Crystal structure of the primary piRNA biogenesis factor Zucchini reveals similarity to the bacterial PLD endonuclease Nuc.
Voigt, F., Reuter, M., Kasaruho, A., Schulz, E.C., Pillai, R.S. & Barabas, O.
RNA. 2012 Dec;18(12):2128-34. doi: 10.1261/rna.034967.112. Epub 2012 Oct 19.
Piwi-interacting RNAs (piRNAs) are a gonad-specific class of small RNAs that associate with the Piwi clade of Argonaute proteins and play a key role in transposon silencing in animals. Since biogenesis of piRNAs is independent of the double-stranded RNA-processing enzyme Dicer, an alternative nuclease that can process single-stranded RNA transcripts has been long sought. A Phospholipase D-like protein, Zucchini, that is essential for piRNA processing has been proposed to be a nuclease acting in piRNA biogenesis. Here we describe the crystal structure of Zucchini from Drosophila melanogaster and show that it is very similar to the bacterial endonuclease, Nuc. The structure also reveals that homodimerization induces major conformational changes assembling the active site. The active site is situated on the dimer interface at the bottom of a narrow groove that can likely accommodate single-stranded nucleic acid substrates. Furthermore, biophysical analysis identifies protein segments essential for dimerization and provides insights into regulation of Zucchini's activity.
DNA recognition and the precleavage state during single-stranded DNA transposition in D. radiodurans.
Hickman, A.B., James, J.A., Barabas, O., Pasternak, C., Ton-Hoang, B., Chandler, M., Sommer, S. & Dyda, F.
EMBO J. 2010 Nov 17;29(22):3840-52. Epub 2010 Oct 1.
Bacterial insertion sequences (ISs) from the IS200/IS605 family encode the smallest known DNA transposases and mobilize through single-stranded DNA transposition. Transposition by one particular family member, ISDra2 from Deinococcus radiodurans, is dramatically stimulated upon massive gamma irradiation. We have determined the crystal structures of four ISDra2 transposase/IS end complexes; combined with in vivo activity assays and fluorescence anisotropy binding measurements, these have revealed the molecular basis of strand discrimination and transposase action. The structures also show that previously established structural rules of target site recognition that allow different specific sequences to be targeted are only partially conserved among family members. Furthermore, we have captured a fully assembled active site including the scissile phosphate bound by a divalent metal ion cofactor (Cd(2)(+)) that supports DNA cleavage. Finally, the observed active site rearrangements when the transposase binds a metal ion in which it is inactive provide a clear rationale for metal ion specificity.
Resetting the site: redirecting integration of an insertion sequence in a predictable way.
Guynet, C., Achard, A., Hoang, B.T., Barabas, O., Hickman, A.B., Dyda, F. & Chandler, M.
Mol Cell. 2009 Jun 12;34(5):612-9.
Target site choice is a complex and poorly understood aspect of DNA transposition despite its importance in rational transposon-mediated gene delivery. Though most transposons choose target sites essentially randomly or with some slight sequence or structural preferences, insertion sequence IS608 from Helicobacter pylori, which transposes using single-stranded DNA, always inserts just 3' of a TTAC tetranucleotide. Our results from studies on the IS608 transposition mechanism demonstrated that the transposase recognizes its target site by co-opting an internal segment of transposon DNA and utilizes it for specific recognition of the target sites through base-pairing. This suggested a way to redirect IS608 transposition to novel target sites. As we demonstrate here, we can now direct insertions in a predictable way into a variety of different chosen target sequences, both in vitro and in vivo.
Molecular shape and prominent role of beta-strand swapping in organization of dUTPase oligomers.
Takacs, E., Barabas, O., Petoukhov, M.V., Svergun, D.I. & Vertessy, B.G.
FEBS Lett. 2009 Mar 4;583(5):865-71. Epub 2009 Feb 11.
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.
Helicobacter Pylori's plasticity zones are novel transposable elements.
Kersulyte, D., Lee, W., Subramaniam, D., Anant, S., Herrera, P., Cabrera, L., Balqui, J., Barabas, O., Kalia, A., Gilman, R.H. & Berg, D.E.
PLoS One. 2009 Sep 3;4(9):e6859.
BACKGROUND: Genes present in only certain strains of a bacterial species can strongly affect cellular phenotypes and evolutionary potentials. One segment that seemed particularly rich in strain-specific genes was found by comparing the first two sequenced Helicobacter pylori genomes (strains 26695 and J99) and was named a "plasticity zone". PRINCIPAL FINDINGS: We studied the nature and evolution of plasticity zones by sequencing them in five more Helicobacter strains, determining their locations in additional strains, and identifying them in recently released genome sequences. They occurred as discrete units, inserted at numerous chromosomal sites, and were usually flanked by direct repeats of 5'AAGAATG, a sequence generally also present in one copy at unoccupied sites in other strains. This showed that plasticity zones are transposable elements, to be called TnPZs. Each full length TnPZ contained a cluster of type IV protein secretion genes (tfs3), a tyrosine recombinase family gene ("xerT"), and a large (>or=2800 codon) orf encoding a protein with helicase and DNA methylase domains, plus additional orfs with no homology to genes of known function. Several TnPZ types were found that differed in gene arrangement or DNA sequence. Our analysis also indicated that the first-identified plasticity zones (in strains 26695 and J99) are complex mosaics of TnPZ remnants, formed by multiple TnPZ insertions, and spontaneous and transposable element mediated deletions. Tests using laboratory-generated deletions showed that TnPZs are not essential for viability, but identified one TnPZ that contributed quantitatively to bacterial growth during mouse infection and another that affected synthesis of proinflammatory cytokines in cell culture. CONCLUSIONS: We propose that plasticity zone genes are contained in conjugative transposons (TnPZs) or remnants of them, that TnPZ insertion is mediated by XerT recombinase, and that some TnPZ genes affect bacterial phenotypes and fitness.
Active site of mycobacterial dUTPase: structural characteristics and a built-in sensor.
Varga, B., Barabas, O., Takacs, E., Nagy, N., Nagy, P. & Vertessy, B.G.
Biochem Biophys Res Commun. 2008 Aug 15;373(1):8-13. Epub 2008 Jun 2.
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.
Methylene substitution at the alpha-beta bridging position within the phosphate chain of dUDP profoundly perturbs ligand accommodation into the dUTPase active site.
Kovari, J., Barabas, O., Varga, B., Bekesi, A., Tolgyesi, F., Fidy, J., Nagy, J. & Vertessy, B.G.
Proteins. 2008 Apr;71(1):308-19.
dUTP pyrophosphatase, a preventive DNA repair enzyme, contributes to maintain the appropriate cellular dUTP/dTTP ratio by catalyzing dUTP hydrolysis. dUTPase is essential for viability in bacteria and eukaryotes alike. Identification of species-specific antagonists of bacterial dUTPases is expected to contribute to the development of novel antimicrobial agents. As a first general step, design of dUTPase inhibitors should be based on modifications of the substrate dUTP phosphate chain, as modifications in either base or sugar moieties strongly impair ligand binding. Based on structural differences between bacterial and human dUTPases, derivatization of dUTP-analogous compounds will be required as a second step to invoke species-specific character. Studies performed with dUTP analogues also offer insights into substrate binding characteristics of this important and structurally peculiar enzyme. In this study, alpha,beta-methylene-dUDP was synthesized and its complex with dUTPase was characterized. Enzymatic phosphorylation of this substrate analogue by pyruvate kinase was not possible in contrast to the successful enzymatic phosphorylation of alpha,beta-imino-dUDP. One explanation for this finding is that the different bond angles and the presence of the methylene group may preclude formation of a catalytically competent complex with the kinase. Crystal structure of E. coli dUTPase:alpha,beta-methylene-dUDP and E. coli dUTPase:dUDP:Mn complexes were determined and analyzed in comparison with previous data. Results show that the "trans" alpha-phosphate conformation of alpha,beta-methylene-dUDP differs from the catalytically competent "gauche" alpha-phosphate conformation of the imino analogue and the oxo substrate, manifested in the shifted position of the alpha-phosphorus by more than 3 A. The three-dimensional structures determined in this work show that the binding of the methylene analogue with the alpha-phosphorus in the "gauche" conformation would result in steric clash of the methylene group with the protein atoms. In addition, the metal ion cofactor was not bound in the crystal of the complex with the methylene analogue while it was clearly visible as coordinated to dUDP, arguing that the altered phosphate chain conformation also perturbs metal ion complexation. Isothermal calorimetry titrations indicate that the binding affinity of alpha,beta-methylene-dUDP toward dUTPase is drastically decreased when compared with that of dUDP. In conclusion, the present data suggest that while alpha,beta-methylene-dUDP seems to be practically nonhydrolyzable, it is not a strong binding inhibitor of dUTPase probably due to the altered binding mode of the phosphate chain. Results indicate that in some cases methylene analogues may not faithfully reflect the competent substrate ligand properties, especially if the methylene hydrogens are in steric conflict with the protein.
Revisiting the mechanism of the autoactivation of the complement protease C1r in the C1 complex: structure of the active catalytic region of C1r.
Kardos, J., Harmat, V., Pallo, A., Barabas, O., Szilagyi, K., Graf, L., Naray-Szabo, G., Goto, Y., Zavodszky, P. & Gal, P.
Mol Immunol. 2008 Mar;45(6):1752-60. Epub 2007 Nov 9.
C1r is a modular serine protease which is the autoactivating component of the C1 complex of the classical pathway of the complement system. We have determined the first crystal structure of the entire active catalytic region of human C1r. This fragment contains the C-terminal serine protease (SP) domain and the preceding two complement control protein (CCP) modules. The activated CCP1-CCP2-SP fragment makes up a dimer in a head-to-tail fashion similarly to the previously characterized zymogen. The present structure shows an increased number of stabilizing interactions. Moreover, in the crystal lattice there is an enzyme-product relationship between the C1r molecules of neighboring dimers. This enzyme-product complex exhibits the crucial S1-P1 salt bridge between Asp631 and Arg446 residues, and intermolecular interaction between the CCP2 module and the SP domain. Based on these novel structural information we propose a new split-and-reassembly model for the autoactivation of the C1r. This model is consistent with experimental results that have not been explained adequately by previous models. It allows autoactivation of C1r without large-scale, directed movement of C1q arms. The model is concordant with the stability of the C1 complex during activation of the next complement components.
In vitro reconstitution of a single-stranded transposition mechanism of IS608.
Guynet, C., Hickman, A.B., Barabas, O., Dyda, F., Chandler, M. & Ton-Hoang, B.
Mol Cell. 2008 Feb 15;29(3):302-12.
Bacterial insertion sequences (IS) play an important role in restructuring their host genomes. IS608, from Helicobacter pylori, belongs to a newly recognized and widespread IS group with a unique transposition mechanism. We have reconstituted the entire set of transposition cleavage and strand transfer reactions in vitro and find that, unlike any other known transposition system, they strictly require single-strand DNA. TnpA, the shortest identified transposase, uses a nucleophilic tyrosine for these reactions. It recognizes and cleaves only the IS608 "top strand." The results support a transposition model involving excision of a single-strand circle with abutted left (LE) and right (RE) IS ends. Insertion occurs site specifically 3' to conserved and essential TTAC tetranucleotide and appears to be driven by LE. This single-strand transposition mode has important implications not only for dispersion of IS608 but also for the other members of this very large IS family.
Mechanism of IS200/IS605 family DNA transposases: activation and transposon-directed target site selection.
Barabas, O., Ronning, D.R., Guynet, C., Hickman, A.B., Ton-Hoang, B., Chandler, M. & Dyda, F.
Cell. 2008 Jan 25;132(2):208-20.
The smallest known DNA transposases are those from the IS200/IS605 family. Here we show how the interplay of protein and DNA activates TnpA, the Helicobacter pylori IS608 transposase, for catalysis. First, transposon end binding causes a conformational change that aligns catalytically important protein residues within the active site. Subsequent precise cleavage at the left and right ends, the steps that liberate the transposon from its donor site, does not involve a site-specific DNA-binding domain. Rather, cleavage site recognition occurs by complementary base pairing with a TnpA-bound subterminal transposon DNA segment. Thus, the enzyme active site is constructed from elements of both protein and DNA, reminiscent of the interdependence of protein and RNA in the ribosome. Our structural results explain why the transposon ends are asymmetric and how the transposon selects a target site for integration, and they allow us to propose a molecular model for the entire transposition reaction.
Active site closure facilitates juxtaposition of reactant atoms for initiation of catalysis by human dUTPase.
Varga, B., Barabas, O., Kovari, J., Toth, J., Hunyadi-Gulyas, E., Klement, E., Medzihradszky, K.F., Tolgyesi, F., Fidy, J. & Vertessy, B.G.
FEBS Lett. 2007 Oct 2;581(24):4783-8. Epub 2007 Sep 12.
Human dUTPase, essential for DNA integrity, is an important survival factor for cancer cells. We determined the crystal structure of the enzyme:alpha,beta-imino-dUTP:Mg complex and performed equilibrium binding experiments in solution. Ordering of the C-terminus upon the active site induces close juxtaposition of the incoming nucleophile attacker water oxygen and the alpha-phosphorus of the substrate, decreasing their distance below the van der Waals limit. Complex interactions of the C-terminus with both substrate and product were observed via a specifically designed tryptophan sensor, suitable for further detailed kinetic and ligand binding studies. Results explain the key functional role of the C-terminus.
Flexible segments modulate co-folding of dUTPase and nucleocapsid proteins.
Nemeth-Pongracz, V., Barabas, O., Fuxreiter, M., Simon, I., Pichova, I., Rumlova, M., Zabranska, H., Svergun, D., Petoukhov, M., Harmat, V., Klement, E., Hunyadi-Gulyas, E., Medzihradszky, K.F., Konya, E. & Vertessy, B.G.
Nucleic Acids Res. 2007;35(2):495-505. Epub 2006 Dec 14.
The homotrimeric fusion protein nucleocapsid (NC)-dUTPase combines domains that participate in RNA/DNA folding, reverse transcription, and DNA repair in Mason-Pfizer monkey betaretrovirus infected cells. The structural organization of the fusion protein remained obscured by the N- and C-terminal flexible segments of dUTPase and the linker region connecting the two domains that are invisible in electron density maps. Small-angle X-ray scattering reveals that upon oligonucleotide binding the NC domains adopt the trimeric symmetry of dUTPase. High-resolution X-ray structures together with molecular modeling indicate that fusion with NC domains dramatically alters the conformation of the flexible C-terminus by perturbing the orientation of a critical beta-strand. Consequently, the C-terminal segment is capable of double backing upon the active site of its own monomer and stabilized by non-covalent interactions formed with the N-terminal segment. This co-folding of the dUTPase terminal segments, not observable in other homologous enzymes, is due to the presence of the fused NC domain. Structural and genomic advantages of fusing the NC domain to a shortened dUTPase in betaretroviruses and the possible physiological consequences are envisaged.
Crystallization and preliminary X-ray studies of dUTPase from Mason-Pfizer monkey retrovirus.
Barabas, O., Nemeth, V. & Vertessy, B.G.
Acta Crystallogr Sect F Struct Biol Cryst Commun. 2006 Apr 1;62(Pt4):399-401. Epub 2006 Mar 25.
Deoxyuridine 5'-triphosphate nucleotidohydrolase from Mason-Pfizer monkey retrovirus (M-PMV dUTPase) is a betaretroviral member of the dUTPase enzyme family. In the mature M-PMV virion, this enzyme is present as the C-terminal domain of the fusion protein nucleocapsid-dUTPase. The homotrimeric organization characteristic of dUTPases is retained in this bifunctional fusion protein. The fusion protein supposedly plays a role in adequate localization of dUTPase activity in the vicinity of nucleic acids during reverse transcription and integration. Here, the nucleocapsid-free dUTPase (48 426 Da) was cocrystallized with a dUTP substrate analogue using the hanging-drop vapour-diffusion method. The obtained crystals belong to the primitive hexagonal space group P6(3), with unit-cell parameters a = 60.6, b = 60.6, c = 63.6 angstroms, alpha = 90, beta = 90, gamma = 120 degrees. Native and PtCl4-derivative data sets were collected using synchrotron radiation to 1.75 and 2.3 angstroms, respectively. Phasing was successfully performed by isomorphous replacement combined with anomalous scattering.
Structural insights into the catalytic mechanism of phosphate ester hydrolysis by dUTPase.
Barabas, O., Pongracz, V., Kovari, J., Wilmanns, M. & Vertessy, B.G.
J Biol Chem 2004 Oct 8;279(41):42907-15. Epub 2004 Jun 17.
dUTPase is essential to keep uracil out of DNA. Crystal structures of substrate (dUTP and alpha,beta-imino-dUTP) and product complexes of wild type and mutant dUTPases were determined to reveal how an enzyme responsible for DNA integrity functions. A kinetic analysis of wild type and mutant dUTPases was performed to obtain relevant mechanistic information in solution. Substrate hydrolysis is shown to be initiated via in-line nucleophile attack of a water molecule oriented by an activating conserved aspartate residue. Substrate binding in a catalytically competent conformation is achieved by (i) multiple interactions of the triphosphate moiety with catalysis-assisting Mg2+, (ii) a concerted motion of residues from three conserved enzyme motifs as compared with the apoenzyme, and (iii) an intricate hydrogen-bonding network that includes several water molecules in the active site. Results provide an understanding for the catalytic role of conserved residues in dUTPases.
Altered active site flexibility and a structural metal-binding site in eukaryotic dUTPase: kinetic characterization, folding, and crystallographic studies of the homotrimeric Drosophila enzyme.
Kovari, J., Barabas, O., Takacs, E., Bekesi, A., Dubrovay, Z., Pongracz, V., Zagyva, I., Imre, T., Szabo, P. & Vertessy, B.G.
J Biol Chem. 2004 Apr 23;279(17):17932-44. Epub 2004 Jan 14.
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.
dUTPase and nucleocapsid polypeptides of the Mason-Pfizer monkey virus form a fusion protein in the virion with homotrimeric organization and low catalytic efficiency.
Barabas, O., Rumlova, M., Erdei, A., Pongracz, V., Pichova, I. & Vertessy, B.G.
J Biol Chem. 2003 Oct 3;278(40):38803-12. Epub 2003 Jul 16.
Betaretroviruses encode dUTPase, an essential factor in DNA metabolism and repair, in the pro open reading frame located between gag and pol. Ribosomal frame-shifts during expression of retroviral proteins provide a unique possibility for covalent joining of nucleocapsid (NC) and dUTPase within Gag-Pro polyproteins. By developing an antibody against the prototype betaretrovirus Mason-Pfizer monkey virus dUTPase, we demonstrate that i) the NC-dUTPase fusion protein exists both within the virions and infected cells providing the only form of dUTPase, and ii) the retroviral protease does not cleave NC-dUTPase either in the virion or in vitro. We show that recombinant betaretroviral NC-dUTPase and dUTPase are both inefficient catalysts compared with all other dUTPases. Dynamic light scattering and gel filtration confirm that the homotrimeric organization, common among dUTPases, is retained in the NC-dUTPase fusion protein. The betaretroviral dUTPase has been crystallized and single crystals contain homotrimers. Oligonucleotide and Zn2+ binding is well retained in the fusion protein, which is the first example of acquisition of a functional nucleic acid binding module by the DNA repair factor dUTPase. Binding of the hexanucleotide ACTGCC or the octanucleotide (TG)4 to NC-dUTPase modulates enzymatic function, indicating that the low catalytic activity may be compensated by adequate localization.
Synthesis and stereochemistry of dispiro substituted pyridazines: application of ellipticity-absorbance ratio spectra for proving enantiomeric relationship by HPLC-CD/UV detection.
Beke, G., Gergely, A., Szasz, G., Szentesi, A., Nyitray, J., Barabas, O., Harmath, V. & Matyus, P.
Chirality. 2002 May 15;14(5):365-71.
The aim of our study was to synthesize vinylic and pyrido-fused pyridazines with a spirano moiety and to investigate their stereochemistry by spectroscopic and HPLC analyses. The vinylic compounds 5 were obtained by Knoevenagel condensation of cyclohexylidene malonates 1 with pyridazinecarbaldehyde 2. Compound 5b exhibits geometric isomerism identified by NMR, HPLC, and X-ray methods. The thermal rearrangement reactions of compounds 5 easily led to the pyridopyridazine derivatives 6. In the case of 6b, possessing both central and axial chirality, both diastereomers and the respective enantiomers were detected. Their stereochemical relationships could be determined by HPLC-CD/UV analyses with application of anisotropy spectra in a novel way.
Theoretical and experimental studies on ring closure reactions of 4(5)-chloro-5(4)-hydroxyalkylamino-6-nitro-3-(2H)-pyridazinones.
Éliás, O., Károlyházy, L., Stájer, G., Fülöp, F., Czakó, K., Harmat, V., Barabás, O., Keserű, K., Mátyus, P.
J. Mol. Structure Theochem. 2001;545:75-96
Novel ring transformations of condensed [1,2,4]triazolo[4,3-b]pyridazine-6(5H)-one-3(2H)-thiones effected by dialkyl-acetylenedicarboxylates.
Simó, M., Csámpai, A., Harmat, V., Barabás, O., Magyarfalvy, G.
Study on ferrocenes, part 9  substrate selective transformations of some ferrocenylhydrazones.
Abrán, Á., Csámpai, A., Kotschy, A., Barabás, O., Sohár, P.
J. Mol. Structure. 2001;569:185-194
Structural basis of solid solution formation during chiral resolution.
Barabás, O., Menyhárd, D.K., Böcskei, Zs., Simon, K., Kiss-Ajzert, I., Takács K. and Hermecz I.
Tetrahedron: Asymmetry. 2000;11:4061-4070