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.

The Role of a Key Amino Acid Position in Species-Specific Proteinaceous dUTPase Inhibition.

Protein inhibitors of key DNA repair enzymes play an important role in deciphering physiological pathways responsible for genome integrity, and may also be exploited in biomedical research. The staphylococcal repressor StlSaPIbov1 protein was described to be an efficient inhibitor of dUTPase homologues showing a certain degree of species-specificity. In order to provide insight into the inhibition mechanism, in the present study we investigated the interaction of StlSaPIbov1 and Escherichia coli dUTPase. Although we observed a strong interaction of these proteins, unexpectedly the E. coli dUTPase was not inhibited. Seeking a structural explanation for this phenomenon, we identified a key amino acid position where specific mutations sensitized E. coli dUTPase to StlSaPIbov1 inhibition. We solved the three-dimensional (3D) crystal structure of such a mutant in complex with the substrate analogue dUPNPP and surprisingly found that the C-terminal arm of the enzyme, containing the P-loop-like motif was ordered in the structure. This segment was never localized before in any other E. coli dUTPase crystal structures. The 3D structure in agreement with solution phase experiments suggested that ordering of the flexible C-terminal segment upon substrate binding is a major factor in defining the sensitivity of E. coli dUTPase for StlSaPIbov1 inhibition.

Structural insights into the tyrosine phosphorylation-mediated inhibition of SH3 domain-ligand interactions.

Src homology 3 (SH3) domains bind proline-rich linear motifs in eukaryotes. By mediating inter- and intramolecular interactions, they regulate the functions of many proteins involved in a wide variety of signal transduction pathways. Phosphorylation at different tyrosine residues in SH3 domains has been reported previously. In several cases, the functional consequences have also been investigated. However, a full understanding of the effects of tyrosine phosphorylation on the ligand interactions and cellular functions of SH3 domains requires detailed structural, atomic-resolution studies along with biochemical and biophysical analyses. Here, we present the first crystal structures of tyrosine-phosphorylated human SH3 domains derived from the Abelson-family kinases ABL1 and ABL2 at 1.6 and 1.4 Å resolutions, respectively. The structures revealed that simultaneous phosphorylation of Tyr89 and Tyr134 in ABL1 or the homologous residues Tyr116 and Tyr161 in ABL2 induces only minor structural perturbations. Instead, the phosphate groups sterically blocked the ligand-binding grooves, thereby strongly inhibiting the interaction with proline-rich peptide ligands. Although some crystal contact surfaces involving phosphotyrosines suggested the possibility of tyrosine phosphorylation-induced dimerization, we excluded this possibility by using small-angle X-ray scattering (SAXS), dynamic light scattering (DLS), and NMR relaxation analyses. Extensive analysis of relevant databases and literature revealed not only that the residues phosphorylated in our model systems are well-conserved in other human SH3 domains, but that the corresponding tyrosines are known phosphorylation sites in vivo in many cases. We conclude that tyrosine phosphorylation might be a mechanism involved in the regulation of the human SH3 interactome.

Molecular dynamics of the cryo-EM CFTR structure.

Cystic fibrosis (CF), a lethal monogenic disease, is caused by mutant variants of the CF transmembrane conductance regulator (CFTR). Recent advances in single molecule cryo-EM methods enabled structural determination of full-length human and zebrafish CFTR, achieving an important milestone for CF drug development. To relate these structures to the gating cycle, we examined its dynamic features using molecular dynamics simulations. Our results show that the nucleotide binding domains (NBDs) in this bottom-open apo conformation exhibit motions related to dimerization and the bottom-closed apo CFTR model indicates opening of NBDs in contrast to transporters. These observations help in understanding the properties of CFTR chloride channel distinct from transporters and in proper interpretation of available structural information on this ABC protein.

Regulation of the Equilibrium between Closed and Open Conformations of Annexin A2 by N-Terminal Phosphorylation and S100A4-Binding.

Annexin A2 (ANXA2) has a versatile role in membrane-associated functions including membrane aggregation, endo- and exocytosis, and it is regulated by post-translational modifications and protein-protein interactions through the unstructured N-terminal domain (NTD). Our sequence analysis revealed a short motif responsible for clamping the NTD to the C-terminal core domain (CTD). Structural studies indicated that the flexibility of the NTD and CTD are interrelated and oppositely regulated by Tyr24 phosphorylation and Ser26Glu phosphomimicking mutation. The crystal structure of the ANXA2-S100A4 complex showed that asymmetric binding of S100A4 induces dislocation of the NTD from the CTD and, similar to the Ser26Glu mutation, unmasks the concave side of ANXA2. In contrast, pTyr24 anchors the NTD to the CTD and hampers the membrane-bridging function. This inhibition can be restored by S100A4 and S100A10 binding. Based on our results we provide a structural model for regulation of ANXA2-mediated membrane aggregation by NTD phosphorylation and S100 binding.

The First Enantioselective Total Synthesis of (-)-trans-Dihydronarciclasine.

A feasible and enantioselective total synthesis of (-)-trans-dihydronarciclasine [(-)-1], a highly biologically active alkaloid, was devised starting from vanillin (8). The key step of this new synthesis was an asymmetric, organocatalytic Michael addition, in which an optically active nitropentanone [(-)-13] was obtained from a butenone derivative (12). Excellent enantioselectivity (>99% ee) was achieved using the (8S,9S)-9-amino(9-deoxy)epiquinine (16) organocatalyst. The target molecule can be prepared in 13 steps from compound (-)-13. The total synthesis has provided a facile and first access to the ent-form of naturally occurring (+)-trans-dihydronarciclasine, a highly potent cytostatic alkaloid.

A Hidden Active Site in the Potential Drug Target Mycobacterium tuberculosis dUTPase Is Accessible through Small Amplitude Protein Conformational Changes.

dUTPases catalyze the hydrolysis of dUTP into dUMP and pyrophosphate to maintain the proper nucleotide pool for DNA metabolism. Recent evidence suggests that dUTPases may also represent a selective drug target in mycobacteria because of the crucial role of these enzymes in maintaining DNA integrity. Nucleotide-hydrolyzing enzymes typically harbor a buried ligand-binding pocket at interdomain or intersubunit clefts, facilitating proper solvent shielding for the catalyzed reaction. The mechanism by which substrate binds this hidden pocket and product is released in dUTPases is unresolved because of conflicting crystallographic and spectroscopic data. We sought to resolve this conflict by using a combination of random acceleration molecular dynamics (RAMD) methodology and structural and biochemical methods to study the dUTPase from Mycobacterium tuberculosis In particular, the RAMD approach used in this study provided invaluable insights into the nucleotide dissociation process that reconciles all previous experimental observations. Specifically, our data suggest that nucleotide binding takes place as a small stretch of amino acids transiently slides away and partially uncovers the active site. The in silico data further revealed a new dUTPase conformation on the pathway to a relatively open active site. To probe this model, we developed the Trp21 reporter and collected crystallographic, spectroscopic, and kinetic data that confirmed the interaction of Trp21 with the active site shielding C-terminal arm, suggesting that the RAMD method is effective. In summary, our computational simulations and spectroscopic results support the idea that small loop movements in dUTPase allow the shuttlingof the nucleotides between the binding pocket and the solvent.

Structural Characterization of Arginine Fingers: Identification of an Arginine Finger for the Pyrophosphatase dUTPases.

Arginine finger is a highly conserved and essential residue in many GTPase and AAA+ ATPase enzymes that completes the active site from a distinct protomer, forming contacts with the γ-phosphate of the nucleotide. To date, no pyrophosphatase has been identified that employs an arginine finger fulfilling all of the above properties; all essential arginine fingers are used to catalyze the cleavage of the γ-phosphate. Here, we identify and unveil the role of a conserved arginine residue in trimeric dUTPases that meets all the criteria established for arginine fingers. We found that the conserved arginine adjacent to the P-loop-like motif enables structural organization of the active site for efficient catalysis via its nucleotide coordination, while its direct electrostatic role in transition state stabilization is secondary. An exhaustive structure-based comparison of analogous, conserved arginines from nucleotide hydrolases and transferases revealed a consensus amino acid location and orientation for contacting the γ-phosphate of the substrate nucleotide. Despite the structurally equivalent position, functional differences between arginine fingers of dUTPases and NTPases are explained on the basis of the unique chemistry performed by the pyrophosphatase dUTPases.

Origin of problems related to Staudinger reduction in carbopeptoid syntheses.

We report the solid phase synthesis of -GG-X-GG- type α/ß-carbopeptoids incorporating RibAFU(ip) (1a, tX) or XylAFU(ip) (2a, cX) sugar amino acids. Though coupling efficacy is moderate, both the lengthier synthetic route using Fmoc derivative (e.g., Fmoc-RibAFU(ip)-OH) and the azido derivative (e.g., N3-RibAFU(ip)-OH) via Staudinger reaction with nBu3P can be successfully applied. Both X-ray diffraction, 1H- and 31P-NMR, and theoretical (QM) data support and explain why the application of Ph3P as Staudinger reagent is "ineffective" in the case of a cis stereoisomer, if cX is attached to the preceding residue with a peptide (-CONH-) bond. The failure of the polypeptide chain elongation with N3-cX originates from the "coincidence" of a steric crowdedness and an electronic effect disabling the mandatory nucleophilic attack during the hydrolysis of a quasi penta-coordinated triphenylphosphinimine. Nevertheless, the synthesis of the above α/ß-chimera peptides as completed now by a new pathway via 1,2-O-isopropylidene-3-azido-3-deoxy-ribo- and -xylo-furanuronic acid (H-RibAFU(ip)-OH 1a and H-XylAFU(ip)-OH 2a) coupled with N-protected α-amino acids on solid phase could serve as useful examples and starting points of further synthetic efforts.

Highly potent dUTPase inhibition by a bacterial repressor protein reveals a novel mechanism for gene expression control.

Transfer of phage-related pathogenicity islands of Staphylococcus aureus (SaPI-s) was recently reported to be activated by helper phage dUTPases. This is a novel function for dUTPases otherwise involved in preservation of genomic integrity by sanitizing the dNTP pool. Here we investigated the molecular mechanism of the dUTPase-induced gene expression control using direct techniques. The expression of SaPI transfer initiating proteins is repressed by proteins called Stl. We found that Φ11 helper phage dUTPase eliminates SaPIbov1 Stl binding to its cognate DNA by binding tightly to Stl protein. We also show that dUTPase enzymatic activity is strongly inhibited in the dUTPase:Stl complex and that the dUTPase:dUTP complex is inaccessible to the Stl repressor. Our results disprove the previously proposed G-protein-like mechanism of SaPI transfer activation. We propose that the transfer only occurs if dUTP is cleared from the nucleotide pool, a condition promoting genomic stability of the virulence elements.

Preventive DNA repair by sanitizing the cellular (deoxy)nucleoside triphosphate pool.

The occurrence of modified bases in DNA is attributed to some major factors: incorporation of altered nucleotide building blocks and chemical reactions or radiation effects on bases within the DNA structure. Several enzyme families are involved in preventing the incorporation of noncanonical bases playing a 'sanitizing' role. The catalytic mechanism of action of these enzymes has been revealed for a number of representatives in clear structural and kinetic detail. In this review, we focus in detail on those examples where clear evidence has been produced using high-resolution structural studies. Comparing the protein fold and architecture of the enzyme active sites, two main classes of sanitizing deoxyribonucleoside triphosphate pyrophosphatases can be assigned that are distinguished by the site of nucleophilic attack. In enzymes associated with attack at the α-phosphorus, it is shown that coordination of the γ-phosphate group is also ensured by multiple interactions. By contrast, enzymes catalyzing attack at the ß-phosphorus atom mainly coordinate the α- and the ß-phosphate only. Characteristic differences are also observed with respect to the role of the metal ion cofactor (Mg(2+) ) and the coordination of nucleophilic water. Using different catalytic mechanisms embedded in different protein folds, these enzymes present a clear example of convergent evolution.

Structure and enzymatic mechanism of a moonlighting dUTPase.

Genome integrity requires well controlled cellular pools of nucleotides. dUTPases are responsible for regulating cellular dUTP levels and providing dUMP for dTTP biosynthesis. In Staphylococcus, phage dUTPases are also suggested to be involved in a moonlighting function regulating the expression of pathogenicity-island genes. Staphylococcal phage trimeric dUTPase sequences include a specific insertion that is not found in other organisms. Here, a 2.1 Šresolution three-dimensional structure of a ϕ11 phage dUTPase trimer with complete localization of the phage-specific insert, which folds into a small ß-pleated mini-domain reaching out from the dUTPase core surface, is presented. The insert mini-domains jointly coordinate a single Mg2+ ion per trimer at the entrance to the threefold inner channel. Structural results provide an explanation for the role of Asp95, which is suggested to have functional significance in the moonlighting activity, as the metal-ion-coordinating moiety potentially involved in correct positioning of the insert. Enzyme-kinetics studies of wild-type and mutant constructs show that the insert has no major role in dUTP binding or cleavage and provide a description of the elementary steps (fast binding of substrate and release of products). In conclusion, the structural and kinetic data allow insights into both the phage-specific characteristics and the generally conserved traits of ϕ11 phage dUTPase.

Crystallization and preliminary crystallographic analysis of dUTPase from the φ11 helper phage of Staphylococcus aureus.

Staphylococcus aureus superantigen-carrying pathogenicity islands (SaPIs) play a determinant role in spreading virulence genes among bacterial populations that constitute a major health hazard. Repressor (Stl) proteins are responsible for the transcriptional regulation of pathogenicity island genes. Recently, a derepressing interaction between the repressor Stl SaPIbov1 and dUTPase from the φ11 helper phage has been suggested [Tormo-Más et al. (2010), Nature (London), 465, 779-782]. Towards elucidation of the molecular mechanism of this interaction, this study reports the expression, purification and X-ray analysis of φ11 dUTPase, which contains a phage-specific polypeptide segment that is not present in other dUTPases. Crystals were obtained using the hanging-drop vapour-diffusion method at room temperature. Data were collected to 2.98 Å resolution from one type of crystal. The crystal of φ11 dUTPase belonged to the cubic space group I23, with unit-cell parameters a = 98.16 Å, α = ß = γ = 90.00°.

Association of RNA with the uracil-DNA-degrading factor has major conformational effects and is potentially involved in protein folding.

Recently, a novel uracil-DNA-degrading factor protein (UDE) was identified in Drosophila melanogaster, with homologues only in pupating insects. Its unique uracil-DNA-degrading activity and a potential domain organization pattern have been described. UDE seems to be the first representative of a new protein family with unique enzyme activity that has a putative role in insect development. In addition, UDE may also serve as potential tool in molecular biological applications. Owing to lack of homology with other proteins with known structure and/or function, de novo data are required for a detailed characterization of UDE structure and function. Here, experimental evidence is provided that recombinant protein is present in two distinct conformers. One of these contains a significant amount of RNA strongly bound to the protein, influencing its conformation. Detailed biophysical characterization of the two distinct conformational states (termed UDE and RNA-UDE) revealed essential differences. UDE cannot be converted into RNA-UDE by addition of the same RNA, implying putatively joint processes of RNA binding and protein folding in this conformational species. By real-time PCR and sequencing after random cloning, the bound RNA pool was shown to consist of UDE mRNA and the two ribosomal RNAs, also suggesting cotranslational RNA-assisted folding. This finding, on the one hand, might open a way to obtain a conformationally homogeneous UDE preparation, promoting successful crystallization; on the other hand, it might imply a further molecular function of the protein. In fact, RNA-dependent complexation of UDE was also demonstrated in a fruit fly pupal extract, suggesting physiological relevance of RNA binding of this DNA-processing enzyme.

Aromatic stacking between nucleobase and enzyme promotes phosphate ester hydrolysis in dUTPase.

Aromatic interactions are well-known players in molecular recognition but their catalytic role in biological systems is less documented. Here, we report that a conserved aromatic stacking interaction between dUTPase and its nucleotide substrate largely contributes to the stabilization of the associative type transition state of the nucleotide hydrolysis reaction. The effect of the aromatic stacking on catalysis is peculiar in that uracil, the aromatic moiety influenced by the aromatic interaction is relatively distant from the site of hydrolysis at the alpha-phosphate group. Using crystallographic, kinetics, optical spectroscopy and thermodynamics calculation approaches we delineate a possible mechanism by which rate acceleration is achieved through the remote π-π interaction. The abundance of similarly positioned aromatic interactions in various nucleotide hydrolyzing enzymes (e.g. most families of ATPases) raises the possibility of the reported phenomenon being a general component of the enzymatic catalysis of phosphate ester hydrolysis.

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.

A novel fruitfly protein under developmental control degrades uracil-DNA.

Uracil in DNA may arise by cytosine deamination or thymine replacement and is removed during DNA repair. Fruitfly larvae lack two repair enzymes, the major uracil-DNA glycosylase and dUTPase, and may accumulate uracil-DNA. We asked if larval tissues contain proteins that specifically recognize uracil-DNA. We show that the best hit of pull-down on uracil-DNA is the protein product of the Drosophila melanogaster gene CG18410. This protein binds to both uracil-DNA and normal DNA but degrades only uracil-DNA; it is termed Uracil-DNA Degrading Factor (UDE). The protein has detectable homology only to a group of sequences present in genomes of pupating insects. It is under detection level in the embryo, most of the larval stages and in the imago, but is strongly upregulated right before pupation. In Schneider 2 cells, UDE mRNA is upregulated by ecdysone. UDE represents a new class of proteins that process uracil-DNA with potential involvement in metamorphosis.