Beyond Chelation: EDTA Tightly Binds Taq DNA Polymerase, MutT and dUTPase and Directly Inhibits dNTPase Activity.

EDTA is commonly used as an efficient chelator of metal ion enzyme cofactors. It is highly soluble, optically inactive and does not interfere with most chemicals used in standard buffers making EDTA a common choice to generate metal-free conditions for biochemical and biophysical investigations. However, the controversy in the literature on metal-free enzyme activities achieved using EDTA or by other means called our attention to a putative effect of EDTA beyond chelation. Here, we show that EDTA competes for the nucleotide binding site of the nucleotide hydrolase dUTPase by developing an interaction network within the active site similar to that of the substrate. To achieve these findings, we applied kinetics and molecular docking techniques using two different dUTPases. Furthermore, we directly measured the binding of EDTA to dUTPases and to two other dNTPases, the Taq polymerase and MutT using isothermal titration calorimetry. EDTA binding proved to be exothermic and mainly enthalpy driven with a submicromolar dissociation constant considerably lower than that of the enzyme:substrate or the Mg:EDTA complexes. Control proteins, including an ATPase, did not interact with EDTA. Our findings indicate that EDTA may act as a selective inhibitor against dNTP hydrolyzing enzymes and urge the rethinking of the utilization of EDTA in enzymatic experiments.

Trading in cooperativity for specificity to maintain uracil-free DNA.

Members of the dUTPase superfamily play an important role in the maintenance of the pyrimidine nucleotide balance and of genome integrity. dCTP deaminases and the bifunctional dCTP deaminase-dUTPases are cooperatively regulated by dTTP. However, the manifestation of allosteric behavior within the same trimeric protein architecture of dUTPases, the third member of the superfamily, has been a question of debate for decades. Therefore, we designed hybrid dUTPase trimers to access conformational states potentially mimicking the ones observed in the cooperative relatives. We studied how the interruption of different steps of the enzyme cycle affects the active site cross talk. We found that subunits work independently in dUTPase. The experimental results combined with a comparative structural analysis of dUTPase superfamily enzymes revealed that subtile structural differences within the allosteric loop and the central channel in these enzymes give rise to their dramatically different cooperative behavior. We demonstrate that the lack of allosteric regulation in dUTPase is related to the functional adaptation to more efficient dUTP hydrolysis which is advantageous in uracil-DNA prevention.

Phosphorylation adjacent to the nuclear localization signal of human dUTPase abolishes nuclear import: structural and mechanistic insights.

Phosphorylation adjacent to nuclear localization signals (NLSs) is involved in the regulation of nucleocytoplasmic transport. The nuclear isoform of human dUTPase, an enzyme that is essential for genomic integrity, has been shown to be phosphorylated on a serine residue (Ser11) in the vicinity of its nuclear localization signal; however, the effect of this phosphorylation is not yet known. To investigate this issue, an integrated set of structural, molecular and cell biological methods were employed. It is shown that NLS-adjacent phosphorylation of dUTPase occurs during the M phase of the cell cycle. Comparison of the cellular distribution of wild-type dUTPase with those of hyperphosphorylation- and hypophosphorylation-mimicking mutants suggests that phosphorylation at Ser11 leads to the exclusion of dUTPase from the nucleus. Isothermal titration microcalorimetry and additional independent biophysical techniques show that the interaction between dUTPase and importin-α, the karyopherin molecule responsible for `classical' NLS binding, is weakened significantly in the case of the S11E hyperphosphorylation-mimicking mutant. The structures of the importin-α-wild-type and the importin-α-hyperphosphorylation-mimicking dUTPase NLS complexes provide structural insights into the molecular details of this regulation. The data indicate that the post-translational modification of dUTPase during the cell cycle may modulate the nuclear availability of this enzyme.

Direct contacts between conserved motifs of different subunits provide major contribution to active site organization in human and mycobacterial dUTPases.

dUTP pyrophosphatases (dUTPases) are essential for genome integrity. Recent results allowed characterization of the role of conserved residues. Here we analyzed the Asp/Asn mutation within conserved Motif I of human and mycobacterial dUTPases, wherein the Asp residue was previously implicated in Mg(2+)-coordination. Our results on transient/steady-state kinetics, ligand binding and a 1.80 A resolution structure of the mutant mycobacterial enzyme, in comparison with wild type and C-terminally truncated structures, argue that this residue has a major role in providing intra- and intersubunit contacts, but is not essential for Mg(2+) accommodation. We conclude that in addition to the role of conserved motifs in substrate accommodation, direct subunit interaction between protein atoms of active site residues from different conserved motifs are crucial for enzyme function.

Molecular shape and prominent role of beta-strand swapping in organization of dUTPase oligomers.

Most dUTP pyrophosphatases (dUTPases) are homotrimers with interfaces formed between subunit surfaces, in the central channel, and by C-terminal beta-strand swapping. Analysis of intersubunit interactions reveals an important cohesive role for the C-terminus. This is reflected in the crystal structure of fruitfly dUTPase displaying a dimeric organization in crystals grown in alcohol solution, where only beta-strand swapping interactions between subunits are retained from the usual trimer structure. Mutations of a suggested hinge proline destabilize human and Escherichia coli dUTPases without preventing trimeric organization. Trimer formation was, however, prevented in the human enzyme by truncating the C-terminus before the swapping arm. The molecular shape of full-length enzymes in solution reveals the localization and variation in flexibility of N- and C-terminal segments.

Active site of mycobacterial dUTPase: structural characteristics and a built-in sensor.

dUTPases are essential to eliminate dUTP for DNA integrity and provide dUMP for thymidylate biosynthesis. Mycobacterium tuberculosis apparently lacks any other thymidylate biosynthesis pathway, therefore dUTPase is a promising antituberculotic drug target. Crystal structure of the mycobacterial enzyme in complex with the isosteric substrate analog, alpha,beta-imido-dUTP and Mg(2+) at 1.5A resolution was determined that visualizes the full-length C-terminus, previously not localized. Interactions of a conserved motif important in catalysis, the Mycobacterium-specific five-residue-loop insert and C-terminal tetrapeptide could now be described in detail. Stacking of C-terminal histidine upon the uracil moiety prompted replacement with tryptophan. The resulting sensitive fluorescent sensor enables fast screening for binding of potential inhibitors to the active site. K(d) for alpha,beta-imido-dUTP binding to mycobacterial dUTPase is determined to be 10-fold less than for human dUTPase, which is to be considered in drug optimization. A robust continuous activity assay for kinetic screening is proposed.

A tradeoff between protein stability and conformational mobility in homotrimeric dUTPases.

Oligomerization directs active site formation in homotrimeric 2'-deoxyuridine triphosphate pyrophosphatases (dUTPases). Stability of the homotrimer is a central determinant in enzyme function. The present comparative studies of bacterial and fruitfly dUTPases with homologous 3D structures by differential scanning microcalorimetry; fluorescence, circular dichorism and infrared spectroscopies, demonstrate that unfolding is a two-state highly cooperative transition in both dUTPases excluding a significantly populated intermediate state of dissociated and folded monomers. The eukaryotic protein is much less resistant against either thermal or guanidine hydrochloride-induced denaturation. Results suggest that hydrophobic packing of the inner threefold channel of the dUTPase homotrimer greatly contributes to stability.

Altered active site flexibility and a structural metal-binding site in eukaryotic dUTPase: kinetic characterization, folding, and crystallographic studies of the homotrimeric Drosophila enzyme.

dUTPase is responsible for preventive DNA repair via exclusion of uracil. Developmental regulation of the Drosophila enzyme is suggested to be involved in thymine-less apoptosis. Here we show that in addition to conserved dUTPase sequence motifs, the gene of Drosophila enzyme codes for a unique Ala-Pro-rich segment. Kinetic and structural analyses of the recombinant protein and a truncation mutant show that the Ala-Pro segment is flexible and has no regulatory role in vitro. The homotrimer enzyme unfolds reversibly as a trimeric entity with a melting temperature of 54 degrees C, 23 degrees C lower than Escherichia coli dUTPase. In contrast to the bacterial enzyme, Mg(2+) binding modulates conformation of fly dUTPase, as identified by spectroscopy and by increment in melting temperature. A single well folded, but inactive, homotrimeric core domain is generated through three distinct steps of limited trypsinolysis. In fly, but not in bacterial dUTPase, binding of the product dUMP induces protection against proteolysis at the tryptic site reflecting formation of the catalytically competent closed conformer. Crystallographic analysis argues for the presence of a stable monomer of Drosophila dUTPase in crystal phase. The significant differences between prototypes of eukaryotic and prokaryotic dUTPases with respect to conformational flexibility of the active site, substrate specificity, metal ion binding, and oligomerization in the crystal phase are consistent with alteration of the catalytic mechanism and hydropathy of subunit interfaces.