Báo cáo khoa học: Physiological truncation and domain organization of a novel uracil-DNA-degrading factor pdf

Physiological truncation and domain organization of a novel uracil-DNA-degrading factor Ma ´ ria Puka ´ ncsik 1 , Ange ´ la Be ´ ke ´ si 1 ,E ´ va Klement 2 ,E ´ va Hunyadi-Gulya ´ s 2 , Katalin F. Medzihradszky 2,3 , Jan Kosinski 4,5 , Janusz M. Bujnicki 4,6 , Carlos Alfonso 7 , Germa ´ n Rivas 7 and Bea ´ ta G. Ve ´ rtessy 1 1 Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, Budapest, Hungary 2 Proteomics Research Group, Biological Research Center, Hungarian Academy of Sciences, Szeged, Hungary 3 Department of Pharmaceutical Chemistry, University of California, San Francisco, CA, USA 4 Laboratory of Bioinformatics and Protein Engineering, International Institute of Molecular and Cell Biology, Warsaw, Poland 5 PhD School, Institute of Biochemistry and Biophysics PAS, Warsaw, Poland 6 Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University, Poznan, Poland 7 Chemical and Physical Biology, Centro de Investigaciones Biolo ´ gicas, Madrid, Spain Introduction The nucleobase uracil is not a normal constituent of DNA, although it provides the same Watson–Crick interaction pattern for adenine as does thymine (i.e. 5-methyl-uracil), and is actually used as the adenine- counterpart base in RNA. Despite its usual absence, there are two physiological ways for uracil to appear Keywords cell death; DNA; nuclease; protein structural modeling; uracil Correspondence B. G. Ve ´ rtessy, Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, H-1113, Budapest, Karolina u ´ t 29, Hungary Fax: +36 1 466 5465 Tel: +36 1 279 3116 E-mail: vertessy@enzim.hu (Received 2 April 2009, revised 16 December 2009, accepted 18 December 2009) doi:10.1111/j.1742-4658.2009.07556.x Uracil in DNA is usually considered to be an error, but it may be used for signaling in Drosophila development via recognition by a novel uracil- DNA-degrading factor (UDE) [(Bekesi A et al. (2007) Biochem Biophys Res Commun 355, 643–648]. The UDE protein has no detectable similarity to any other uracil-DNA-binding factors, and has no structurally or func- tionally described homologs. Here, a combination of theoretical and experi- mental analyses reveals the domain organization and DNA-binding pattern of UDE. Sequence alignments and limited proteolysis with different prote- ases show extensive protection by DNA at the N-terminal duplicated con- served motif 1A ⁄ 1B segment, and a well-folded domain within the C-terminal half encompassing conserved motifs 2–4. Theoretical structure prediction suggests that motifs 1A and 1B fold as similar a-helical bundles, and reveals two conserved positively charged surface patches that may bind DNA. CD spectroscopy also supports the presence of a-helices in UDE. Full functionality of a physiologically occurring truncated isoform in Tribolium castaneum lacking one copy of the N-terminal conserved motif 1 is revealed by activity assays of a representative truncated construct of Drosophila melanogaster UDE. Gel filtration and analytical ultracentrifuga- tion results, together with analysis of predicted structural models, suggest a possible dimerization mechanism for preserving functionality of the truncated isoform. Structured digital abstract l MINT-7385914: UDE (uniprotkb:Q961C4) and UDE (uniprotkb:Q961C4) bind (MI:0407)by cosedimentation in solution ( MI:0028) Abbreviations DmUDE, Drosophila melanogaster uracil-DNA-degrading factor; Dm rc UDE, recombinant Drosophila melanogaster uracil-DNA-degrading factor; MQAP, model quality assessment program; TcUDE, Tribolium castaneum truncated uracil-DNA-degrading factor isoform; UDE, uracil-DNA-degrading factor; UDG, uracil-DNA glycosylase. FEBS Journal 277 (2010) 1245–1259 ª 2010 The Authors Journal compilation ª 2010 FEBS 1245 in DNA: cytosine deamination and thymine replace- ment. Cytosine-to-uracil transitions via hydrolytic deamination are among the most frequently occurring spontaneous mutations. These generate premutagenic U:G mispairs [1,2]. Thymine replacement by uracil can occur if the cellular dUTP ⁄ dTTP ratio increases, as most DNA polymerases will incorporate either uracil or thymine against adenine, based solely on the avail- ability of the corresponding building block nucleotides [3,4]. Thymine-replacing uracil moieties are not muta- genic, as they provide the same genomic information, but may perturb the binding of factors that require the 5-methyl group on the thymine ring for recognition. There are also two mechanisms to ensure uracil-free DNA: prevention and excision. dUTPases prevent uracil incorporation into DNA by removing dUTP from the DNA polymerase pathway [5]. Uracil in DNA, produced by either cytosine deamination or uracil misincorporation, is excised by uracil-DNA gly- cosylases (UDGs) in the base excision repair pathway [6,7]. Among the different UDGs, the protein product of the ung gene (termed UNG) is by far the most effi- cient in catalyzing uracil excision [8]. UNG is responsi- ble for most of the repair process, as its mutation in Escherichia coli, mouse and human has been found to induce a considerable increase in uracil content [9–11]. Null mutations in the dUTPase gene (dut) result in a nonviable phenotype that can be rescued by a second null mutation in the ung gene. The dut ) ung ) genotype presents mass uracil incorporation into DNA [9,12]. Interestingly, an analogous situation, with simulta- neous lack of dUTPase and UNG activities, arises in Drosophila larvae under physiological conditions. On the one hand, the ung gene coding for the major UDG enzyme is not present in the Drosophila genome [13]. On the other hand, it has been shown that the dUTPase level is under the limit of detection in larval tissues, and that the enzyme is present exclusively in the imaginal disks [14]. Simultaneous lack of UNG and dUTPase may lead to accumulation of uracil- substituted DNA in fruitfly larval tissues. A specific protein termed uracil-DNA-degrading factor (UDE), which recognizes and degrades uracil-DNA, was identified in Drosophila late larvae and pupae, strengthening the hypothesis that Drosophila melanog- aster may use uracil-DNA as a signal to switch on metamorphosis-related cell death [15–17]. UDE is the first member of a new protein family whose members recognize uracil-DNA. It has no glycosylase activity, and its sequence does not show any appreciable similarity to those of other nucleases or uracil-DNA-recognizing proteins [15] (Fig. 1). Sig- nificantly similar protein sequences were found only in translated genomes of other pupating insects, but no structural or functional data have been published on any of these putative proteins. In all of these sequences of homologous proteins, four distinct conserved sequence motifs could be identified (motifs 1–4), the first of which is substantially longer and is usually present in two copies (motifs 1A and 1B). Comparison of these motifs with motifs in UDGs does not offer any clue regarding the structure and function of UDE, as no apparent similarity could be observed (Fig. 1B–E) [18]. Investigation of this protein may therefore offer new insights into the physiological role and catalytic mech- anism of nucleases. To this end, in the present study we probed the domain organization of UDE from D. melanogaster, expressed as a recombinant protein (Dm rc UDE), by limited proteolysis, and revealed that a specific trun- cated fragment lacking the N-terminus may fold into a stable conformation. Interestingly, we also identified such a truncated physiologically occurring UDE iso- form from the pupating insect Tribolium castaneum (TcUDE) [19]. The TcUDE isoform lacks one copy of the N-terminal duplicated first motif. We generated the respective segment from Dm rc UDE by chemical cleav- age with hydroxylamine, and found that this truncated segment retains catalytic specificity and activity. The structural results therefore offer an explanation for the physiological existence of the truncated isoform. De novo modeling was performed using rosetta, and a 3D structural model was constructed for the tan- demly duplicated N-terminal motifs 1A and 1B. The model suggests that both motifs comprise similar three-helical bundles, with the same topology and rela- tive orientation of a-helices. A high content of helical secondary structure in UDE was also independently confirmed by CD. The predictions, together with the domain organization studies, offer a model of DNA binding to an extended surface on the protein along the conserved motifs. Results Identification of a physiologically occurring truncated isoform of UDE blast searches indicated that UDE has detectable homologs only in pupating insects (Fig. 1A). The mul- tiple sequence alignment shows four conserved motifs (Fig. 1A,E). The first extended UDE motif is present in two highly similar copies. The UDE homolog from T. castaneum contains only one copy of motif 1, sug- gesting that lack of the first motif may still result in a functional protein (Fig. 1A). Protein function preserved in a truncated isoform M. Puka ´ ncsik et al. 1246 FEBS Journal 277 (2010) 1245–1259 ª 2010 The Authors Journal compilation ª 2010 FEBS MUG/TDG AUDG UDGX SMUG UNG DRUDG 1 2 3 Common α/β fold 1A 1B 2 3 4 UDG families Motif 1 Motif 2 Motif 3 UNG VhhLGQDPYH F hFhhWG hhcppH PSP MUG/TDG hhhxGINPGL F/Y hhhFxG haVhPppSh UDGX hhhhGShPxx Y hhhxpG hhxLPSTSx AUDG hhhhGExPGx F hhhxhG hhhxaHPSh DRUDG xLxLLExPGP f VVhxLG xhxxxHPSh SMUG hhFhGMNPGP F hhVhVG VxxLxH PSP UDE motifs 1A/1B GFKDxxxAxxTLxxLxxRDxpYpxxxhxGLhxxAKRVLxxTKxExKhxxIKxAhxxhEpaL 2 GpYKcLRp 3 TWDIxRN 4 KxFpxcxxxPTxxHLxxIxWAYSxpxxKhK UDE UDG families B A D E C Fig. 1. Sequence alignment of UDE homologs in D. melanogaster and T. castaneum, and conserved motifs in UDE and members of the UDG superfamily. (A) Alignment of D. melanogaster and T. castaneum UDE homologs. Gray background: conserved motifs. Red letters: strictly conserved residues. (B) Evolutionary relationship and organization of conserved motifs among UDG proteins [18]. Gray background: uracil-DNA-recognizing proteins present in D. melanogaster. (C) Organization of conserved motifs in UDE. (D, E) Consensus sequences of UDG (D) and UDE (E) motifs. Upper-case letters: conserved residues. Lower-case letters: residues with conserved characteristics (h, hydro- phobic; a, aromatic; p, polar ⁄ charged). Nonconserved positions are indicated by x. A conserved F ⁄ Y residue, overlapping with the uracil ring, is invariably present C-terminal to motif 1 in UDGs. Underlined Asp ⁄ Glu residues in UDG motif 1 are involved in catalysis; the underlined His in UDG motif 3 is suggested to stabilize reaction intermediates. Note the lack of detectable similarities between UDE and UDG motifs. M. Puka ´ ncsik et al. Protein function preserved in a truncated isoform FEBS Journal 277 (2010) 1245–1259 ª 2010 The Authors Journal compilation ª 2010 FEBS 1247 To confirm the in silico prediction of the UDE-like protein product in Tribolium, extracts of the insect larvae were investigated by western blot, using the polyclonal antiserum produced against Dm rc UDE. As expected from the high sequence similarity, the antise- rum recognized the Tribolium protein (TcUDE) as well (Fig. 2). The blot clearly indicates that larval extract from T. castaneum contains a single protein that reacts with the UDE-specific antibody. This positive band is found at a position corresponding to a much lower molecular mass than that of Dm rc UDE and that of the physiological form of D. melanogaster uracil-DNA- degrading factor (DmUDE). The altered position of TcUDE was in agreement with the genomic data (Fig. 1A), and led to the conclusion that the physiolog- ically occurring TcUDE lacks the N-terminal segment. These results suggest that an isoform of UDE lacking motif 1A may fold on its own, and may form a func- tional protein. Domain organization studies using limited proteolysis To delineate the domain organization of the UDE pro- tein more precisely, limited proteolysis experiments were performed. Three proteases with different specificities were used. Experiments were conducted with Dm rc UDE alone, and also in the presence of added DNA to study potential DNA-binding protein segments. Trypsin was selected first, as the UDE protein con- tains many potential tryptic cleavage sites (i.e. Lys and Arg residues) scattered throughout the sequence. Fig- ure 3A indicates fast initial fragmentation leading to loss of 5–7 kDa fragments from either the N-terminus or the C-terminus, or both. This initial fragmentation is not affected by the presence of DNA. Flexibility of the N-terminal segment (residues 1–47) is also sug- gested by the drastic overrepresentation of basic resi- dues, leading to an extremely high pI (11.5) for this segment. At later stages of proteolysis, DNA protec- tion is evident, as a specific fragment persists stably in the presence of DNA, whereas this fragment is rapidly degraded in the absence of DNA. Several smaller frag- ments are produced in relatively large amounts in the absence of DNA, whereas these peptides are practi- cally absent in the presence of DNA. The data suggest the presence of an inner folded core, which is sug- gested to participate in DNA binding, on the basis of DNA-binding-induced stabilization. The large number of potential tryptic cleavage sites prevented straight- forward identification of the fragments, observed on SDS ⁄ PAGE, by MS. For further characterization and localization of pro- tein segments involved in DNA binding to UDE, two additional sets of experiments were conducted, using highly specific chymotrypsin [20] and Asp-N endopro- teinase. These enzymes have considerably fewer poten- tial cleavage sites in the protein. In both cases, protection by DNA is again evident (Fig. 3B,C). Figure 3B shows that, in the absence of DNA, initial chymotryptic cleavage removes a segment of about 9.6 kDa from UDE, whereas in the presence of DNA, the removed peptide is much smaller, around 3 kDa. MS analysis of the initially cleaved fragments revealed that the C-terminus remained intact, and the two pep- tide bonds most sensitive to chymotrypsin could there- fore be localized at the N-terminus at Trp10 and Tyr69 in the presence and in the absence of DNA, respectively (Fig. 3D). DNA binding is therefore asso- ciated with significant protection at the Tyr69-Arg70 peptide bond located within the conserved motif 1A. In addition, DNA-binding-induced conformational changes are also reflected at the Phe104-Glu105 and Tyr311-Ile312 peptide bonds, which become exposed in the presence of DNA (Fig. 3D). To characterize the potential involvement of the C-terminal region of UDE in DNA binding, Asp-N endoproteinase was also used for limited proteolysis, as the C-terminus of the protein is rather rich in Asp residues (Fig. 3C,D). When it is digested by Asp-N endoproteinase, the primary cleavage removes a short fragment of about 3.4 kDa, independently of the 55 kDa 36 kDa DmUDE Dm rc UDE TcUDE 1A 1B 2 3 4 1 2 3 4 DmUDE TcUDE Fig. 2. Immunodetection of UDE homolog from T. castaneum. Western blot indicates that polyclonal anti-DmUDE serum recog- nizes the UDE homolog from T. castaneum that appeared at a lower position than physiological DmUDE or Dm rc UDE. Lane 1: D. melanogaster larval extract. Lane 2: purified Dm rc UDE. Lane 3: T. castaneum larval extract. Protein function preserved in a truncated isoform M. Puka ´ ncsik et al. 1248 FEBS Journal 277 (2010) 1245–1259 ª 2010 The Authors Journal compilation ª 2010 FEBS presence of DNA. This loss is in good agreement with a C-terminal cleavage (at Asp333) leading to the loss of 2.6 kDa; cleavage at the first N-terminal Asp (Asp44) would remove a peptide of 6.6 kDa, which is much larger than estimated from the gel electropho- retic analysis. It is evident that, in the absence of DNA, additional cleavages can also occur, yielding 23–25 and 17 kDa polypeptides, as observed on SDS ⁄ PAGE. Binding of DNA induces significant pro- tection against all of these cleavages, except at the Asp333 site, which shows the same highly exposed character for Asp-N endoproteinase digestion in the presence and in the absence of DNA. The results of proteolytic experiments are summa- rized in Fig. 3D. It is obvious that the segment encom- passing motifs 2–4 is a well-folded part of the protein, even in the absence of DNA that lacks exposed prote- olytic sites [despite the presence of numerous potential tryptic, chymotryptic and Asp-N sites (Figs 1A and 3D)]. Motifs 1A and 1B, on the other hand, are signifi- cantly more prone to proteolysis, especially in the absence of DNA. DNA binding provides significant protection against proteolytic cleavage along motifs 1A and 1B, indicating either DNA-binding-induced conformational changes or covering of otherwise exposed proteolytic sites by DNA binding to these segments. Motif 1A is dispensable for UDE function To produce a specific truncated DmUDE isoform mimicking the physiologically occurring protein in T. castaneum, we selected a chemical agent, hydroxyl- amine, that cleaves peptide bonds exclusively between 36 kDa 28 kDa 17 kDa 11 kDa 55 kDa w/o U-DNA w/U-DNA MM 0´ 30´ 60´ 120´ 180´ 0´ 30´ 60´ 120´ 1A 1B 2 3 4 His – tag W10 Y69 W107 F136 Y156 F194 F198 Y311 D44 D66 D126 D179 D193 D333 N111 36 kDa 28 kDa 17 kDa 11 kDa 55 kDa w/o U-DNA w/U-DNA 0´ 60´ 180´ 300´ MM 0´ 60´ 180´ 300´ 36 kDa w/o U-DNA w/U-DNA 0´ 15´ 30´ 60´ 0´ 15´ 30´ 60´ MM 45 kDa 29 kDa 24 kDa 20 kDa 14.2 kDa F104 Trypsin digestion Chymotrypsin digestion Asp-N proteinase digestion AC B D Fig. 3. Initial domain analysis of DmUDE by limited proteolysis. (A) Tryptic digestion pattern. Arrows indicate fragments that are preferen- tially produced in the absence of DNA; the star shows the detected position of stable fragment persisting in the presence of DNA. (B, C) Limited digestion patterns obtained using high-specificity chymotrypsin and Asp-N endoproteinase. The timescale of limited digestion and the presence or absence of added ligand are indicated at the top of the gel. MM, molecular markers. (D) Summary of cleavage sites identi- fied by MS. Top row: chymotryptic sites. Bottom row: Asp-N sites. Solid arrows indicate cleavage sites that are similarly observable in both the presence and the absence of DNA. Dashed arrows indicate sites protected in the presence of DNA. Dotted arrows indicate cleavage sites detected only in the presence of DNA. The cleavage site of hydroxylamine is marked with a bold arrow. M. Puka ´ ncsik et al. Protein function preserved in a truncated isoform FEBS Journal 277 (2010) 1245–1259 ª 2010 The Authors Journal compilation ª 2010 FEBS 1249 Asn and Gly [21]. There is only one such peptide bond in DmUDE, at Asn111-Gly112 (Fig. 3D), located between motifs 1A and 1B. Figure 4A shows that, in agreement with the previously determined exposed character of the linker segment between motifs 1A and 1B, hydroxylamine cleaved the protein into an N-ter- minal Met1–Asn111 and a C-terminal Gly112–Glu355 fragment, as verified by MS. The molecular masses of the cleavage products are 14 and 28 kDa as calculated from the sequence, whereas values of 16 and 32 kDa were estimated from the SDS ⁄ PAGE gels. The C-ter- minal fragment closely corresponds to the physiologi- cal TcUDE isoform. The presence of the N-terminal His-tag on Dm rc UDE allowed straightforward separa- tion of N-terminal and C-terminal hydroxylamine- cleaved segments by Ni 2+ –nitrilotriacetic acid chroma- tography (Fig. 4A). To check whether the removal of motif 1A alters the specific function of the protein, we performed catalytic assays and electrophoretic mobility shift assays with the purified Gly112–Glu355 C-terminal fragment. Fig- ure 4B shows that the C-terminal segment preserves catalytic activity and specificity for uracil-substituted DNA that do not depend on the presence or absence of available divalent metal ions. The gel shift indicates the DNA-binding capability of the C-terminal frag- ment, and also demonstrates the specific DNA-cleaving activity (Fig. 4C). N111 His- tag 1A 1B 2 3 4 Dm rc UDE N-terminal M1-N111 C-terminal G112-E355 Intact HA digested Purified C-term Intact G112- E355 M1-N111 ds U-oligo ss U-oligo ds U-oligo ss U-oligo 0′ 30′ 60′ 120′ 0′ 30′ 60′ 120′ 0′ 30′ 60′ 120′ 31-mer 0′ 30′ 60′ 120′ 0 50 100 G112-E355 Dm rc UDE 31-me r U-DNA Control DNA 0′ 30′ 60′ 90′ 0′ 30′ 60′ G112-E355 Dm rc UDE G112-E355 Dm rc UDE Full-length Dm rc UDE A BC DE Fig. 4. (A) Production and characterization of the truncated UDE isoform. Cleavage with hydroxylamine (HA) generates the expected fragments. In the schematic repre- sentation, the single cleavage site at Asn111 between the 1A and 1B motifs is marked with an arrow. Gel images show gelectrophoretic analysis of hydroxylamine cleavage and purification of the C-terminal motif to homogeneity. (B) Electrophoretic mobility shift assay. The concentration of Dm rc UDE Gly112–Glu355 segment used in the experiment is given at the top of the lanes (lgÆmL )1 ). Uracil-DNA plasmid, 20 lgÆmL )1 , was used in all mixtures. (C–E) Truncated UDE lacking motif 1A retains uracil-DNA-degrading activity. (C) Uracil-DNA or control DNA linearized plasmid was incubated for the indicated time periods with truncated Dm rc UDE (Gly112–Glu355 segment). Note degradation (as well as shift) of the uracil-containing DNA plasmid substrate. (D, E) Activities of full-length UDE and Gly112–Glu355 truncated Dm rc UDE constructs were compared using uracil- containing fluorescently labeled synthetic double-stranded (ds) and single-stranded (ss) oligonucleotide substrates (incubation times are indicated). Note the specific degradation product very close to the 31mer standard position, indicating that cleavage of the oligonucleotide only occurred at the uracil- containing position. The catalytic activity of the truncated enzyme is still present, but is detectable only on single-stranded substrate. Protein function preserved in a truncated isoform M. Puka ´ ncsik et al. 1250 FEBS Journal 277 (2010) 1245–1259 ª 2010 The Authors Journal compilation ª 2010 FEBS To clearly identify the cleavage site of the UDE pro- tein and its truncated form on uracil-containing DNA substrate, we performed cleavage experiments using synthetic 60mer single-stranded and double-stranded oligonucleotides, containing one single uracil moiety in one of the strands, at the 32nd position. The uracil- containing strand was labeled with a fluorescent dye to aid visualization of the reaction (Fig. 4D,E). Quaternary protein structure of full-length and truncated proteins To determine whether the absence of the N-terminus has any effect on the quaternary structure organization of UDE, the native molecular masses for the full-length protein and the C-terminal fragment were determined by analytical gel filtration. The full-length protein eluted at a position corresponding to 52 kDa, which is somewhat larger than the full-length mono- mer calculated molecular mass of 41.446 kDa. This alteration may indicate partial rapid equilibrium dimerization and ⁄ or the anomalous gel permeation behavior may suggest that the proteins contain signifi- cant amounts of natively unfolded, highly flexible seg- ments. To check this suggestion, we performed an in silico analysis using several servers for sequence- based prediction of structural disorder [22–24]. The results are shown in Fig. 5, and indicate that the dif- ferent predictors suggest, in agreement, considerably high flexibility at the N-terminus and C-terminus, as well as in the region between motifs 1A and 1B. Inter- estingly, the C-terminal Gly112–Glu355 fragment eluted from the gel filtration column at practically the same position as observed for the full-length UDE, corresponding to 52 kDa. As the calculated molecular mass of the monomeric Gly112–Glu355 fragment is 28 kDa, the elution profile strongly suggests that this fragment forms a dimer. Analytical ultracentrifugation was also applied to corroborate the results from the gel filtration studies. The sedimentation equilibrium technique is reported to be optimal for determining native molecular masses [25]. In fact, our results with full-length Dm rc UDE indicate that the determined molecular mass was 42.8 ± 2 kDa, in very close agreement with the mass calculated from the amino acid sequence (Fig. 6). For the truncated Gly112–Glu355 construct, the deter- mined native molecular mass was 49 ± 1.2 kDa, cor- responding rather closely to a dimer of the truncated segment (for which the calculated masses are 28 kDa for the monomer and 56 kDa for the dimer). These results, in agreement with the gel filtration data, argue for a native monomer of the full-length protein and a native dimer for the truncated construct. Sedimentation velocity experiments revealed that full-length Dm rc UDE has a main sedimenting species (82% of the loading concentration) with a standard sedimentation value of 2.6S ± 0.1S, which, together 0 50 100 150 200 250 300 350 –0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 RONN IUPred DISOPRED Number of amino acids Disorder probability Fig. 5. Disorder profile of Dm rc UDE. The plot shows sequence position against probability of disorder. Segments of the sequence at the N-terminus and C-terminus and between motif 1A and motif 1B were classified as disordered by three predictor programs ( IUPRED, RONN, and DISOPRED). 6.95 7.00 7.05 7.10 7.15 7.20 –0.03 0.00 0.03 02468101214 0.0 0.2 0.4 0.6 0.8 1.0 C (s) Sedimentation coefficient (S) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 Residuals A 280 Radius (cm) Fig. 6. Determination of UDE oligomer status by analytical ultracen- trifugation. Top panel: sedimentation equilibrium gradients of 0.53 mgÆmL )1 full-length Dm rc UDE (s) and 0.8 mgÆmL )1 for Dm rc UDE Gly112–Glu355 (h) at 13 400 g as described in Experi- mental procedures. The solid line shows the fit of the experimental data to single ideal species. Bottom panel: residual distribution as a function of the sedimentation distance (this plot corresponds to the difference between the experimental data and the fitted data for each point). Inset panel: sedimentation coefficient distributions of full-length Dm rc UDE (solid line) and Dm rc UDE Gly112–Glu355 (dashed line). M. Puka ´ ncsik et al. Protein function preserved in a truncated isoform FEBS Journal 277 (2010) 1245–1259 ª 2010 The Authors Journal compilation ª 2010 FEBS 1251 with the sedimentation equilibrium data, is compatible with a protein monomer whose hydrodynamic behav- ior deviates from the expected for a globular species [calculated frictional ratio (f ⁄ f 0 ) = 1.6]; the rest of the protein sediments as faster oligomeric species. The truncated Gly112–Glu355 protein construct showed significant polydispersity, with main peaks at 2.5S, 4.3S, and 6.0S, representing approximately 70%, 20%, and 6%, respectively, of the loading concentration. The 2.5S peak is compatible with a protein globular monomer (f ⁄ f 0 = 1.3). These data argue for potential monomer self-association into dimers and higher-order oligomers. Structure prediction of UDE reveals a pseudosymmetrical arrangement of two a-helical bundles For structural prediction, the DmUDE full-length sequence was submitted to the genesilico metaserv- er [26], which is the gateway providing a unified inter- face to several servers for secondary and tertiary structure predictions. The analysis of predictions of domain composition suggested that UDE contains an N-terminal helical region of approximately 30 residues and at least three structural domains corresponding to motifs 1A and 1B, and the C-terminus, encompassing motifs 2, 3 and 4. The C-terminus of 40–50 residues and the loop connecting motifs 1A and 1B (between residues 109 and 137) are predicted to be mostly dis- ordered. All three domains are predicted to be mainly helical, although the secondary structure predictions for the third domain were uncertain, as there was no agreement between alternative servers. The fold recognition analysis did not reveal any con- fident matches with known protein structures, suggest- ing that the UDE 3D structure may exhibit a novel fold. Therefore, to predict at least partially the tertiary structure of UDE, we performed de novo modeling of the region encompassing motifs 1A and 1B, using the rosetta program [27]. In total, about 500 000 differ- ent models (also known as decoys) were generated, and 10% of the lowest-energy structures were clustered on the basis of their similarity. The representatives of the best clusters were refined with the rosetta full atom refinement protocol, and scored with the model quality assessment programs (MQAPs) proq [28] and metamqap [29]. Evaluation of the largest clusters revealed that both motifs 1A and 1B comprise similar three-helical bundles, with the same topology and rela- tive orientation of the helices. Nevertheless, the top clusters differed in relative orientation of the two heli- cal bundles to each other (data not shown). Among these clusters, one single cluster contained the specific topology that exhibited pseudosymmetrical orientation of the two motifs. Importantly, members of this cluster exhibited low energy levels and were well scored by MQAPs (proq – predicted LGscore in the range 1.2–3.2, and metamqap – predicted rmsd in the range 3–4.2 A ˚ , for the five lowest-energy representatives of the cluster), which indicates a high probability that they resemble the currently unknown native structure. Figure 7 depicts the predicted model in several different orientations. The two homologous motifs (1A and 1B) form a four-helix bundle interaction surface (Fig. 7A,B). On the surface of the model, a well-con- served, positively charged surface is well defined. This may serve as the nucleic acid-binding surface, in agree- ment with the limited proteolysis data. Estimation of secondary structural elements by CD spectroscopy To verify structural predictions, CD spectroscopy mea- surements were performed, as CD spectra in the far- UV wavelength (190–240 nm) range are very indicative of different secondary structural elements [30]. Spectra of the intact protein and of the C-terminal fragment Gly112–Glu355 showed double negative maxima at 208 and 222 nm, which are characteristic for the pres- ence of a-helices (Fig. 8). Quantitative evaluation of the spectral data was performed with k2d and selcon [24,31,32]. The estimated percentages of protein sec- ondary structures from CD spectra reveal 37% a-heli- ces and 18–26% b-structure. Discussion The potential signaling role of deoxyuridine moieties in genomes of pupating insects was first suggested by Deutsch et al. [16], on the basis of the lack of UDG activity in these insects. The hypothesis stating that uracil-DNA might be present transiently in larval stages and that its degradation at the end of larval stages may contribute to cell death during metamor- phosis was much debated, owing to independent find- ings from several laboratories showing the presence of UDG activity in some developmental stages of Dro- sophila [33–37]. This debate was resolved by the fully annotated Drosophila genome, which clearly indicated the lack of the major UDG gene ung but the presence of several other genes that encode catalytically much less efficient UDGs. The absence of dUTPase in larval stages [14] and our recent discovery of the strictly regu- lated UDE [15] reinforced the hypothesis on the possi- ble role of uracil-DNA in Drosophila and suggested a Protein function preserved in a truncated isoform M. Puka ´ ncsik et al. 1252 FEBS Journal 277 (2010) 1245–1259 ª 2010 The Authors Journal compilation ª 2010 FEBS role for UDE in programmed cell death during meta- morphosis. Functional analysis of UDE identified this protein as a novel uracil-recognizing factor [15], with no similarities to either UDGs [18] or the Exo- III ⁄ Mth212 nuclease [38]. Multiple sequence alignments of UDE homologs from all available pupating insect genomes indicated the presence of conserved motifs in most species, with the same distribution (Fig. 1). The UDE homolog in T. castaneum lacks one copy of the N-terminal duplicated first motif (Figs 1 and 2). TcUDE showed reactivity with the antiserum produced against Dm rc UDE, suggesting that the truncated TcUDE isoform is a well-folded UDE-like protein. It was also observable on the blot that the physiological forms of the proteins from both Drosophila and Tribo- lium extracts were detected at much higher electro- phoretic positions than expected from the calculated molecular mass values: molecular masses estimated 200 210 220 230 240 250 260 –10 000 –8000 –6000 –4000 –2000 0 2000 4000 6000 Intact Dm rc UDE G112-E355 Dm rc UDE Wavelength (nm) Θ MRE (deg cm 2 ·dmol –1 ) Fig. 8. CD spectra of intact UDE (solid line) and C-terminal frag- ment (dashed line) confirm the presence of a-helices. MRE, mean residue molar ellipticity. Orange – strictly conserved Yellow – conserved Green – variable Cartoon model colored by motifs (blue and red – protease cleavage sites) Cartoon model colored by sequence conservation Surface model colored by sequence conservation Surface model colored by electrostatic potential AB Motif 1A Motif 1B CD –3 kT/e + 3 kT/e Orange – strictly conserved Yellow – conserved Green – variable Fig. 7. Structural model of DmUDE duplication fragment. Structures are shown in two views: front (upper panel) and top (bottom panel). (A) Cartoon representation. Duplicated motifs 1A and 1B are colored green and orange, respectively, and the nonconserved linker is colored gray. Peptide bonds protected from proteolytic cleavage on DNA binding are colored blue. The peptide bond between residues 104 and 105, cleaved only on DNA binding, is colored red. Note that the duplicated fragments are only approximately symmetrical, as the model is of low resolution and the local conformation of the backbone is uncertain. (B, C) Sequence conservation mapped onto the ribbon diagram (B) or the molecular surface (C) (conserved residues are colored orange and yellow; variable residues are colored green). (D) Electrostatic potential mapped onto the molecular surface (positively and negatively charged regions are colored blue and red, respectively). Arrows indicate the positively charged conserved patches that may accommodate DNA. M. Puka ´ ncsik et al. Protein function preserved in a truncated isoform FEBS Journal 277 (2010) 1245–1259 ª 2010 The Authors Journal compilation ª 2010 FEBS 1253 UDE sequence BLAST Multiple sequence alignment T. cas predicted protein product Western blotting Secondary structure prediction Modeling Identification of conserved surface patches Mapping of electrostatic potential Prediction of DNA binding site Limited proteolysis Peptide identification by MS Trypsin Asp-N endoproteinase Chymotrypsin Hydroxylamine cleavage Analysis of C-ter fragment DNA binding by EMSA DNA cleavage assay Quaternary structure by gel flitration Theoretical analysis Experimental analysis Domain organization Analytical ultracentrifugation Circular dichroism Fig. 10. Flowchart scheme of bioinformatics and experimental approaches. Motifs 2,3,4 Motifs 2,3,4 Motifs 2,3,4 Motif 1 B Motif 1 A Motif 1 Motif 1 DmUDE TcUDE AB C Fig. 9. Structural models of DmUDE pseu- dodimer (A) and TcUDE dimer (B). Struc- tures are shown in cartoon representation and colored by motif (motif 1A in DmUDE and motif 1 in TcUDE, dark red; motif 1B, dark gray; nonconserved segments, light gray). Residues 1–11 of TcUDE are not shown (the conformation of this fragment is very uncertain). C-terminal parts correspond- ing to motifs 2, 3 and 4 are shown schemat- ically only. (C) Alignment between motif 1 residues for DmUDE and TcUDE. Identical and conserved residues are colored red and green, respectively. The helical prediction is indicated. Note the numerous conserved hydrophobic and polar residues that may form the dimerization surface. Protein function preserved in a truncated isoform M. Puka ´ ncsik et al. 1254 FEBS Journal 277 (2010) 1245–1259 ª 2010 The Authors Journal compilation ª 2010 FEBS [...]... et al M Puka BSA and 1 mm EDTA Protein and DNA were mixed, and the mixtures were loaded on agarose gel Analytical gel filtration analysis Analytical gel filtration was conducted on Superdex 200HR column calibrated with BSA, ovalbumin, chymotrypsin, and RNase (molecular masses of 67, 43, 25 and 13.7 kDa, respectively) Calibrating proteins or UDE samples were applied in a total volume of 500 lL, at a concentration... at a concentration of 1–6 mgÆmL)1 Analytical ultracentrifugation analysis An Optima XL -A analytical ultracentrifuge (BeckmanCoulter, Palo Alto, CA, USA) was used to perform the analytical ultracentrifugation experiments Detection was performed by means of a UV–visible absorbance detection system Experiments were conducted at 20 °C, using an AnTi50 eight-hole rotor and epon–charcoal standard doublesector... complementary oligonucleotide was 5¢-CAC TCA GAT GTT GAT TTC GAG GTG AAG TAG TGC GAC CGC ATC GCC CAG TTC ATT TGC GAG-3¢ (with the adenine position opposite to uracil in the double-stranded oligonucleotide underlined) For preparation of double-stranded substrates, equal amounts of uracil-containing oligonucleotide and its complementary strand were incubated at 95 °C for 5 min For the assay, 25 pmol of single-stranded... recombinant construct included a His6 tag and a linker segment at the C-terminus Western blotting Western blotting was performed as described in [15], using anti-DmUDE serum at 1 : 180 000 dilution as primary antibody, and peroxidase-labeled secondary antibody Extracts from D melanogaster and T castaneum larvae were prepared with the addition of protease inhibitor cocktail (Sigma-Aldrich, Budapest, Hungary)... manufacturer’s suggestion (Sigma-Aldrich) MS Analysis of the limited proteolysis fragments was performed either without fractionation or after 1D SDS ⁄ PAGE separation The unfractionated fragments were analyzed on a Bruker Reflex III MALDI-TOF mass spectrometer in a sinapinic acid matrix in positive linear mode SDS ⁄ PAGE-separated fragments were in-gel digested by trypsin, and the digests were analyzed... formation of a functional protein This hypothesis was confirmed by producing the respective truncated isoform with chemical cleavage from DmrcUDE (Fig 4) We therefore conclude that the physiological form of TcUDE could have the same unique function and the same putative physiological role Native molecular mass estimation by gel filtration and analytical ultracentrifugation indicated that the truncated... melanogaster All resulting structures were scored with proq [28] and metamqap [29], and the final model was selected on the basis of the scores and evaluation of the approximate pseudosymmetry between duplicated fragments The electrostatic potential was calculated using apbs [48] and mapped on the molecular surface with pymol [49] FEBS Journal 277 (2010) 1245–1259 ª 2010 The Authors Journal compilation... by LC-MS ⁄ MS analysis as in [40–42] Catalytic assay For plasmid substrates, uracil-containing plasmid DNA was prepared by amplification of normal plasmid DNA 1256 (pSUPERIOR-puro; Invitrogene, Csertex, Budapest, Hungary) in the dut)ung) K12 CJ236 E coli strain [15] Control plasmid was prepared in the XL1Blue E coli strain Plasmids were purified with a Qiagen plasmid isolation kit, and linearized with... labeled at the 5¢-end with Cye3 fluorescent dye, and contained one single uracil moiety at the 32nd position Its complementary strand (to be used for constructing the double-stranded substrate) did not contain either uracil or fluorescent label The uracil-containing oligonucleotide labeled with Cye3 was 5¢-CTC GCA AAT GAA CTG GGC GAT GCG GTC GCA CUA CTT CAC CTC GAA ATC AAC ATC TGA GTG-3¢ (with the uracil... The significance of UDE is two-fold: (a) it may be developed into a versatile molecular biotechnological tool [39]; and (b) its targeting may yield species-specific insecticides to be used against, for example, malaria mosquitoes Here, we employed a multidisciplinary set of theoretical and experimental approaches (schematically described in Fig 10) to reveal structural and functional characteristics . Physiological truncation and domain organization of a novel uracil-DNA-degrading factor Ma ´ ria Puka ´ ncsik 1 , Ange ´ la Be ´ ke ´ si 1 ,E ´ va Klement 2 ,E ´ va Hunyadi-Gulya ´ s 2 , Katalin. MS Trypsin Asp-N endoproteinase Chymotrypsin Hydroxylamine cleavage Analysis of C-ter fragment DNA binding by EMSA DNA cleavage assay Quaternary structure by gel flitration Theoretical analysis Experimental analysis Domain organization Analytical ultracentrifugation Circular dichroism Fig melanogaster uracil-DNA-degrading factor; Dm rc UDE, recombinant Drosophila melanogaster uracil-DNA-degrading factor; MQAP, model quality assessment program; TcUDE, Tribolium castaneum truncated