- Email: [email protected]
A Universal Type IA Topoisomerase Fold
doi:10.1016/j.jmb.2006.04.021
J. Mol. Biol. (2006) 359, 805–812
A Universal Type IA Topoisomerase Fold Michel Duguet†, Marie-Claude Serre and Claire Bouthier de La Tour* Laboratoire d’Enzymologie des Acides Nucle´iques, Institut de Ge´ne´tique et Microbiologie Universite´ Paris-Sud, Unite´ Mixte de Recherche 8621 Centre National de la Recherche Scientifique, 91405 Orsay France
A class of enzymes, called DNA topoisomerases, is responsible for controlling the topological state of cellular DNA. Among these, type IA topoisomerases form a vast family that is present in all living organisms, including higher eukaryotes, in which they play important roles in genome stability. The known 3D structures of three of these enzymes indicate that they share a common toroidal architecture. We previously showed that the toroidal structure could be split off from the core enzyme of Thermotoga maritima topoisomerase I by limited proteolysis. This structure is produced by the association of two tandemly repeated elementary folds in a head-totail orientation. By using a combination of structural and sequence data analysis, we show that the elementary fold of about 150 amino acid residues, referred to as the topofold, is likely to be present in the whole topoisomerase IA family. Within each enzyme, the successive topofolds share two conserved sequence motifs located at the base of the ring, and referred to as the MI and MII motifs. However, the overall sequences of the folds have largely diverged. By contrast, secondary and tertiary structures appear remarkably conserved. We suggest that this twofold repeat has evolved by gene duplication/fusion from an ancestral topofold. q 2006 Elsevier Ltd. All rights reserved.
*Corresponding author
Keywords: Thermotoga maritima; topoisomerase IA; structural fold; evolution
Introduction Several major cellular processes, such as DNA replication, recombination, transcription and chromosome condensation/decondensation, generate intertwinings between DNA strands or DNA duplexes. A class of enzymes, DNA topoisomerases, play an important role in these processes by modifying the topological state of DNA.1,2 Among these, type IA topoisomerases are of particular interest, since their in vivo functions are still poorly understood,2 despite extensive studies. Moreover, the eukaryotic version of these enzymes (topoisomerase III) has recently been the focus of considerable interest, since this enzyme seems to play an essential role in eukaryal genome stability and telomere maintenance, through interaction with various helicases of the RecQ family.3,4 It is known that a defect in these helicases leads to several genetic diseases, such as Bloom and Werner syndromes.4 Type IA topoisomerases form a well-defined superfamily present in all three domains of life, † This paper is dedicated to the memory of Michel Duguet, who died on October 14th, 2005. Abbreviations used: XXX, ?. E-mail address of the corresponding author: [email protected]
Bacteria, Archaea, and Eukarya.1 As illustrated in Figure 1, five subfamilies can be distinguished among the type IA superfamily. These include: (i) bacterial topoisomerase I, likely involved in the control of DNA supercoiling;5 (ii) bacterial topoisomerase III presumed to play a prominent role in episome segregation;6 (iii) the topoisomerase domain of reverse gyrases, present in both thermophilic bacteria and archaea;7 (iv) archaeal topoisomerase III;8 and (v) eukaryal topoisomerase III, whose precise function is unknown.9 All these enzymes share a common mechanism: they catalyse the cleavage of a single DNA strand, forming a transient covalent bond specifically between the 5 0 DNA end and the active site tyrosine. This is followed by the passage of another DNA segment through the break and the final rejoining of the DNA ends.1 This overall reaction cycle results in a modification of DNA topology (i.e. linking number) strictly by steps of 1.10 Within the type IA superfamily, extensive sequence similarity was found in a region spanning about 600 amino acid residues, termed the core region, that corresponds to the 67 kDa amino-terminal fragment (Top 67) of Escherichia coli topoisomerase I.11 Recently, we showed that the core region from Thermotoga maritima topoisomerase IA is able to sustain a complete topoisomerization cycle by itself.12
0022-2836/$ - see front matter q 2006 Elsevier Ltd. All rights reserved.
806
Universal Type IA Topoisomerase Fold
Figure 1. Phylogenic tree and three-dimensional structures of topoisomerases IA. The tree derives from multiple alignment of 49 type IA topoisomerase sequences produced by the ClustalX program (see Methods). The five subfamilies that are distinguished are: bacterial Topo I (green), reverse gyrases (blue), bacterial Topo III (violet), archaeal Topo III (red), eukaryal Topo III (orange). Bacterial species abbreviations are: Aae, Aquifex aeolicus; Ban, Bacillus anthracis; Bha, Bacillus halodurans; Bsu, Bacillus subtilis; Dra, Deinococcus radiodurans; Eco, Escherichia coli; Fis, Fervidobacterium islandicum; Hin, Haemophilus influenzae; Mle, Mycobacterium leprae; Pmu, Pasteurella multocida; Rpr, Ricketssia prowazekii; Syn, Synechocystis sp.; Tma, Thermotoga maritima; Tte, Thermoanaerobacter tengcongensis; Vch, Vibrio cholerae. Archaeal species abbreviations are: Afu, Archaeoglobus fulgidus; Ape, Aeropyrum pernix; Mja, Methanococcus jannaschii; Mka, Methanopyrus kandleri; Neq, Nanoarchaeum equitans; Pab, Pyrococcus abyssi; Pfu, Pyrococcus furiosus; Pho, Pyrococcus horikoshii; Sac, Sulfolobus acidocaldarius; Sso, Sulfolobus solfataricus; Sto, Sulfolobus tokodai. Eukaryal species abbreviations are: Ath, Arabidopsis thaliana; Cel, Caenorhabditis elegans; Dme, Drosophila melanogaster; Hsa, Homo sapiens; Mus, Mus musculus; Sce, Saccharomyces cerevisiae; Spo, Schizosaccharomyces pombe. Structures of E. coli Topo I (Top67 fragment), E. coli Topo III, and A. fulgidus Reverse gyrases are shown as ribbon representations using Swiss PDB viewer. Accession numbers are 1CY4, 1MW8 for Topo I, 1D6M for Topo III, and 1GKU for reverse gyrase. The characteristic topoisomerase IA toroid structure is coloured red. The two RecA folds of reverse gyrase16 are in light and dark blue, respectively, and the latch domain is in green. Green dots indicate the active site tyrosine residues.
Sequence similarity within the core region is also reflected by structural data. Currently, the structures, or partial structures, of three type IA topoisomerases have been determined, belonging to three different subfamilies: Escherichia coli topoisomerase I Top 67, E. coli topoisomerase III and Archaeoglobus fulgidus reverse gyrase.13–15 These structures are illustrated in Figure 1 and show that the core region (red) presents striking similarities: in all cases, the structure exhibits a typical toroidal shape with a central hole able to accommodate single-stranded or doublestranded DNA. In the case of reverse gyrase, an additional helicase-like domain is located at the amino-terminal region of the protein.16 This domain is composed of two closely associated RecA folds (blue) and a latch domain (green) as shown in Figure 1.15 The two RecA folds are involved in ATP binding and may participate in the conformational changes required for reverse gyrase supercoiling activity.15 The structures of topoisomerases IA were further enriched by the resolution of complexes with mono- or oligonucleotides. This led the authors to propose more precise models of the topoisomerization mechanism.17–19
Limited proteolysis of the T. maritima topoisomerase I core region (Top 65) led to the identification of two hotspots of proteolytic cleavage within the 540 amino acid residue core sequence.20 In the present work, we show that these two cleavage hotspots are within a domain of about 150 amino acid residues, with a typical ring fold that we call topofold. This ring fold architecture was noticed in the Top67 fragment of E. coli topoisomerase I,21 but its universal conservation could not be inferred at that time. Analyzing the three known structures of topoisomerases IA, we found independently that in all cases the toroidal shape of these enzymes is formed by two successive topofolds arranged in a head-to-tail orientation. Using a combination of structural and sequence data analysis, we propose that this organisation can be extended to the whole topoisomerase IA family. Comparison of the two tandemly repeated topofolds found in each topoisomerase indicates that these folds are strikingly similar in 3D structure. Further analysis at the amino acid sequence level revealed that two motifs are conserved between the successive folds, although the overall sequence has
Universal Type IA Topoisomerase Fold
largely diverged. We propose that this twofold repeat pattern, present in the whole type IA topoisomerase superfamily, evolved by gene duplication and fusion from an ancestral topofold.
Results and Discussion Limited proteolysis of T. maritima topoisomerase I reveals a domain of about 150 amino acid residues with a typical ring fold The structure of the type IA topoisomerase core region (Figure 2(b)) forms a ring clamp fold that is classically divided into four structural domains (I to IV).13 The active site is at the interface between
807 domain I (TOPRIM domain22) and domain III, while the single-stranded DNA to be cleaved is accommodated in the groove at the interface of domains I and IV. Finally, domain II forms the top of the arch. Importantly, two of the four domains (II and IV) do not follow the path of the polypeptide chain.13 The chain (Figure 2(b)) first goes through the entire domain I (residues 1–158 for E. coli Topo I) and about one-third of domain IV (159–213). Then the chain goes into half of domain II (214–278) and the entire domain III (279–404), before forming the second half of domain II (405–471). Finally, the chain spans the rest of domain IV (472 to end). The results of proteolytic cleavage by trypsin of the core region from T. maritima topoisomerase I led us to define new features in the topoisomerase IA
Figure 2. Generation and description of the topofolds from T. maritima Top65 and E. coli Top67. (a) The structure of T. maritima Top65 (left) was predicted from that of E. coli Top67 by using the Swiss Model protein modelling package (see Methods). Amino and carboxy ends are indicated. The two proteolytic cleavage points at K156 and K325 are shown in heavy red lines and define a fragment (right) called the topofold. The predicted secondary structure elements of the topofold, helices H1 to H5 and strands S1 to S3, are indicated. (b) Isolation of the topofold structures from E. coli Top67 (accession number 1CY4) was performed by using the Swiss PDB Viewer program. Upper left: the complete structure of Top67 with the four structural domains I to IV coloured in red, blue, green and magenta, respectively. Domain I of the original structure was removed from residues 1–158, as well as two regions of domain IV (residues 159–186 and 540–596) to generate the structure shown in the upper centre. The structure was then split into halves to isolate fold 1 (residues 187–346) and fold 2 (residues 375–540) in their natural orientation. The bottom part of the Figure compares the two folds after a 1808 rotation of fold 2 around a vertical axis. Secondary structure elements H1 to H5 and S1 to S4 are indicated. The inset in the centre gives the positions of the folds within the 596 residues of E. coli Top 67.
808 structure.20 As shown in Figure 2(a), only two cleavage points were accessible to trypsin at positions 156 and 325. Modelling of the core T. maritima structure allowed us to identify a domain of about 150 amino acid residues in size with a horseshoe structure. This structural domain is formed by the continuous polypeptide chain from residue 157 to residue 325 (Figure 2(a), right). We subsequently called this domain, defined by its typical 3D-structure, the topofold. As seen below, this fold is present in two tandem copies and conserved in the whole type IA topoisomerase superfamily. The toroidal shape of topoisomerases IA is constituted by two successive topofolds in a head to tail orientation The high level of conservation of structures of topoisomerase I from E. coli and T. maritima suggested that a topofold could be defined in the former. To delimitate such a fold, we used the wellestablished structure of the 67 kDa fragment from E. coli topoisomerase I,13 and we based our search on structure comparison with the topofold previously defined in T. maritima. Interestingly, two successive topofolds, fold 1 (residues 187–346) and fold 2 (residues 375–540) can be defined within the E. coli topoisomerase I structure (Figure 2(b)). The two folds can be revealed by using the Swiss PDB viewer program: the entire domain I (1–158) and two regions of domain IV (159–186 and 540–596) are removed, and the remaining structure is split into halves called fold 1 (187–346) and fold 2 (375–540) (Figure 2(b), right). For fold 1, the chain starts in domain IV (purple), goes through domain II (blue) and ends in domain III (green). Symmetrically, for fold 2 the chain starts in domain III (green), goes through domain II (blue) and ends in domain IV (purple). After 1808 rotation of fold 2, the two topofolds exhibit remarkably similar structures (Figure 2(b), bottom). They both start with a long a helix (H1) followed by two antiparallel b strands (S1 and S2). Then the chain forms a short b strand S2 0 (fold 1) or a short a helix H1 0 (fold 2), followed either by helix H2 (fold 1) or by a long unstructured segment (fold 2). In both cases, the chain continues down the arch through a long twisted b strand S3, connected to two successive helices, H3 and H4. This is followed either by a short b strand S4 and a loop (fold 1), or by two short b strands S4, S4 0 (fold 2). Finally, the chain is terminated by helix H5. The structure of the E. coli topofold is globally similar to that predicted for the T. maritima topofold (Figure 2(a) and (b)). These findings show that the toroidal structure of bacterial topoisomerases I results from the association of two tandem topofolds in a head-to-tail orientation. Importantly, the folds are separated by a short segment (residue 347–374 in E. coli topoisomerase I) that contains a disordered loop and a conserved histidine residue (H365). It was suggested that this loop could serve as a door,
Universal Type IA Topoisomerase Fold
allowing entry or exclusion of nucleotides from a binding site close to the active site tyrosine residue.17 As we show below, this organization is probably present in the whole type IA topoisomerase superfamily. The organisation into two successive topofolds is found in the three known type IA structures: each fold contains a conserved tyrosine at the base of the ring We next asked if the topofolds found in E. coli and T. maritima topoisomerases I are common to all three type IA topoisomerases of known structures; Figure 3 shows that this is indeed the case. In each of the three structures, two folds can be defined that appear remarkably similar (compare fold 1 to fold 2 in the three species). In any case, the overall organization, i.e. the succession of helices and strands described for E. coli Topo I (Figure 2(b)) is largely conserved. Moreover, the CAP-like motifs found in a number of DNA-binding proteins and described for both type IA and type II topoisomerases,21,23 are present within the topofolds. In each fold, they are represented by helices H3 and H4, and by strand S4 (Figure 2). Another important feature is the presence of the active site tyrosine within fold 1 in all three structures (see green dots, Figures 1 and 3). As expected, its location is very similar in all cases, at the beginning of a loop, immediately after a short b strand following helix H4 (directly after H4 for Topo III). Remarkably, a highly conserved tyrosine is found in exactly the same location within fold 2 (Figure 3). In a multiple alignment of 71 type IA topoisomerases, this tyrosine is present in 64 sequences and replaced by phenylalanine in five cases and by leucine in two cases (not shown). These exceptions are found in two subfamilies, some archaeal and some bacterial topoisomerases III, including E. coli Topo III, where the tyrosine is replaced by phenylalanine. We also noted the presence of a conserved arginine (black dots) two residues downstream of the active site tyrosine in fold 1 (Figure 3). This arginine is involved in the modulation of the nucleophilic attack of the phosphate group of the scissile DNA chain by the active site tyrosine.19 In fold 2, a conserved arginine (only four changes out of 71 sequences) is found two residues upstream of the tyrosine (Figure 3). It has been shown that for E. coli Topo I, this arginine (R507) contacts single-stranded DNA. 19 A detailed examination of the three known type IA topoisomerases structures reveals that in all six topofolds the tyrosine and the arginine residues point in the same direction (not shown). These observations give more credence to the similarity between folds 1 and 2. The finding of two tandem topofolds within members of three different subfamilies of topoisomerase IA supports the idea that this organisation is a probably a structural signature for the whole superfamily.
Universal Type IA Topoisomerase Fold
809
Figure 3. Comparison of the topofolds structures from different type IA topoisomerases. The ribbon structures of the two topofold repeats in E. coli Topo I (left), E. coli Topo III (right), and A. fulgidus reverse gyrase (bottom) were generated by using the Swiss PDB Viewer program. In each case, fold 1 and fold 2 are shown and the numbers indicate the position in the protein sequence of the amino acid residue at the beginning and the end of each fold. Green dots give the location of conserved tyrosine residues (or Phe in fold 2 of Topo III) within each fold. Black dots indicate the conserved arginine residues near the tyrosine residues. Coloured segments in red (motif I, MI) and magenta (motif II, M II) indicate conserved amino acid motifs in each fold. For E. coli Topo I and Topo III, sequences in the one-letter code indicate the location of short acidic regions found in a number of type IA topoisomerases.
Comparison of secondary and primary structures of the two successive topofolds The probable presence of the two topofold repeat patterns within the whole type IA superfamily further supported the idea that it evolved by duplication of an ancestral fold, and that this duplication occurred before the divergence between Bacteria, Archaea and Eukarya. We therefore looked for similarities between the topofolds at the structural level and at the amino acid sequence level. Reflecting the similarity of the 3D structures between folds 1 and 2, the secondary structure of the folds is very similar, as illustrated in Figure 4 for E. coli topoisomerases I and III. The same observation is made for A. fulgidus reverse gyrase (not shown). Analysis of the similarities at the sequence level was more difficult, since this necessitated the identification of the boundaries of the folds. For the three type IA topoisomerases of known structure, this was based on the structural similarities (Figure 3) and the positioning of helices and strands (Figure 4). To identify the fold boundaries in a variety of type IA enzymes, a multiple alignment of 71 sequences was produced by using the Clustal X program (not shown). We found that fold 1 from all the various species were reasonably aligned (30% identity, 40% similarity for the most distant) and their boundaries could be defined by comparison with those of E. coli Topo I, Topo III and A. fulgidus reverse gyrase. The same approach was
used for every fold 2. We then looked for sequence similarities between each fold 1/fold 2 pair in a number of species. As expected from a very ancient duplication event, only a weak similarity was found between the two folds of a same species. For instance, the tandem topofolds of E. coli Topo I (Figure 4) exhibit only 14% identity and 24% similarity in the approximately 150 amino acid residue sequence. Interestingly, these scores were slightly higher for M. kandleri archaeal Topo III (22% identity, 32% similarity) and A. fulgidus Topo III (17.5% identity, 30% similarity), possibly reflecting their deeper phylogenetic position (Figure 1). On the other hand, homologous folds of the different type IA subfamilies appear more similar to each other, as expected from a duplication event that took place before their divergent evolution. For example, fold 1 from E. coli Topo I and human Topo IIIa exhibit 27.7% identity and 39.1% similarity, and fold 2 from the same species exhibit 27.4% identity and 42.3% similarity, which is consistent with the higher level of structural similarity observed between homologous folds (Figure 3). A careful examination of the sequences revealed two motifs that are clearly conserved between fold 1 and fold 2 in the whole topoisomerase IA superfamily. Figure 5 shows an alignment of these motifs for 20 topofolds (ten fold 1, ten fold 2) from ten different topoisomerases. These motifs are found near the extremities of each fold, motif I within helix H1, and motif II within helix H4. Their spatial location is also indicated in Figure 3 (MI and MII) and Figure 4
810
Universal Type IA Topoisomerase Fold
Figure 4. Alignment of folds 1 and 2 and comparison of their secondary structure. Upper scheme, E. coli Topo I; lower scheme, E. coli Topo III. Alignments (ClustalX) were visualized by using the Macboxshade 2.11 program (Methods). Identical residues in the alignment are boxed in red and similar residues are boxed in blue. For folds 1 and 2, a helices are represented by blue cylinders and bstrands by yellow arrows, above and under the alignment. For Topo III, 19 amino acid residues were deleted to allow alignment of helices H4 from the two folds. Green and black dots point to the positions of conserved tyrosine and arginine residues described in the text. Red bars indicate motifs (MI in H1, MII in H4) conserved within both fold 1 and fold 2 in various type IA topoisomerases. The green bars indicate acidic regions.
(red bars). Figure 5 shows that in the E. coli Top67 structure the motifs are located at the base of the horseshoe. Because of the head-to-tail orientation of the two topofolds, motif I of fold 1 is on the right, near motif II of fold 2, while motif I of fold 2 is on the left, close to motif II of fold 1, adjacent to the active site tyrosine (Figure 5, centre). Moreover, in the three topoisomerase structures, motifs I and II of each topofold appear in the same relative locations (Figure 3). Although the tyrosine residues in each fold have nearly the same spatial location, they do not exactly match in the alignments of Figure 4. The tyrosine of fold 1 is immediately outside motif II (red bars), while the tyrosine of fold 2 is the penultimate residue of motif II. This may be due to the fact that pairwise alignments are less reliable than multiple alignments when performed on divergent proteins or motif samples. Non-exclusively, this may be due to the accumulation of sequence changes since the divergence of folds 1 and 2. The presence of these two conserved sequence motifs (MI and MII) at the base of the topofolds emphasizes their potential importance to the maintenance of the typical architecture of the type IA topoisomerases. Other additional traces of a common origin for folds 1 and 2 can be considered. First, is the previously mentioned presence of the pairs Tyr-X-Arg and
Arg-X-Tyr in folds 1 and 2, respectively, found in nearly all type IA topoisomerase sequences and in the same spatial location. Although the tyrosine of fold 2 is not involved in the trans-esterification reaction, and is replaced by phenylalanine in a few cases, its conservation suggests that it plays another important role, perhaps in interacting with single-stranded DNA, as is the case for the nearby arginine.19 The second feature is the presence of short acidic regions in a number of type IA topoisomerases. Remarkably, these regions (identified by green bars in Figure 4 and indicated in the structures, Figure 3) are found in equivalent sequence regions within each fold (see motifs EQTQ/GDED for Topo I and QDEE/DEEN for Topo III). The role of these acidic regions in either catalysis or structure maintenance remains to be investigated. The observations described here reveal that the universal toroidal architecture of type IA topoisomerases is formed from two tandem domains, the topofolds, arranged in a head-to-tail fashion. The high level of structural similarity and the finding of traces of conservation at the sequence level suggest that the fold repeat results from a duplicationfusion event of an ancestral fold. With the accumulating data from genome sequencing, such a situation is more and more frequently described
811
Universal Type IA Topoisomerase Fold
Figure 5. Conserved sequence motifs between the folds. Alignment (by using the Clustal X and MacBoxshade 2.11 programs) of two motifs within folds 1 and 2 from ten different species, representing the five families of topoisomerase IA: AaeI, Aquifex aeolicus Topo I; AfuIII, Archaeoglobus fulgidus Topo III; AfuRG, Archaeoglobus fulgidus reverse gyrase; BsuIII, Bacillus subtilis Topo III; CelIII, Caenorhabditis elegans Topo III; EcoI, Escherichia coli Topo I; EcoIII, Escherichia coli Topo III; HsaIIIa, Homo sapiens Topo IIIa; MkaIII, Methanopyrus kandleri Topo III; NeqRG, Nanoarchaeum equitans reverse gyrase. Residues strictly conserved in at least 12 sequences out of 20 are in yellow. Similar residues are in black above a blue background. The position of the motifs within the w150 residues of the topofold is shown on the bottom of the Figure Right: location of motifs I and II within the E. coli Topo I fold 1 (I-1 and I-2) in yellow and fold 2 (II-1 and II-2) in green. The central structure gives the position of the motifs within the whole E. coli Top67 structure. The green dots locate the active site tyrosine. The rest of the structure outside the folds is in grey.
for a number of multidomain proteins. In the case of type IA topoisomerases, the ancestral precursor domains, although not immediately visible as independent domains in the topoisomerase structure, could be revealed by a combination of comparative structural and sequence analysis. Another example of this has been described in the case of enzymes with a b/a barrel scaffold,24 and one can imagine that many proteins are built in this way. Finally, the description of the type IA topoisomerase structure based on a logic different from that of the four canonical structural domains, opens up new possibilities to model interactions with other molecules. In particular, it might be useful to design novel inhibitors of type IA topoisomerases, as potential antibiotics, and to target the eukaryotic type IA enzymes that are presently the subject of intense interest.
Methods Thermotoga maritima topoisomerase I limited proteolysis The recombinant T. maritima topoisomerase I was purified to homogeneity from E. coli, using the previously
described procedure,25 and submitted to limited proteolysis by trypsin as described by Cossard et al.20
Multiple sequence alignments Multiple alignments of type IA topoisomerases were produced by using the ClustalX package (F. Jeanmougin IGBMC, Strasbourg). Alignments were visualized by using the Macboxshade 2.11 program (M. D. Baron, Institute of Animal Health, UK). An alignment of 71 complete topoisomerase IA sequences was first made and the conserved sequence motifs were used manually to identify the core domains that contain these motifs.
Phylogenetic trees A selection of 49 type IA topoisomerase core sequences was used to produce a multiple alignment with the ClustalX program. Distance trees were derived from this alignment by using the Phylip package (version 4.1†). Confidence limits for each node of the distance trees were determined using a bootstrap approach (Condense program). † http://evolution.genetics.washington.edu/phylip. html
812
Universal Type IA Topoisomerase Fold
Three-dimensional protein structures Ribbon representations of topoisomerases IA structures were obtained using the SWISS PDB viewer program (version 3.6b2) with the published accession numbers 1CY4 and 1MW8 for E. coli Top 67, 1D6M for E. coli Topo III and 1GKU for A. fulgidus reverse gyrase. The structure of T. maritima type IA topoisomerase was predicted from the other known structures using the Swiss Model protein modelling package†. The modelled structure was confirmed by low-resolution crystallographic data (V. Lamour, T. Viard, C.B. de la T., D. Moras and M. D., unpublished results).
Acknowledgements We thank B. Labedan and O. Lespinet for useful advice, and B. Holland and S. Sommer for critical reading of the manuscript. The Laboratoire d’Enzymologie des Acides Nucleiques was supported by funds from CNRS and Universite´ Paris-Sud.
References 1. Champoux, J. J. (2001). DNA topoisomerases: structure, function, and mechanism. Annu. Rev. Biochem. 70, 369–413. 2. Wang, J. C. (2002). Cellular roles of DNA topoisomerases: a molecular perspective. Nature Rev. Mol. Cell Biol. 3, 430–440. 3. Duguet, M. (1997). When helicase and topoisomerase meet!. J. Cell Sci. 110, 1345–1350. 4. Khakhar, R. R., Cobb, J. A., Bjergbaek, L., Hickson, I. D. & Gasser, S. M. (2003). RecQ helicases: multiple roles in genome maintenance. Trends Cell Biol. 13, 493–501. 5. Drlica, K. (1992). Control of bacterial DNA supercoiling. Mol. Microbiol. 6, 425–433. 6. DiGate, R. J. & Marians, K. J. (1988). Identification of a potent decatenating enzyme from Escherichia coli. J. Biol. Chem. 263, 13366–13373. 7. Bouthier de la Tour, C., Portemer, C., Huber, R., Forterre, P. & Duguet, M. (1991). Reverse gyrase in thermophilic eubacteria. J. Bacteriol. 173, 3921–3923. 8. Slesarev, A. I., Zaitzev, D. A., Kopylov, V. M., Stetter, K. O. & Kozyavkin, S. A. (1991). DNA topoisomerase III from extremely thermophilic archaebacteria. ATPindependent type I topoisomerase from Desulfurococcus amylolyticus drives extensive unwinding of closed circular DNA at high temperature. J. Biol. Chem. 266, 12321–12328. 9. Wallis, J. W., Chrebet, G., Brodsky, G., Rolfe, M. & Rothstein, R. (1989). A hyper-recombination mutation in S. cerevisiae identifies a novel eukaryotic topoisomerase. Cell, 58, 409–419.
10. Dekker, N. H., Rybenkov, V. V., Duguet, M., Crisona, N. J., Cozzarelli, N. R., Bensimon, D. & Croquette, V. (2002). The mechanism of type IA topoisomerases. Proc. Natl Acad. Sci. USA, 99, 12126–12131. 11. Lima, C. D., Wang, J. C. & Mondragon, A. (1993). Crystallization of a 67 kDa fragment of Escherichia coli DNA topoisomerase I. J. Mol. Biol. 232, 1213–1216. 12. Viard, T., Cossard, R., Duguet, M. & de La Tour, C. B. (2004). Thermotoga maritima-Escherichia coli chimeric topoisomerases. Answers about involvement of the carboxyl-terminal domain in DNA topoisomerase I-mediated catalysis. J. Biol. Chem. 279, 30073–30080. 13. Lima, C. D., Wang, J. C. & Mondragon, A. (1994). Three-dimensional structure of the 67K N-terminal fragment of E. coli DNA topoisomerase I. Nature, 367, 138–146. 14. Mondragon, A. & DiGate, R. (1999). The structure of Escherichia coli DNA topoisomerase III. Structure, 7, 1373–1383. 15. Rodriguez, A. C. & Stock, D. (2002). Crystal structure of reverse gyrase: insights into the positive supercoiling of DNA. EMBO J. 21, 418–426. 16. Confalonieri, F., Elie, C., Nadal, M., de La Tour, C., Forterre, P. & Duguet, M. (1993). Reverse gyrase: a helicase-like domain and a type I topoisomerase in the same polypeptide. Proc. Natl Acad. Sci. USA, 90, 4753–4757. 17. Feinberg, H., Changela, A. & Mondragon, A. (1999). Protein-nucleotide interactions in E. coli DNA topoisomerase I. Nature Struct. Biol. 6, 961–968. 18. Changela, A., DiGate, R. J. & Mondragon, A. (2001). Crystal structure of a complex of a type IA DNA topoisomerase with a single-stranded DNA molecule. Nature, 411, 1077–1081. 19. Perry, K. & Mondragon, A. (2003). Structure of a complex between E. coli DNA topoisomerase I and single-stranded DNA. Structure, 11, 1349–1358. 20. Cossard, R., Viard, T., Lamour, V., Duguet, M. & Bouthier de La Tour, C. (2004). Proteolytic cleavage of the hyperthermophilic topoisomerase I from Thermotoga maritima does not impair its enzymatic properties. Biochim. Biophys. Acta, 1700, 161–170. 21. Murzin, A. G. (1994). New protein folds. Curr. Opin. Struct. Biol. 4, 441–449. 22. Aravind, L., Leipe, D. D. & Koonin, E. V. (1998). Toprim—a conserved catalytic domain in type IA and II topoisomerases. DnaG-type primases, OLD family nucleases and RecR proteins. Nucl. Acids Res. 26, 4205–4213. 23. Berger, J. M. (1998). Structure of DNA topoisomerases. Biochim. Biophys. Acta, 1400, 3–18. 24. Lang, D., Thoma, R., Henn-Sax, M., Sterner, R. & Wilmanns, M. (2000). Structural evidence for evolution of the beta/alpha barrel scaffold by gene duplication and fusion. Science, 289, 1546–1550. 25. Viard, T., Lamour, V., Duguet, M. & Bouthier de la Tour, C. (2001). Hyperthermophilic topoisomerase I from Thermotoga maritima. A very efficient enzyme that functions independently of zinc binding. J. Biol. Chem. 276, 46495–46503.
Edited by I.B. Holland (Received 14 February 2006; received in revised form 5 April 2006; accepted 6 April 2006) Available online 25 April 2006 † http://swissmodel.expasy.org/
J. Mol. Biol. (2006) 359, 805–812
A Universal Type IA Topoisomerase Fold Michel Duguet†, Marie-Claude Serre and Claire Bouthier de La Tour* Laboratoire d’Enzymologie des Acides Nucle´iques, Institut de Ge´ne´tique et Microbiologie Universite´ Paris-Sud, Unite´ Mixte de Recherche 8621 Centre National de la Recherche Scientifique, 91405 Orsay France
A class of enzymes, called DNA topoisomerases, is responsible for controlling the topological state of cellular DNA. Among these, type IA topoisomerases form a vast family that is present in all living organisms, including higher eukaryotes, in which they play important roles in genome stability. The known 3D structures of three of these enzymes indicate that they share a common toroidal architecture. We previously showed that the toroidal structure could be split off from the core enzyme of Thermotoga maritima topoisomerase I by limited proteolysis. This structure is produced by the association of two tandemly repeated elementary folds in a head-totail orientation. By using a combination of structural and sequence data analysis, we show that the elementary fold of about 150 amino acid residues, referred to as the topofold, is likely to be present in the whole topoisomerase IA family. Within each enzyme, the successive topofolds share two conserved sequence motifs located at the base of the ring, and referred to as the MI and MII motifs. However, the overall sequences of the folds have largely diverged. By contrast, secondary and tertiary structures appear remarkably conserved. We suggest that this twofold repeat has evolved by gene duplication/fusion from an ancestral topofold. q 2006 Elsevier Ltd. All rights reserved.
*Corresponding author
Keywords: Thermotoga maritima; topoisomerase IA; structural fold; evolution
Introduction Several major cellular processes, such as DNA replication, recombination, transcription and chromosome condensation/decondensation, generate intertwinings between DNA strands or DNA duplexes. A class of enzymes, DNA topoisomerases, play an important role in these processes by modifying the topological state of DNA.1,2 Among these, type IA topoisomerases are of particular interest, since their in vivo functions are still poorly understood,2 despite extensive studies. Moreover, the eukaryotic version of these enzymes (topoisomerase III) has recently been the focus of considerable interest, since this enzyme seems to play an essential role in eukaryal genome stability and telomere maintenance, through interaction with various helicases of the RecQ family.3,4 It is known that a defect in these helicases leads to several genetic diseases, such as Bloom and Werner syndromes.4 Type IA topoisomerases form a well-defined superfamily present in all three domains of life, † This paper is dedicated to the memory of Michel Duguet, who died on October 14th, 2005. Abbreviations used: XXX, ?. E-mail address of the corresponding author: [email protected]
Bacteria, Archaea, and Eukarya.1 As illustrated in Figure 1, five subfamilies can be distinguished among the type IA superfamily. These include: (i) bacterial topoisomerase I, likely involved in the control of DNA supercoiling;5 (ii) bacterial topoisomerase III presumed to play a prominent role in episome segregation;6 (iii) the topoisomerase domain of reverse gyrases, present in both thermophilic bacteria and archaea;7 (iv) archaeal topoisomerase III;8 and (v) eukaryal topoisomerase III, whose precise function is unknown.9 All these enzymes share a common mechanism: they catalyse the cleavage of a single DNA strand, forming a transient covalent bond specifically between the 5 0 DNA end and the active site tyrosine. This is followed by the passage of another DNA segment through the break and the final rejoining of the DNA ends.1 This overall reaction cycle results in a modification of DNA topology (i.e. linking number) strictly by steps of 1.10 Within the type IA superfamily, extensive sequence similarity was found in a region spanning about 600 amino acid residues, termed the core region, that corresponds to the 67 kDa amino-terminal fragment (Top 67) of Escherichia coli topoisomerase I.11 Recently, we showed that the core region from Thermotoga maritima topoisomerase IA is able to sustain a complete topoisomerization cycle by itself.12
0022-2836/$ - see front matter q 2006 Elsevier Ltd. All rights reserved.
806
Universal Type IA Topoisomerase Fold
Figure 1. Phylogenic tree and three-dimensional structures of topoisomerases IA. The tree derives from multiple alignment of 49 type IA topoisomerase sequences produced by the ClustalX program (see Methods). The five subfamilies that are distinguished are: bacterial Topo I (green), reverse gyrases (blue), bacterial Topo III (violet), archaeal Topo III (red), eukaryal Topo III (orange). Bacterial species abbreviations are: Aae, Aquifex aeolicus; Ban, Bacillus anthracis; Bha, Bacillus halodurans; Bsu, Bacillus subtilis; Dra, Deinococcus radiodurans; Eco, Escherichia coli; Fis, Fervidobacterium islandicum; Hin, Haemophilus influenzae; Mle, Mycobacterium leprae; Pmu, Pasteurella multocida; Rpr, Ricketssia prowazekii; Syn, Synechocystis sp.; Tma, Thermotoga maritima; Tte, Thermoanaerobacter tengcongensis; Vch, Vibrio cholerae. Archaeal species abbreviations are: Afu, Archaeoglobus fulgidus; Ape, Aeropyrum pernix; Mja, Methanococcus jannaschii; Mka, Methanopyrus kandleri; Neq, Nanoarchaeum equitans; Pab, Pyrococcus abyssi; Pfu, Pyrococcus furiosus; Pho, Pyrococcus horikoshii; Sac, Sulfolobus acidocaldarius; Sso, Sulfolobus solfataricus; Sto, Sulfolobus tokodai. Eukaryal species abbreviations are: Ath, Arabidopsis thaliana; Cel, Caenorhabditis elegans; Dme, Drosophila melanogaster; Hsa, Homo sapiens; Mus, Mus musculus; Sce, Saccharomyces cerevisiae; Spo, Schizosaccharomyces pombe. Structures of E. coli Topo I (Top67 fragment), E. coli Topo III, and A. fulgidus Reverse gyrases are shown as ribbon representations using Swiss PDB viewer. Accession numbers are 1CY4, 1MW8 for Topo I, 1D6M for Topo III, and 1GKU for reverse gyrase. The characteristic topoisomerase IA toroid structure is coloured red. The two RecA folds of reverse gyrase16 are in light and dark blue, respectively, and the latch domain is in green. Green dots indicate the active site tyrosine residues.
Sequence similarity within the core region is also reflected by structural data. Currently, the structures, or partial structures, of three type IA topoisomerases have been determined, belonging to three different subfamilies: Escherichia coli topoisomerase I Top 67, E. coli topoisomerase III and Archaeoglobus fulgidus reverse gyrase.13–15 These structures are illustrated in Figure 1 and show that the core region (red) presents striking similarities: in all cases, the structure exhibits a typical toroidal shape with a central hole able to accommodate single-stranded or doublestranded DNA. In the case of reverse gyrase, an additional helicase-like domain is located at the amino-terminal region of the protein.16 This domain is composed of two closely associated RecA folds (blue) and a latch domain (green) as shown in Figure 1.15 The two RecA folds are involved in ATP binding and may participate in the conformational changes required for reverse gyrase supercoiling activity.15 The structures of topoisomerases IA were further enriched by the resolution of complexes with mono- or oligonucleotides. This led the authors to propose more precise models of the topoisomerization mechanism.17–19
Limited proteolysis of the T. maritima topoisomerase I core region (Top 65) led to the identification of two hotspots of proteolytic cleavage within the 540 amino acid residue core sequence.20 In the present work, we show that these two cleavage hotspots are within a domain of about 150 amino acid residues, with a typical ring fold that we call topofold. This ring fold architecture was noticed in the Top67 fragment of E. coli topoisomerase I,21 but its universal conservation could not be inferred at that time. Analyzing the three known structures of topoisomerases IA, we found independently that in all cases the toroidal shape of these enzymes is formed by two successive topofolds arranged in a head-to-tail orientation. Using a combination of structural and sequence data analysis, we propose that this organisation can be extended to the whole topoisomerase IA family. Comparison of the two tandemly repeated topofolds found in each topoisomerase indicates that these folds are strikingly similar in 3D structure. Further analysis at the amino acid sequence level revealed that two motifs are conserved between the successive folds, although the overall sequence has
Universal Type IA Topoisomerase Fold
largely diverged. We propose that this twofold repeat pattern, present in the whole type IA topoisomerase superfamily, evolved by gene duplication and fusion from an ancestral topofold.
Results and Discussion Limited proteolysis of T. maritima topoisomerase I reveals a domain of about 150 amino acid residues with a typical ring fold The structure of the type IA topoisomerase core region (Figure 2(b)) forms a ring clamp fold that is classically divided into four structural domains (I to IV).13 The active site is at the interface between
807 domain I (TOPRIM domain22) and domain III, while the single-stranded DNA to be cleaved is accommodated in the groove at the interface of domains I and IV. Finally, domain II forms the top of the arch. Importantly, two of the four domains (II and IV) do not follow the path of the polypeptide chain.13 The chain (Figure 2(b)) first goes through the entire domain I (residues 1–158 for E. coli Topo I) and about one-third of domain IV (159–213). Then the chain goes into half of domain II (214–278) and the entire domain III (279–404), before forming the second half of domain II (405–471). Finally, the chain spans the rest of domain IV (472 to end). The results of proteolytic cleavage by trypsin of the core region from T. maritima topoisomerase I led us to define new features in the topoisomerase IA
Figure 2. Generation and description of the topofolds from T. maritima Top65 and E. coli Top67. (a) The structure of T. maritima Top65 (left) was predicted from that of E. coli Top67 by using the Swiss Model protein modelling package (see Methods). Amino and carboxy ends are indicated. The two proteolytic cleavage points at K156 and K325 are shown in heavy red lines and define a fragment (right) called the topofold. The predicted secondary structure elements of the topofold, helices H1 to H5 and strands S1 to S3, are indicated. (b) Isolation of the topofold structures from E. coli Top67 (accession number 1CY4) was performed by using the Swiss PDB Viewer program. Upper left: the complete structure of Top67 with the four structural domains I to IV coloured in red, blue, green and magenta, respectively. Domain I of the original structure was removed from residues 1–158, as well as two regions of domain IV (residues 159–186 and 540–596) to generate the structure shown in the upper centre. The structure was then split into halves to isolate fold 1 (residues 187–346) and fold 2 (residues 375–540) in their natural orientation. The bottom part of the Figure compares the two folds after a 1808 rotation of fold 2 around a vertical axis. Secondary structure elements H1 to H5 and S1 to S4 are indicated. The inset in the centre gives the positions of the folds within the 596 residues of E. coli Top 67.
808 structure.20 As shown in Figure 2(a), only two cleavage points were accessible to trypsin at positions 156 and 325. Modelling of the core T. maritima structure allowed us to identify a domain of about 150 amino acid residues in size with a horseshoe structure. This structural domain is formed by the continuous polypeptide chain from residue 157 to residue 325 (Figure 2(a), right). We subsequently called this domain, defined by its typical 3D-structure, the topofold. As seen below, this fold is present in two tandem copies and conserved in the whole type IA topoisomerase superfamily. The toroidal shape of topoisomerases IA is constituted by two successive topofolds in a head to tail orientation The high level of conservation of structures of topoisomerase I from E. coli and T. maritima suggested that a topofold could be defined in the former. To delimitate such a fold, we used the wellestablished structure of the 67 kDa fragment from E. coli topoisomerase I,13 and we based our search on structure comparison with the topofold previously defined in T. maritima. Interestingly, two successive topofolds, fold 1 (residues 187–346) and fold 2 (residues 375–540) can be defined within the E. coli topoisomerase I structure (Figure 2(b)). The two folds can be revealed by using the Swiss PDB viewer program: the entire domain I (1–158) and two regions of domain IV (159–186 and 540–596) are removed, and the remaining structure is split into halves called fold 1 (187–346) and fold 2 (375–540) (Figure 2(b), right). For fold 1, the chain starts in domain IV (purple), goes through domain II (blue) and ends in domain III (green). Symmetrically, for fold 2 the chain starts in domain III (green), goes through domain II (blue) and ends in domain IV (purple). After 1808 rotation of fold 2, the two topofolds exhibit remarkably similar structures (Figure 2(b), bottom). They both start with a long a helix (H1) followed by two antiparallel b strands (S1 and S2). Then the chain forms a short b strand S2 0 (fold 1) or a short a helix H1 0 (fold 2), followed either by helix H2 (fold 1) or by a long unstructured segment (fold 2). In both cases, the chain continues down the arch through a long twisted b strand S3, connected to two successive helices, H3 and H4. This is followed either by a short b strand S4 and a loop (fold 1), or by two short b strands S4, S4 0 (fold 2). Finally, the chain is terminated by helix H5. The structure of the E. coli topofold is globally similar to that predicted for the T. maritima topofold (Figure 2(a) and (b)). These findings show that the toroidal structure of bacterial topoisomerases I results from the association of two tandem topofolds in a head-to-tail orientation. Importantly, the folds are separated by a short segment (residue 347–374 in E. coli topoisomerase I) that contains a disordered loop and a conserved histidine residue (H365). It was suggested that this loop could serve as a door,
Universal Type IA Topoisomerase Fold
allowing entry or exclusion of nucleotides from a binding site close to the active site tyrosine residue.17 As we show below, this organization is probably present in the whole type IA topoisomerase superfamily. The organisation into two successive topofolds is found in the three known type IA structures: each fold contains a conserved tyrosine at the base of the ring We next asked if the topofolds found in E. coli and T. maritima topoisomerases I are common to all three type IA topoisomerases of known structures; Figure 3 shows that this is indeed the case. In each of the three structures, two folds can be defined that appear remarkably similar (compare fold 1 to fold 2 in the three species). In any case, the overall organization, i.e. the succession of helices and strands described for E. coli Topo I (Figure 2(b)) is largely conserved. Moreover, the CAP-like motifs found in a number of DNA-binding proteins and described for both type IA and type II topoisomerases,21,23 are present within the topofolds. In each fold, they are represented by helices H3 and H4, and by strand S4 (Figure 2). Another important feature is the presence of the active site tyrosine within fold 1 in all three structures (see green dots, Figures 1 and 3). As expected, its location is very similar in all cases, at the beginning of a loop, immediately after a short b strand following helix H4 (directly after H4 for Topo III). Remarkably, a highly conserved tyrosine is found in exactly the same location within fold 2 (Figure 3). In a multiple alignment of 71 type IA topoisomerases, this tyrosine is present in 64 sequences and replaced by phenylalanine in five cases and by leucine in two cases (not shown). These exceptions are found in two subfamilies, some archaeal and some bacterial topoisomerases III, including E. coli Topo III, where the tyrosine is replaced by phenylalanine. We also noted the presence of a conserved arginine (black dots) two residues downstream of the active site tyrosine in fold 1 (Figure 3). This arginine is involved in the modulation of the nucleophilic attack of the phosphate group of the scissile DNA chain by the active site tyrosine.19 In fold 2, a conserved arginine (only four changes out of 71 sequences) is found two residues upstream of the tyrosine (Figure 3). It has been shown that for E. coli Topo I, this arginine (R507) contacts single-stranded DNA. 19 A detailed examination of the three known type IA topoisomerases structures reveals that in all six topofolds the tyrosine and the arginine residues point in the same direction (not shown). These observations give more credence to the similarity between folds 1 and 2. The finding of two tandem topofolds within members of three different subfamilies of topoisomerase IA supports the idea that this organisation is a probably a structural signature for the whole superfamily.
Universal Type IA Topoisomerase Fold
809
Figure 3. Comparison of the topofolds structures from different type IA topoisomerases. The ribbon structures of the two topofold repeats in E. coli Topo I (left), E. coli Topo III (right), and A. fulgidus reverse gyrase (bottom) were generated by using the Swiss PDB Viewer program. In each case, fold 1 and fold 2 are shown and the numbers indicate the position in the protein sequence of the amino acid residue at the beginning and the end of each fold. Green dots give the location of conserved tyrosine residues (or Phe in fold 2 of Topo III) within each fold. Black dots indicate the conserved arginine residues near the tyrosine residues. Coloured segments in red (motif I, MI) and magenta (motif II, M II) indicate conserved amino acid motifs in each fold. For E. coli Topo I and Topo III, sequences in the one-letter code indicate the location of short acidic regions found in a number of type IA topoisomerases.
Comparison of secondary and primary structures of the two successive topofolds The probable presence of the two topofold repeat patterns within the whole type IA superfamily further supported the idea that it evolved by duplication of an ancestral fold, and that this duplication occurred before the divergence between Bacteria, Archaea and Eukarya. We therefore looked for similarities between the topofolds at the structural level and at the amino acid sequence level. Reflecting the similarity of the 3D structures between folds 1 and 2, the secondary structure of the folds is very similar, as illustrated in Figure 4 for E. coli topoisomerases I and III. The same observation is made for A. fulgidus reverse gyrase (not shown). Analysis of the similarities at the sequence level was more difficult, since this necessitated the identification of the boundaries of the folds. For the three type IA topoisomerases of known structure, this was based on the structural similarities (Figure 3) and the positioning of helices and strands (Figure 4). To identify the fold boundaries in a variety of type IA enzymes, a multiple alignment of 71 sequences was produced by using the Clustal X program (not shown). We found that fold 1 from all the various species were reasonably aligned (30% identity, 40% similarity for the most distant) and their boundaries could be defined by comparison with those of E. coli Topo I, Topo III and A. fulgidus reverse gyrase. The same approach was
used for every fold 2. We then looked for sequence similarities between each fold 1/fold 2 pair in a number of species. As expected from a very ancient duplication event, only a weak similarity was found between the two folds of a same species. For instance, the tandem topofolds of E. coli Topo I (Figure 4) exhibit only 14% identity and 24% similarity in the approximately 150 amino acid residue sequence. Interestingly, these scores were slightly higher for M. kandleri archaeal Topo III (22% identity, 32% similarity) and A. fulgidus Topo III (17.5% identity, 30% similarity), possibly reflecting their deeper phylogenetic position (Figure 1). On the other hand, homologous folds of the different type IA subfamilies appear more similar to each other, as expected from a duplication event that took place before their divergent evolution. For example, fold 1 from E. coli Topo I and human Topo IIIa exhibit 27.7% identity and 39.1% similarity, and fold 2 from the same species exhibit 27.4% identity and 42.3% similarity, which is consistent with the higher level of structural similarity observed between homologous folds (Figure 3). A careful examination of the sequences revealed two motifs that are clearly conserved between fold 1 and fold 2 in the whole topoisomerase IA superfamily. Figure 5 shows an alignment of these motifs for 20 topofolds (ten fold 1, ten fold 2) from ten different topoisomerases. These motifs are found near the extremities of each fold, motif I within helix H1, and motif II within helix H4. Their spatial location is also indicated in Figure 3 (MI and MII) and Figure 4
810
Universal Type IA Topoisomerase Fold
Figure 4. Alignment of folds 1 and 2 and comparison of their secondary structure. Upper scheme, E. coli Topo I; lower scheme, E. coli Topo III. Alignments (ClustalX) were visualized by using the Macboxshade 2.11 program (Methods). Identical residues in the alignment are boxed in red and similar residues are boxed in blue. For folds 1 and 2, a helices are represented by blue cylinders and bstrands by yellow arrows, above and under the alignment. For Topo III, 19 amino acid residues were deleted to allow alignment of helices H4 from the two folds. Green and black dots point to the positions of conserved tyrosine and arginine residues described in the text. Red bars indicate motifs (MI in H1, MII in H4) conserved within both fold 1 and fold 2 in various type IA topoisomerases. The green bars indicate acidic regions.
(red bars). Figure 5 shows that in the E. coli Top67 structure the motifs are located at the base of the horseshoe. Because of the head-to-tail orientation of the two topofolds, motif I of fold 1 is on the right, near motif II of fold 2, while motif I of fold 2 is on the left, close to motif II of fold 1, adjacent to the active site tyrosine (Figure 5, centre). Moreover, in the three topoisomerase structures, motifs I and II of each topofold appear in the same relative locations (Figure 3). Although the tyrosine residues in each fold have nearly the same spatial location, they do not exactly match in the alignments of Figure 4. The tyrosine of fold 1 is immediately outside motif II (red bars), while the tyrosine of fold 2 is the penultimate residue of motif II. This may be due to the fact that pairwise alignments are less reliable than multiple alignments when performed on divergent proteins or motif samples. Non-exclusively, this may be due to the accumulation of sequence changes since the divergence of folds 1 and 2. The presence of these two conserved sequence motifs (MI and MII) at the base of the topofolds emphasizes their potential importance to the maintenance of the typical architecture of the type IA topoisomerases. Other additional traces of a common origin for folds 1 and 2 can be considered. First, is the previously mentioned presence of the pairs Tyr-X-Arg and
Arg-X-Tyr in folds 1 and 2, respectively, found in nearly all type IA topoisomerase sequences and in the same spatial location. Although the tyrosine of fold 2 is not involved in the trans-esterification reaction, and is replaced by phenylalanine in a few cases, its conservation suggests that it plays another important role, perhaps in interacting with single-stranded DNA, as is the case for the nearby arginine.19 The second feature is the presence of short acidic regions in a number of type IA topoisomerases. Remarkably, these regions (identified by green bars in Figure 4 and indicated in the structures, Figure 3) are found in equivalent sequence regions within each fold (see motifs EQTQ/GDED for Topo I and QDEE/DEEN for Topo III). The role of these acidic regions in either catalysis or structure maintenance remains to be investigated. The observations described here reveal that the universal toroidal architecture of type IA topoisomerases is formed from two tandem domains, the topofolds, arranged in a head-to-tail fashion. The high level of structural similarity and the finding of traces of conservation at the sequence level suggest that the fold repeat results from a duplicationfusion event of an ancestral fold. With the accumulating data from genome sequencing, such a situation is more and more frequently described
811
Universal Type IA Topoisomerase Fold
Figure 5. Conserved sequence motifs between the folds. Alignment (by using the Clustal X and MacBoxshade 2.11 programs) of two motifs within folds 1 and 2 from ten different species, representing the five families of topoisomerase IA: AaeI, Aquifex aeolicus Topo I; AfuIII, Archaeoglobus fulgidus Topo III; AfuRG, Archaeoglobus fulgidus reverse gyrase; BsuIII, Bacillus subtilis Topo III; CelIII, Caenorhabditis elegans Topo III; EcoI, Escherichia coli Topo I; EcoIII, Escherichia coli Topo III; HsaIIIa, Homo sapiens Topo IIIa; MkaIII, Methanopyrus kandleri Topo III; NeqRG, Nanoarchaeum equitans reverse gyrase. Residues strictly conserved in at least 12 sequences out of 20 are in yellow. Similar residues are in black above a blue background. The position of the motifs within the w150 residues of the topofold is shown on the bottom of the Figure Right: location of motifs I and II within the E. coli Topo I fold 1 (I-1 and I-2) in yellow and fold 2 (II-1 and II-2) in green. The central structure gives the position of the motifs within the whole E. coli Top67 structure. The green dots locate the active site tyrosine. The rest of the structure outside the folds is in grey.
for a number of multidomain proteins. In the case of type IA topoisomerases, the ancestral precursor domains, although not immediately visible as independent domains in the topoisomerase structure, could be revealed by a combination of comparative structural and sequence analysis. Another example of this has been described in the case of enzymes with a b/a barrel scaffold,24 and one can imagine that many proteins are built in this way. Finally, the description of the type IA topoisomerase structure based on a logic different from that of the four canonical structural domains, opens up new possibilities to model interactions with other molecules. In particular, it might be useful to design novel inhibitors of type IA topoisomerases, as potential antibiotics, and to target the eukaryotic type IA enzymes that are presently the subject of intense interest.
Methods Thermotoga maritima topoisomerase I limited proteolysis The recombinant T. maritima topoisomerase I was purified to homogeneity from E. coli, using the previously
described procedure,25 and submitted to limited proteolysis by trypsin as described by Cossard et al.20
Multiple sequence alignments Multiple alignments of type IA topoisomerases were produced by using the ClustalX package (F. Jeanmougin IGBMC, Strasbourg). Alignments were visualized by using the Macboxshade 2.11 program (M. D. Baron, Institute of Animal Health, UK). An alignment of 71 complete topoisomerase IA sequences was first made and the conserved sequence motifs were used manually to identify the core domains that contain these motifs.
Phylogenetic trees A selection of 49 type IA topoisomerase core sequences was used to produce a multiple alignment with the ClustalX program. Distance trees were derived from this alignment by using the Phylip package (version 4.1†). Confidence limits for each node of the distance trees were determined using a bootstrap approach (Condense program). † http://evolution.genetics.washington.edu/phylip. html
812
Universal Type IA Topoisomerase Fold
Three-dimensional protein structures Ribbon representations of topoisomerases IA structures were obtained using the SWISS PDB viewer program (version 3.6b2) with the published accession numbers 1CY4 and 1MW8 for E. coli Top 67, 1D6M for E. coli Topo III and 1GKU for A. fulgidus reverse gyrase. The structure of T. maritima type IA topoisomerase was predicted from the other known structures using the Swiss Model protein modelling package†. The modelled structure was confirmed by low-resolution crystallographic data (V. Lamour, T. Viard, C.B. de la T., D. Moras and M. D., unpublished results).
Acknowledgements We thank B. Labedan and O. Lespinet for useful advice, and B. Holland and S. Sommer for critical reading of the manuscript. The Laboratoire d’Enzymologie des Acides Nucleiques was supported by funds from CNRS and Universite´ Paris-Sud.
References 1. Champoux, J. J. (2001). DNA topoisomerases: structure, function, and mechanism. Annu. Rev. Biochem. 70, 369–413. 2. Wang, J. C. (2002). Cellular roles of DNA topoisomerases: a molecular perspective. Nature Rev. Mol. Cell Biol. 3, 430–440. 3. Duguet, M. (1997). When helicase and topoisomerase meet!. J. Cell Sci. 110, 1345–1350. 4. Khakhar, R. R., Cobb, J. A., Bjergbaek, L., Hickson, I. D. & Gasser, S. M. (2003). RecQ helicases: multiple roles in genome maintenance. Trends Cell Biol. 13, 493–501. 5. Drlica, K. (1992). Control of bacterial DNA supercoiling. Mol. Microbiol. 6, 425–433. 6. DiGate, R. J. & Marians, K. J. (1988). Identification of a potent decatenating enzyme from Escherichia coli. J. Biol. Chem. 263, 13366–13373. 7. Bouthier de la Tour, C., Portemer, C., Huber, R., Forterre, P. & Duguet, M. (1991). Reverse gyrase in thermophilic eubacteria. J. Bacteriol. 173, 3921–3923. 8. Slesarev, A. I., Zaitzev, D. A., Kopylov, V. M., Stetter, K. O. & Kozyavkin, S. A. (1991). DNA topoisomerase III from extremely thermophilic archaebacteria. ATPindependent type I topoisomerase from Desulfurococcus amylolyticus drives extensive unwinding of closed circular DNA at high temperature. J. Biol. Chem. 266, 12321–12328. 9. Wallis, J. W., Chrebet, G., Brodsky, G., Rolfe, M. & Rothstein, R. (1989). A hyper-recombination mutation in S. cerevisiae identifies a novel eukaryotic topoisomerase. Cell, 58, 409–419.
10. Dekker, N. H., Rybenkov, V. V., Duguet, M., Crisona, N. J., Cozzarelli, N. R., Bensimon, D. & Croquette, V. (2002). The mechanism of type IA topoisomerases. Proc. Natl Acad. Sci. USA, 99, 12126–12131. 11. Lima, C. D., Wang, J. C. & Mondragon, A. (1993). Crystallization of a 67 kDa fragment of Escherichia coli DNA topoisomerase I. J. Mol. Biol. 232, 1213–1216. 12. Viard, T., Cossard, R., Duguet, M. & de La Tour, C. B. (2004). Thermotoga maritima-Escherichia coli chimeric topoisomerases. Answers about involvement of the carboxyl-terminal domain in DNA topoisomerase I-mediated catalysis. J. Biol. Chem. 279, 30073–30080. 13. Lima, C. D., Wang, J. C. & Mondragon, A. (1994). Three-dimensional structure of the 67K N-terminal fragment of E. coli DNA topoisomerase I. Nature, 367, 138–146. 14. Mondragon, A. & DiGate, R. (1999). The structure of Escherichia coli DNA topoisomerase III. Structure, 7, 1373–1383. 15. Rodriguez, A. C. & Stock, D. (2002). Crystal structure of reverse gyrase: insights into the positive supercoiling of DNA. EMBO J. 21, 418–426. 16. Confalonieri, F., Elie, C., Nadal, M., de La Tour, C., Forterre, P. & Duguet, M. (1993). Reverse gyrase: a helicase-like domain and a type I topoisomerase in the same polypeptide. Proc. Natl Acad. Sci. USA, 90, 4753–4757. 17. Feinberg, H., Changela, A. & Mondragon, A. (1999). Protein-nucleotide interactions in E. coli DNA topoisomerase I. Nature Struct. Biol. 6, 961–968. 18. Changela, A., DiGate, R. J. & Mondragon, A. (2001). Crystal structure of a complex of a type IA DNA topoisomerase with a single-stranded DNA molecule. Nature, 411, 1077–1081. 19. Perry, K. & Mondragon, A. (2003). Structure of a complex between E. coli DNA topoisomerase I and single-stranded DNA. Structure, 11, 1349–1358. 20. Cossard, R., Viard, T., Lamour, V., Duguet, M. & Bouthier de La Tour, C. (2004). Proteolytic cleavage of the hyperthermophilic topoisomerase I from Thermotoga maritima does not impair its enzymatic properties. Biochim. Biophys. Acta, 1700, 161–170. 21. Murzin, A. G. (1994). New protein folds. Curr. Opin. Struct. Biol. 4, 441–449. 22. Aravind, L., Leipe, D. D. & Koonin, E. V. (1998). Toprim—a conserved catalytic domain in type IA and II topoisomerases. DnaG-type primases, OLD family nucleases and RecR proteins. Nucl. Acids Res. 26, 4205–4213. 23. Berger, J. M. (1998). Structure of DNA topoisomerases. Biochim. Biophys. Acta, 1400, 3–18. 24. Lang, D., Thoma, R., Henn-Sax, M., Sterner, R. & Wilmanns, M. (2000). Structural evidence for evolution of the beta/alpha barrel scaffold by gene duplication and fusion. Science, 289, 1546–1550. 25. Viard, T., Lamour, V., Duguet, M. & Bouthier de la Tour, C. (2001). Hyperthermophilic topoisomerase I from Thermotoga maritima. A very efficient enzyme that functions independently of zinc binding. J. Biol. Chem. 276, 46495–46503.
Edited by I.B. Holland (Received 14 February 2006; received in revised form 5 April 2006; accepted 6 April 2006) Available online 25 April 2006 † http://swissmodel.expasy.org/