Crystal structure of a deletion mutant of human thymidylate synthase Delta (7-29) and its ternary complex with Tomudex and dUMP.

The crystal structures of a deletion mutant of human thymidylate synthase (TS) and its ternary complex with dUMP and Tomudex have been determined at 2.0 A and 2.5 A resolution, respectively. The mutant TS, which lacks 23 residues near the amino terminus, is as active as the wild-type enzyme. The ternary complex is observed in the open conformation, similar to that of the free enzyme and to that of the ternary complex of rat TS with the same ligands. This is in contrast to Escherichia coli TS, where the ternary complex with Tomudex and dUMP is observed in the closed conformation. While the ligands interact with each other in identical fashion regardless of the enzyme conformation, they are displaced by about 1.0 A away from the catalytic cysteine in the open conformation. As a result, the covalent bond between the catalytic cysteine sulfhydryl and the base of dUMP, which is the first step in the reaction mechanism of TS and is observed in all ternary complexes of the E. coli enzyme, is not formed. This displacement results from differences in the interactions between Tomudex and the protein that are caused by differences in the environment of the glutamyl tail of the Tomudex molecule. Despite the absence of the closed conformation, Tomudex inhibits human TS ten-fold more strongly than E. coli TS. These results suggest that formation of a covalent bond between the catalytic cysteine and the substrate dUMP is not required for effective inhibition of human TS by cofactor analogs and could have implications for drug design by eliminating this as a condition for lead compounds.

In vitro selection of an RNA sequence that interacts with high affinity with thymidylate synthase.

Previous studies have shown that the repressive effect of thymidylate synthase (TS) mRNA translation is mediated by direct binding of TS itself to two cis-acting elements on its cognate mRNA. To identify the optimal RNA nucleotides that interact with TS, we in vitro synthesized a completely degenerate, linear RNA pool of 25 nt and employed in vitro selection to isolate high affinity RNA ligands that bind human TS protein. After 10 rounds of selection and amplification, a single RNA molecule was selected that bound TS protein with nearly 20-fold greater affinity than native, wild-type TS RNA sequences. Secondary structure analysis of this RNA sequence predicted it to possess a stem-loop structure. Deletion and/or modification of the UGU loop element within the RNA sequence decreased binding to TS by up to 1000-fold. In vivo transfection experiments revealed that the presence of the selected RNA sequence resulted in a significant increase in the expression of a heterologous luciferase reporter construct in human colon cancer H630 and TS-overexpressing HCT-C:His-TS+ cells, but not in HCT-C18 cells expressing a functionally inactive TS. In addition, the presence of this element in H630 cells leads to induced expression of TS protein. An immunoprecipitation method using RT-PCR confirmed a direct interaction between human TS protein and the selected RNA sequence in transfected human cancer H630 cells. This study identified a novel RNA sequence from a degenerate RNA library that specifically interacts with TS.

High-level expression of Escherichia coli and Bacillus subtilis thymidylate synthases.

Procedures are described for the preparation of highly purified thymidylate synthases from Escherichia coli and Bacillus subtilis. The yields in each case are quite high with about 350 mg of pure protein obtained from 1 liter of cells. Basically all that is required to obtain pure enzyme is an induction step from a high-expression vector, followed by a DE-52 column elution. Both enzymes appeared to be fairly stable in that incubation at 43 degrees C for 10 min resulted in the loss of 50% of the E. coli thymidylate synthase activity, while 50 degrees C for 10 min was required to obtain the same effect with the B. subtilis enzyme. In the presence of the substrate, dUMP, each protein was stabilized further by 6 to 7 degrees C, which was increased to 9 to 10 degrees C on addition of dihydrofolate. It was shown also that the E. coli thymidylate synthase could be maintained at 4 degrees C for at least 4 months with little or no loss in activity provided that mercaptoethanol was not present. The presence of the latter led to a progressive loss in activity until little activity could be detected after 18 weeks, which was due, in part, to the formation of a disulfide bond with the active site cysteine. Addition of dithiothreitol restored the enzyme activity to its original state.

A T4-phage deoxycytidylate deaminase mutant that no longer requires deoxycytidine 5'-triphosphate for activation.

A deoxycytidylate (dCMP) deaminase encoded in T4-bacteriophage DNA that is induced on phage infection of Escherichia coli was shown earlier (Maley, G. F., Duceman, B. W., Wang, A. M., Martinez, J. M., and Maley, F. (1990) J. Biol. Chem. 265, 47-51) to be similar in size, properties, and amino acid composition to the T2-phage-induced deaminase. Neither enzyme is active in the absence of dCTP or its natural activator, 5-hydroxymethyl-dCTP. However, on changing the arginine (Arg) at residue 115 of the T4-deaminase to either a glutamate (R115E) or a glutamine (R115Q), the resulting mutant enzymes were active in the absence of dCTP, with each mutant possessing a turnover number or k(cat) that is about 15% that of the wild-type deaminase. When compared on the basis of specific activity, however, the mutants are about 40-50% of the wild-type (WT)-enzyme's specific activity. Molecular weight analysis on the wild-type and mutant deaminases using HPLC size exclusion chromatography revealed that the wild-type deaminase was basically a hexamer, particularly in the presence of dCTP, regardless of the extent of dilution. Under similar conditions, R115E remained a dimer, whereas R115Q and F112A varied from hexamers to dimers particularly at concentrations normally present in the assay solution. Activity measurements appear to support the conclusion that the hexameric form of the enzyme is activated by dCTP, while the dimer is not. Another feature emphasizing the difference between the WT and mutant deaminases was observed on their denaturation-renaturation in EDTA, which revealed the mutants to be restored to 50% of their original activities with the WT deaminase only marginally restored.

Characterization of a cis-acting regulatory element in the protein coding region of thymidylate synthase mRNA.

Thymidylate synthase (TS) functions as an RNA-binding protein by interacting with two different sequences on its own mRNA. One site is located in the 5'-upstream region of human TS mRNA while the second site is located within the protein coding region corresponding to nt 434-634. In this paper, a 70 nt RNA sequence, corresponding to nt 480-550, was identified that binds TS protein with an affinity similar to that of full-length TS mRNA and TS434-634 RNA. In vitro translation studies confirmed that this sequence is critical for the translational autoregulatory effects of TS. To document in vivo biological significance, TS sequences contained within this region were cloned onto the 5'-end of a luciferase reporter plasmid and transient transfection experiments were performed using H630 human colon cancer cells. In cells transfected with p644/TS434-634 or p644/TS480-550, luciferase activity was decreased 2.5-fold when compared to cells transfected with p644 plasmid alone. Luciferase mRNA levels were identical for each of these conditions as determined by RNase protection and RT-PCR analysis. Immunoprecipitation of TS ribonucleoprotein complexes revealed a direct interaction between TS protein and TS480-550 RNA in transfected H630 cells. Treatment with 5-fluorouridine resulted in a nearly 2-fold increase in luciferase activity only in cells transfected with p644/TS434-634 and p644/TS480-550. This study identifies a 70 nt TS response element in the protein coding region of TS mRNA with in vitro and in vivo translational regulatory activity.

Crystal structures of rat thymidylate synthase inhibited by Tomudex, a potent anticancer drug.

Two crystal structures of rat thymidylate synthase (TS) complexed with dUMP and the anticancer drug Tomudex (ZD1694) have been determined to resolutions of 3.3 and 2.6 A. Tomudex is one of several new antifolates targeted to TS and the first to be approved for clinical use. The structures represent the first views of any mammalian TS bound to ligands and suggest that the rat protein undergoes a ligand-induced conformational change similar to that of the Escherichia coli protein. Surprisingly, Tomudex does not induce the "closed" conformation in rat TS that is seen on binding to E. coli TS, resulting in inhibitor atoms that differ in position by more than 1.5 A. Several species-specific differences in sequence may be the reason for this. Phe 74 shifts to a new position in the rat complex and is in van der Waals contact with the inhibitor, while in the E. coli protein the equivalent amino acid (His 51) hydrogen bonds to the glutamate portion of the inhibitor. Amino acids Arg 101, Asn 106, and Met 305 make no contacts with the inhibitor in the open conformation, unlike the equivalent residues in the E. coli protein (Thr 78, Trp 83, and Val 262). dUMP binding is similar in both proteins, except that there is no covalent adduct to the active site cysteine (Cys 189) in the rat structures. Two insertions in the rat protein are clearly seen, but the N-termini (residues 1-20) and C-termini (residues 301-307) are disordered in both crystal forms.

Thymidylate synthase protein and p53 mRNA form an in vivo ribonucleoprotein complex.

A thymidylate synthase (TS)-ribonucleoprotein (RNP) complex composed of TS protein and the mRNA of the tumor suppressor gene p53 was isolated from cultured human colon cancer cells. RNA gel shift assays confirmed a specific interaction between TS protein and the protein-coding region of p53 mRNA, and in vitro translation studies demonstrated that this interaction resulted in the specific repression of p53 mRNA translation. To demonstrate the potential biological role of the TS protein-p53 mRNA interaction, Western immunoblot analysis revealed nearly undetectable levels of p53 protein in TS-overexpressing human colon cancer H630-R10 and rat hepatoma H35(F/F) cell lines compared to the levels in their respective parent H630 and H35 cell lines. Polysome analysis revealed that the p53 mRNA was associated with higher-molecular-weight polysomes in H35 cells compared to H35(F/F) cells. While the level of p53 mRNA expression was identical in parent and TS-overexpressing cell lines, the level of p53 RNA bound to TS in the form of RNP complexes was significantly higher in TS-overexpressing cells. The effect of TS on p53 expression was also investigated with human colon cancer RKO cells by use of a tetracycline-inducible system. Treatment of RKO cells with a tetracycline derivative, doxycycline, resulted in 15-fold-induced expression of TS protein and nearly complete suppression of p53 protein expression. However, p53 mRNA levels were identical in transfected RKO cells in the absence and presence of doxycycline. Taken together, these findings suggest that TS regulates the expression of p53 at the translational level. This study identifies a novel pathway for regulating p53 gene expression and expands current understanding of the potential role of TS as a regulator of cellular gene expression.

High-level expression of human thymidylate synthase.

A method is presented for expressing human thymidylate synthase (TS) to the extent of 25-30% of the protein in Escherichia coli. By this procedure, 200-400 mg of pure enzyme can be obtained from a 2-liter culture of cells. The key to the level of expression appears to be related to the conversion of purine bases in the third, fourth, and fifth codons of the TS cDNA to thymine, without altering the encoded protein product. Conversion of the penultimate proline to a leucine did not diminish expression, but while the isolated native enzyme contained only proline on its amino-terminal end, the mutated enzyme was found to contain methionine on its amino terminus. By contrast, the expression of the unmodified TS cDNA represented only about 0.1-0.2% of the total cellular protein. Unlike recombinant rat and human TSs, the respective enzymes purified to homogeneity from eukaryotic cells were blocked at the amino ends and possessed 2- to 4-fold lower specific activities. To determine at what level the impairment of expression occurred, an in vitro transcription, translation system was employed and the results showed that while transcription was unaffected, the translation of native TS mRNA was reduced by at least 20-fold relative to modified TS mRNA using a rabbit reticulocyte translation system. Thus, it appears that at least for the TS gene, expression is greatly influenced by the GC content of the 5' coding region of the gene in both prokaryote and eukaryote systems.

Protein-protein interactions involving T4 phage-coded deoxycytidylate deaminase and thymidylate synthase.

The enzymes deoxycytidylate deaminase (EC) and thymidylate synthase (EC) are functionally associated with one another, since they catalyze sequential reactions. In T4 coliphage infection the two enzymes are found in dNTP synthetase, a multienzyme complex for deoxyribonucleotide biosynthesis. Protein-protein interactions involving the phage-coded forms of these two enzymes have been explored in three experiments that use the respective purified protein as an affinity ligand. First, an extract of radiolabeled T4 proteins was passed through a column of immobilized enzyme (either dTMP synthase or dCMP deaminase), and the specifically bound proteins were identified. Second, two mutant form of dCMP deaminase (H90N and H94N), altered in presumed zinc-binding sites, were analyzed similarly, with the results suggesting that some, but not all, interactions require normal structure near the catalytic site. Third, affinity chromatography using either enzyme as the immobilized ligand, revealed interactions between the two purified enzymes in the absence of other proteins. In these experiments we noted a significant effect of dCTP, an allosteric modifier of dCMP deaminase, upon the interactions.

Chromosomal location and structural organization of the human deoxycytidylate deaminase gene.

Deoxycytidylate deaminase is an allosteric enzyme whose impairment can lead to deoxynucleotide imbalances that affect the fidelity of DNA synthesis. A DNA fragment encompassing the gene for deoxycytidylate deaminase has been isolated from a human lung fibroblast genomic library and sequenced in both directions through 26,764 base pairs. The previously isolated cDNA, which was used to establish the amino acid sequence for this enzyme (Weiner, K.X.B., Weiner, R.S., Maley, F., and Maley, G.F. (1993) J. Biol. Chem. 268, 12983-12989) was instrumental in isolating this gene. The gene consists of five exons of about 100 base pairs each, separated by four introns. The most striking feature of the genomic structure is that the second and third exons are separated by an intron of about 20 kilobases. The chromosomal location of the deaminase gene was determined by fluorescence in situ hybridization as 4q35, which is the extreme end of this chromosome. The position of this gene on chromosome 4, in addition to the role of its product in limiting potentially detrimental mutations, suggests that the normal operation of both the gene and its product is important to the well being of the organism.

Isolation and expression of rat thymidylate synthase cDNA: phylogenetic comparison with human and mouse thymidylate synthases.

Two cDNA clones representing rat hepatoma thymidylate synthase (rTS) were isolated from a lambda ZAP II cDNA library using as a probe a fragment of the human TS cDNA. The two were identical except that one was missing 50 bp and the other 23 bp corresponding to the 5' coding region of the protein. The missing region was obtained by screening a rat genomic library. The open reading frame of rTS cDNA encoded 921 bp encompassing a protein of 307 amino acids with a calculated molecular mass of 35,015 Da. Rat hepatoma TS appears identical to normal rat thymus TS and the two sequences differ from mouse TS in the same eight amino acid residues. Six of these differences are in the first 21 amino acids from the amino-end. The human enzyme differed from rat and mouse TS at 17 residues where the latter two were identical, with most changes being conservative in nature. The three species differed completely at only four sites. Because the mouse TS shares four amino acids with human TS at sites which differ from rTS and a comparable situation does not exist between rTS and human TS, it is suggested that mouse TS is closer to human TS phylogenetically than rTS. The polymerase chain reaction was used to subclone the protein coding region of rTS into a high expression vector, which expressed rTS in Escherichia coli to the extent of 10 to 20% of its cellular protein. Although the amino-end of the amplified TS was unblocked, that isolated from a FUdR-resistant rat hepatoma cell line contained mostly N-acetylmethionine on its N-terminal end, a finding that may have significant regulatory consequences, which are discussed. The TS level in the resistant cell line was 60 to 70-fold higher than normal which was found to be associated with both multiple gene copies and an expanded TS mRNA pool.

Characterization of a specific interaction between Escherichia coli thymidylate synthase and Escherichia coli thymidylate synthase mRNA.

Previous studies have shown that human TS mRNA translation is controlled by a negative autoregulatory mechanism. In this study, an RNA electrophoretic gel mobility shift assay confirmed a direct interaction between Escherichia coli (E.coli) TS protein and its own E.coli TS mRNA. Two cis-acting sequences in the E.coli TS mRNA protein-coding region were identified, with one site corresponding to nucleotides 207-460 and the second site corresponding to nucleotides 461-807. Each of these mRNA sequences bind TS with a relative affinity similar to that of the full-length E.coli TS mRNA sequence (IC50 = 1 nM). A third binding site was identified, corresponding to nucleotides 808-1015, although its relative affinity for TS (IC50 = 5.1 nM) was lower than that of the other two cis-acting elements. E.coli TS proteins with mutations in amino acids located within the nucleotide-binding region retained the ability to bind RNA while proteins with mutations at either the nucleotide active site cysteine (C146S) or at amino acids located within the folate-binding region were unable to bind TS mRNA. These studies suggest that the regions on E.coli TS defined by the folate-binding site and/or critical cysteine sulfhydryl groups may represent important RNA binding domains. Further evidence is presented which demonstrates that the direct interaction with TS results in in vitro repression of E.coli TS mRNA translation.

Thymidylate synthase binds to c-myc RNA in human colon cancer cells and in vitro.

Using an immunoprecipitation-reverse transcription-PCR technique, we characterized a thymidylate synthase (TS) ribonucleoprotein complex in cultured human colon cancer cells that consists of TS protein and the mRNA of the nuclear oncogene c-myc. TS protein is complexed in intact cells with the C-terminal coding region of c-myc mRNA that includes nucleotide positions 1625 to 1790. RNA electrophoretic gel mobility shift assays confirm a specific interaction between TS protein and c-myc mRNA and provide additional evidence that the C-terminal coding region represents an important cis-acting regulatory element. Further evidence demonstrates that the in vitro translational efficiency of c-myc mRNA is inhibited as a result of its direct interaction with TS protein. In addition, the presence of exogenous c-myc mRNA specifically relieves the inhibitory effects of TS protein on TS mRNA translation.

Crystal structure of thymidylate synthase from T4 phage: component of a deoxynucleoside triphosphate-synthesizing complex.

Thymidylate synthase from phage T4 (T4TS) is part of a complex of several enzymes required for coordinate DNA synthesis in infected Escherichia coli cells. It has been proposed that similar complexes of enzymes related to DNA synthesis are also functional in eukaryotes [Pardee, A. B. (1989) Science 246, 603-608]. To delineate the role of structure in the function of this complex, we have solved the structure of T4TS as a basis for mapping the complex by mutagenesis. The 3.1 A structure of the unliganded enzyme was determined by molecular replacement and refined to 19.9% for all data. Three inserts and one deletion in the coding region are unique to T4TS, and all sites lie on one side of the enzyme surface, possibly encoding unique T4 specific intermolecular interactions during the infective cycle. The crystal structure is generally in the open, unliganded conformation seen in unliganded E. coli TS, as opposed to the closed, ternary complex conformation, except that the critically important C-terminus is inserted into the active site hydrogen bonded to residue Asn85, as seen in functional ternary complex structures. Other differences between E. coli TS and T4TS appear to explain the enhanced binding of folyl polyglutamate to the latter.

The effect of reducing reagents on binding of thymidylate synthase protein to thymidylate synthase messenger RNA.

Human thymidylate synthase (TS) protein specifically binds to its own TS mRNA and functions as a translational repressor. In the presence of reducing agents, the RNA binding activity of TS protein is significantly enhanced. In contrast, treatment of TS protein with the oxidizing agent diamide inhibits RNA binding. Scatchard analysis reveals that in the presence of the reducing agent 2-mercaptoethanol, the TS protein/TS mRNA interaction changes from low (Kd = 66 nM) to high (Kd = 2.6 nM) apparent affinity. The catalytic activity of TS is increased by up to 6.5-fold in the presence of 2-mercaptoethanol. These studies demonstrate that the interaction between TS protein and its target TS mRNA is sensitive to the presence of reducing reagents and is dependent upon a reversible sulfhydryl switch mechanism.

Identification of a site necessary for allosteric regulation in T4-phage deoxycytidylate deaminase.

An allosteric inhibitor of dCMP deaminase, dTTP, forms a photolabile covalent bond with T4-phage dCMP deaminase in the presence of UV light at 254 nm. The importance of the methyl group in this process is supported by the findings that dUTP, also an allosteric inhibitor, does not photofix to the enzyme and that tritium is released from [methyl-3H dTTP during the course of the photofixation. That the bond formed is photolabile is demonstrated by the fact that tritium is released by about 10-fold over the amount of nucleotide that is photofixed. The amino acid that covalently binds dTTP in T4-dCMP deaminase was identified as Phe112. On conversion of Phe112 to an alanine by site-directed mutagenesis, there was a dramatic change in the enzyme's response to its allosteric effectors when measured early in the reaction, in that the mutant enzyme was as active as the wild-type even in the absence of dCTP and was only weakly inhibited by dTTP. However, after 10-15% of the substrate had been deaminated, the reaction rate fell off rather markedly, indicating either that an inhibitor was being accumulated on the enzyme or that the enzyme was being irreversibly inactivated with time. That the latter was not the case was shown by the addition of dCTP to the reaction, which restored the rate to that expected when it was present initially. Furthermore, we showed that, consistent with the observed loss of allosteric regulation by dCTP and dTTP, the affinity of the mutant enzyme for dTTP and dCTP as determined by binding studies was greatly reduced relative to the wild-type enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Water-mediated substrate/product discrimination: the product complex of thymidylate synthase at 1.83 A.

In an irreversible enzyme-catalyzed reaction, strong binding of the products would lead to substantial product inhibition. The X-ray crystal structure of the product complex of thymidylate synthase (1.83-A resolution, R factor = 0.183 for all data between 7.0 and 1.83 A) identifies a bound water molecule that serves to disfavor binding of the product nucleotide, dTMP. This water molecule is hydrogen bonded to absolutely conserved Tyr 146 (using the Lactobacillus casei numbering system) and is displaced by the C7 methyl group of the reaction product thymidylate. The relation between this observation and kinetic and thermodynamic values is discussed. The structure reveals a carbamate modified N-terminus that binds in a highly conserved site, replaced by side chains that can exploit the same site in other TS sequences. The enzyme-products complex is compared to the previously determined structure of enzyme-substrate-cofactor analog. This comparison reveals changes that occur between the first covalent complex formed between enzyme and substrate with an inhibitory cofactor analog and the completed reaction. The almost identical arrangement of ligands in these two structures contributes to our model for the TS reaction and verifies the physiological relevance of the mode in which potent inhibitors bind to this target for rational drug design.

Identification of a thymidylate synthase ribonucleoprotein complex in human colon cancer cells.

Translation of thymidylate synthase (TS) mRNA is controlled by its own protein product, TS, in an autoregulatory manner. Direct binding of TS protein to two different cis-acting elements on the TS mRNA is associated with this translational regulation. In this study, an immunoprecipitation-reverse transcription-PCR technique was used to identify a TS ribonucleoprotein (RNP) complex in cultured human colon cancer cells. Using antibodies specific for TS protein, we show that TS is complexed in vivo with its own TS RNA. Furthermore, evidence demonstrating a direct interaction between the mRNA of the nuclear oncogene c-myc and TS protein is presented.

Primary structure of human deoxycytidylate deaminase and overexpression of its functional protein in Escherichia coli.

The cDNA encoding human dCMP deaminase was isolated from a lambda ZAPII expression library using an antibody generated against highly purified HeLa cell dCMP deaminase. The cloned cDNA consists of 1856 base pairs and encodes a protein of 178 amino acids with a calculated molecular mass of 19,985 daltons. The sequence of several cyanogen bromide-cleaved peptides derived from HeLa cell dCMP deaminase are all contained within the deduced amino acid sequence. A zinc binding region is present in the enzyme, similar to that reported for cytidine deaminase (Yang, E. C., Carlow, D., Wolfenden, R., and Short, S. A. (1992) Biochemistry 31, 4168-4174). Northern blot analysis revealed a predominant messenger RNA species of 1.9 kilobases. Expression of the active protein to about 10% of Escherichia coli's total protein was achieved by subcloning the open reading frame into a high expression system using the polymerase chain reaction. Polyacrylamide gel electrophoresis revealed a prominent protein band which comigrated with affinity purified HeLa dCMP deaminase, while Western blot analysis yielded an immunoreactive band which comigrated with the single immunoreactive affinity column purified dCMP deaminase band. The enzyme which possesses a kcat of 1.02 x 10(3) s-1 was purified to homogeneity in over 60% yield. The overexpression of dCMP deaminase should permit more exacting studies on the regulation of this important allosteric enzyme which provides substrate for DNA synthesis.

Overcoming inclusion body formation in a high-level expression system.

Attempts at overexpressing T4-phage deoxycytidylate deaminase using the pET3c/BL21(DE3)/pLysS system resulted in this enzyme being part of an inactive inclusion-body complex. However, by employing an enriched growth medium it was found that the deaminase could be induced in a soluble active form to at least 20% of this organism's cellular protein. Insoluble inclusion bodies were obtained with less rich media. This procedure was employed successfully with other highly expressed proteins that formed inclusion bodies. The use of a rich growth medium during the course of protein induction may be a valuable adjunct to limiting inclusion body formation with this as well as other expression systems.