexes (versus unliganded TS). The active site loop of human TS (hTS) has a unique conformation resulted from a rotation by 180 degrees relative to its orientation in bacterial TSs. In this conformation, the enzyme must be inactive, because the catalytic cysteine is no longer positioned in the ligand-binding pocket. The ordered solvent structure obtained from high resolution crystallographic data (2.0 A) suggests that the inactive loop conformation promotes mRNA binding and intracellular degradation of the enzyme. This hypothesis is supported by fluorescence studies, which indicate that in solution both active and inactive forms of hTS are present. The binding of phosphate ion shifts the equilibrium toward the inactive conformation; subsequent dUMP binding reverses the equilibrium toward the active form. Thus, TS inhibition via stabilization of the inactive conformation should lead to less resistance than is observed with presently used drugs, which are analogs of its substrates, dUMP and CH(2)H(4)folate, and bind in the active site, promoting the active conformation. The presence of an extension at the N terminus of native hTS has no significant effect on kinetic properties or crystal structure.

DNA-binding sequence of the human prostate-specific homeodomain protein NKX3.1.

NKX3.1 is a member of the NK class of homeodomain proteins and is most closely related to Drosophila NK-3. NKX3.1 has predominantly prostate-specific expression in the adult human. Previous studies suggested that NKX3.1 exerts a growth-suppressive effect on prostatic epithelial cells and controls differentiated glandular functions. Using a binding site selection assay with recombinant NKX3.1 protein we identified a TAAGTA consensus binding sequence that has not been reported for any other NK class homeoprotein. By electromobility shift assay we demonstrated that NKX3.1 preferentially binds the TAAGTA sequence rather than the binding site for Nkx2.1 (CAAGTG) or Msx1 (TAATTG). Using mutated binding sites in competitive gel shift assays, we analyzed the nucleotides in the TAAGTA consensus sequence that are important for NKX3.1 binding. The consensus binding site of a naturally occurring polymorphic NKX3.1 protein with arginine replaced by cysteine at position 52 was identical to the wild-type binding sequence. The binding affinities of wild-type and polymorphic NKX3.1 for the TAAGTA consensus site were very similar, with values of 20 and 22 nM, respectively. Wild-type and polymorphic NKX3.1 specifically repressed transcription of luciferase from a reporter vector with three copies of the NKX3.1-binding site upstream from a thymidine kinase promoter. The data show that among NK family proteins NKX3.1 binds a novel DNA sequence and can behave as an in vitro transcriptional repressor.

Genetic complementation and resistance to 5-fluoro-2'-deoxyuridine in thymidine auxotrophs expressing a highly defective mutant of human thymidylate synthase.

A mutant human thymidylate synthase (TS) has been created in which a glutamine residue at position 214 has been replaced by glutamate. Glutamine at position 214 is postulated to be involved in maintaining the enzyme in a conformation that facilitates the binding of the substrate dUMP. Although the kcat/Km of the mutant protein for the substrate, dUMP, is 10(3) lower than that of wild-type TS, the mutant TS confers thymidine prototrophy on a TS-deficient bacterial strain when expressed at high levels. In the present investigation, a TS-deficient Chinese hamster lung cell line was transfected with DNA encoding the defective protein. Thymidine prototrophs were isolated that expressed the defective protein at levels that were physiologically relevant. The activities of the enzymes expressed endogenously in representative prototrophs were consistent with the activities observed for the purified proteins. At similar levels of TS expression, thymidine prototrophs expressing Glu214 TS were 8-fold more resistant to 5-fluoro-2'-deoxyuridine (FdUrd) cytotoxicity than are prototrophs expressing Gln214 TS. FdUrd is a prodrug of the tight-binding TS inhibitor, 5-fluoro-2'-deoxyuridine-5'-monophosphate (FdUMP). The resistance to FdUrd was associated with a significant decrease in the binding of FdUMP to the purified mutant enzyme. The data are consistent with the interpretation that TSs that are highly defective are capable of sufficient dTMP production for cell survival and optimal growth, yet may confer resistance to TS-directed inhibitors.

Substitution at residue 214 of human thymidylate synthase alters nucleotide binding and isomerization of ligand-protein complexes.

Based on crystal structures of bacterial thymidylate synthases (TS), a glutamine corresponding to residue 214 in human TS (hTS) is located in a region that is postulated to be critical for conformational changes that occur upon ligand binding. Previous steady-state kinetic studies indicated that replacement of glutamine at position 214 (Gln214) of hTS by other residues results in a decrease in nucleotide binding and catalysis, with only minor effects on folate binding (D. J. Steadman et al. (1998) Biochemistry 37, 7089-7095). The data suggested that Gln214 maintains the enzyme in a conformation that facilitates nucleotide binding. In the present study, transient-state kinetic analysis was utilized to determine rate constants that govern specific steps along the catalytic pathway of hTS, which provides the first detailed kinetic mechanism for hTS. Analysis of the reaction mechanisms of mutant TSs revealed that substitution at position 214 significantly affects nucleotide binding and the rate of chemical conversion of bound substrates to products, which is consistent with the results of steady-state kinetic analysis. Furthermore, it is shown that substitution at position 214 affects the rate of isomerization, presumably from an open to a closed form of the enzyme-substrate complex. Although the affinity of the initial binding of CH2H4folate is not substantially affected, Kiso, the ratio of the forward rate of isomerization (kiso) to the reverse rate of isomerization (kr, iso), is 2-6-fold lower for the mutants at position 214 compared to Q214, with the greatest effects on kiso. In addition, the binding of the folate analogue, CB3717, to dUMP binary complexes of mutant enzymes was characterized by a slow isomerization phase that was not detected in binding studies utilizing wild-type hTS. The data are consistent with the hypothesis that Gln214 is located at a structurally critical region of the enzyme.

A structural role for glutamine 214 in human thymidylate synthase.

Studies of the crystal structures of thymidylate synthase (TS) have revealed that a kink is present in beta-sheets that form the core of the enzyme. The beta-kink is proposed to serve as a "hinge" during conformational changes that occur in the enzyme after ligand binding at the active site. A residue in one of the beta-bulges that form the kink, glutamine at position 214 of human TS, is highly conserved in all TSs and is postulated to interact with nucleotide ligands that bind at the active site. To examine the role of this residue, glutamine at position 214 was replaced by residues that differ in volume, hydrophobicity, electrostatic charge, and hydrogen bonding potential. Genetic complementation studies utilizing a TS-deficient bacterial strain revealed that residues with large side chain volumes or that are prohibited in beta-bulges created loss of function proteins. Kinetic studies indicated that residue hydrophobicity is not correlated with catalytic activity. Residues that are predicted to alter the charge at position 214 created enzymes with kcat/Km values at least 10(3) lower than those of the wild type. Kinetic and ligand binding studies indicated that residue 214 is involved in nucleotide binding; however, hydrogen bonding potential does not contribute significantly to nucleotide binding energy. The data are consistent with the hypothesis that residue 214 is involved in maintaining the enzyme in a conformation that facilitates nucleotide binding and catalysis.