Abstract
T-even type bacteriophages are virulent phages commonly used as model organisms, playing a crucial role in understanding various biological processes. One such process involves the regulation of DNA topology during phage replication upon host infection, governed by type IIA DNA topoisomerases. In spite of various studies on prokaryotic and eukaryotic counterparts, viral topoisomerase II remains insufficiently understood, especially the unique domain composition of T4 phage. In this study, we determine the cryo-EM structures of topoisomerase II from T4 and T6 phages, including full-length structures of both apo and DNA-binding states which have never been determined before. Together with other conformational states, these structures provide an explicit blueprint of mechanisms of phage topoisomerase II. Particularly, the asymmetric dimeric interactions observed in cryo-EM structures of T6 phage topoisomerase II ATPase domain and central domain bound with DNA shed light on the asynchronous ATP usage and asynchronous cleavage of the G-segment DNA, respectively. The elucidation of phage topoisomerase II’s structures and functions not only enhances our understanding of mechanisms and evolutionary parallels with prokaryotic and eukaryotic homologs but also highlights its potential as a model for developing type IIA topoisomerase inhibitors.
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Introduction
Type IIA DNA topoisomerase plays a pivotal role in the life cycle of prokaryotes, eukaryotes and viruses, facilitating the regulation of DNA topology through induction of transient double-strand breaks (DSBs) in DNA during various biological processes such as DNA replication, transcription, recombination and chromosome condensation1,2,3. The structural and functional characteristics of type IIA DNA topoisomerases from prokaryotic organisms (such as Escherichia coli Gyrase), and eukaryotic organisms (such as human Topo IIα and Topo IIβ) have been extensively studied in previous research since their identifications4,5,6,7. A salient feature in the architecture of type IIA DNA topoisomerases is the presence of two highly conserved domains that are integral to their catalytic functions: (1) the ATPase domain, commonly as a homodimer, is essential for the binding of ATP and divalent metal ions (usually Mg2+), providing the energy required for the conformational changes within the enzyme8,9. (2) the DNA binding/cleavage domain (also called the central domain and organized as a homodimer), endows with the ability to bind and cleave a double-strand DNA (dsDNA), known as the G-segment, then generate a transient DNA break allowing the passage of another DNA duplex, referred to as the T-segment DNA5,10. By orchestrating these domains, type IIA DNA topoisomerases reconnect the cleaved DNA strands, thereby changing the degree of DNA superhelix without disrupting the genomic integrity10,11.
In canonical mechanism of type IIA topoisomerase, the two strands of the G-segment DNA duplex are cleaved simultaneously in the presence of divalent metal ions (usually Mg2+) and ATP12,13. However, evidence from cleavage assay of eukaryotic Topo II in the absence of ATP suggests an alternative mechanism of the asynchronous G-segment DNA cleavage, supported by the single-strand nicked plasmid DNA product14,15. But so far, structural studies have not yet supported this mechanism, and no such native single-strand nicked DNA-Topo II complex structure has been determined.
Recently, structures of a virus-encoded topoisomerase II (African swine fever virus Topo II) in various conformations were reported by several research groups16,17,18. The enzyme resembles human Topo II with a domain-swapped homodimeric configuration while lacking an additional C-terminal domain, which suggests a more primitive form of topoisomerase II16. However, structures and mechanisms of topoisomerase II encoded by other viruses, such as bacteriophages, remains limited, especially considering the unique domain composition observed in T4 bacteriophage topoisomerase II (three subunits while lacking CTD)19. Here, we explored the structural and functional characteristics of topoisomerase II encoded by bacteriophages T4 and T6 (hereafter referred as T4 Topo II and T6 Topo II), and discussed their potential roles in the corresponding T-even type bacteriophages.
Bacteriophages as crucial model organisms represent a distinct class of viruses reliant on bacterial hosts for multiplication20,21. There are mainly seven types of Escherichia coli infecting phages, three even-numbered T-phages (e.g., T2, T4 and T6) and four odd-numbered T-phages (e.g., T1, T3, T5, T7)22,23. Out of these, T-even type phages are good examples of virulent phages that lead to host rupture through cell lysis and encode their own topoisomerase II23,24. These T-even type phages are similar in structure, antigenic response and genetics, with a relatively large dsDNA genome more than 160 kbp containing about 300 open reading frames25,26. The large genome is characterized by circularly permuted linear concatemers replicated through strand invasion recombination and the glycosylated hydroxymethyl cytosine (HMC) DNA, which is crucial to the high expression and replication of its genome25,26,27. Its own encoding Topo II probably plays a significant role in resolving knots or supercoils during DNA replication19,28. In the case of the T4 phage, a notable peculiarity is that it encodes a Topo II composed of three subunits (gp39, gp60 and gp52), standing as the singular instance of a known Topo II with a trimeric subunit composition19,24. In contrast, T2 and T6 phages exhibit a difference in their Topo II with only two subunits (gp39 and gp52), potentially resulting from a naturally occurring “gene-fusion” or “gene-separation” event24,29. Explicit structural and functional studies will enhance our comprehension of these evolutionary connections.
In this work, we determine crystal and cryo-EM structures of T4 and T6 Topo II both in the presence and absence of dsDNA, particularly T6 Topo II with more complete structure and more varied conformations. The first cryo-EM structure of the ATPase domain of type IIA topoisomerase displays a unique dimeric conformation, distinguished from crystal structures, suggesting a potential for asynchronous ATP hydrolysis. Notably, the high-resolution structure of T6 Topo II central domain bound with DNA provides evidence for an asymmetric dimeric interaction with DNA, indicating an alternative mechanism of asynchronous DNA cleavage, which has been hypothesized in prokaryotic and eukaryotic Topo II but not yet found in viral counterparts14,30,31. These structures, together with in-depth enzymatic assays, provide a comprehensive insight into the ingenious mechanisms, conformational changes, and evolutionary patterns intrinsic to type IIA topoisomerase.
Results
Cryo-EM reconstruction of T4&T6 Bacteriophage Topo II complexes
To investigate the structures and functions of the T-even bacteriophage topoisomerase II, T4 & T6 bacteriophage Topo II subunit gp39 (gp39, gp60 for T4) and gp52 were overexpressed in E. Coli BL21 (DE3), and purified separately using Ni-NTA affinity chromatography before assembly at a 1:1 stoichiometric ratio (Fig. 1a). The holoenzymes were further purified by size-exclusion chromatography and tested for relaxation activity (Supplementary Fig. 1a-d). The pre-cleavage nucleoprotein complexes were constructed by adding 52 bp DNA oligonucleotides at a molar ratio of 1:2 (Fig. 1b)32, and the non-hydrolysable homolog of ATP, AMPPNP was added to stabilize the ATPase domain33,34. In addition, for T6 Topo II, the antineoplastic agent m-AMSA (final concentration of 1 mM) was added to obtain the DNA-cleavage complex. The details for sample preparations are summarized in Supplementary Fig. 2.
a Schematic representation of the T2/4/6 phage Topo II domain composition. T4 phage Topo II is the sole type IIA topoisomerase which contains three subunits, gp39, gp60 and gp52. Its TOPRIM domain is separated by a unique insertion of ~70 amino acids. In contrast, T2 and T6 phage Topo II contains two subunits, gp39 and gp52 (The dashed line shown for T4 Topo II between the gp39 and gp60 subunit is artificial and was introduced here to improve complex stability). b 52 bp DNA duplex used in this study (Sequence modeled in high-resolution structures is highlighted in orange, and the red triangles represent DNA cleavage sites in the m-AMSA bound structure). c–h The final cryo-EM density maps of T4 & T6 Topo II complexes in distinct conformations solved at relatively high resolution (The maps are colored as in a, and the DNA is colored in orange). Briefly, two relatively high resolution cryo-EM structures were obtained from the apo state T6 Topo II sample (gp39 and gp52 heterotetramer, Mg2+ and AMPPNP were added to improve protein stability), the T6 apo central domain at 3.9 Å (c) and the ATPase domain at 3.4 Å (d). T6 central domain with an intact DNA / a cleaved DNA complex structure at 2.8 Å (f) or 3.2 Å (g) was obtained from the T6 Topo II–DNA sample or T6 Topo II-DNA-m-AMSA sample, respectively (Likewise, Mg2+ and AMPPNP were added to improve protein stability). Similarly, T4 Topo II apo central domain at 3.6 Å (e) or central domain with an intact DNA at 3.2 Å (h) was obtained from the apo T4 Topo II sample or T4 Topo II-DNA sample, respectively (gp39-gp60 and gp52 heterotetramer). i 2D classes of T6 apo and DNA-bound full-length structure and T4 central-open structure showing the flexibility of the ATPase domain as well as the DNA binding/cleavage domain. j–l The final cryo-EM density maps of T6 apo and DNA-bound full-length Topo II complexes and T4 central-open state solved at marginal resolution.
The apo, pre-cleavage and cleaved complexes were plunged-frozen on an amorphous nickel-titanium alloy film (R1.2/1.3, Au, 300 mesh) at the concentration of 0.8 mg/ml, respectively (AMPPNP and Mg2+ were added to the apo protein for stabilizing the ATPase domain. For simplicity, we used “apo protein” to indicate the protein structure with only AMPPNP and Mg2+)35. The single-particle cryo-EM images were collected with a 300 kV FEI Titan Krios electron microscope with a GIF-Quantum energy filter (Gatan) and K2-summit Detector (Supplementary Fig. 3 & Table 1). After motion correction, frame alignment, manual inspection, 2D classification, Ab-initio reconstruction, hetero, homo and non-uniform refinement, a total of 4 high-resolution, 2 moderate resolution and 3 marginal resolution structures were obtained from dozens of datasets. These structures included T4 Topo II apo central domain solved at 3.6 Å using ~279k particles, T4 Topo II central domain with DNA solved at 3.2 Å using ~270k particles, T6 Topo II apo central domain solved at 3.9 Å using ~125k particles, T6 Topo II ATPase domain with AMPPNP solved at 3.4 Å using ~148k particles, T6 Topo II central domain with an intact DNA (pre-cleavage state) solved at 2.8 Å using ~714k particles, T6 Topo II central domain with a cleaved DNA and m-AMSA solved at 3.2 Å using ~459k particles, as well as T6 Topo II apo full-length structure solved at 6.8 Å using ~69k particles, T6 Topo II DNA-bound full-length structure solved at 6.7 Å using ~11k particles and T4 Topo II central-open structure solved at 6.1 Å using ~39k particles (Fig. 1c–l and Supplementary Fig. 4).
Model building, refinement and validation were completed by using COOT36 and PHENIX37. For the high-resolution and moderate resolution structures, Alphafold2 predicted structures were used as initial model for further manual building and refinement38. For the marginal resolution structures, the modeled high-resolution structures were fitted into the corresponding maps and further refined by COOT and Phenix. Statistics of cryo-EM data collection, image processing and model building are summarized in Supplementary Table 1. All information about the final structure models is provided in Supplementary Table 3.
Overall structures and functional characterizations of T4&T6 Bacteriophage Topo II
Overall, five distinct kinds of type IIA topoisomerases share similar but not identical domain architectures. Eukaryotic and its nucleocytovirus Topo II contain only one polypeptide, while others are composed of two or three subunits. For eukaryotic Topo II, prokaryotic DNA gyrase and Topo IV, they have additional CTDs (Supplementary Fig. 5). Cryo-EM structures of the T6 phage apo & DNA-bound full-length Topo II combined with the high-resolution ATPase domain and DNA binding/cleavage domain (central domain) show clearly the domain-swapped or intertwined hetero-tetrameric architecture (A2B2), resembling eukaryotic Topo II or procaryotic gyrases34,39 (Fig. 2a, b and Supplementary Fig. 6a, b). Compared by the overall structure, T6 Topo II is more similar to eukaryotic Topo II without the additional CTD β-pinwheel and GyrA-box, while it is more similar to prokaryotic gyrase with two split subunits39,40 (Supplementary Fig. 6a, b). The subunit gp39 consists of GHKL, Transducer and TOPRIM domains, while the other subunit gp52 is composed of WHD, Tower and Coiled-coil domains. The gp39 homodimer is sitting in an orthogonal orientation above the gp52 homodimer like mortise and tenon joint structure of Chinese conventional wooden building (Supplementary Fig. 6a). In addition, the C-terminus of gp39 and N-terminus of gp52 show extensive electrostatic and hydrophobic interactions, in which the C-terminus of gp39 resembles a hook hanging from the α-helix of WHD domain, and the N-terminus of gp52 domain is like an arm wrapping around the waist of the TOPRIM domain, similar to previous reports (Supplementary Fig. 6c)39,41. This interface is basically conserved among prokaryotic gyrases and Topo IV while the two terminuses fused together through an extended α-helix and a loop in eukaryotic Topo IIs and some viral Topo II39,40,41,42,43 (Supplementary Fig. 6d and 15).
a Structure of the T6 apo full-length Topo II. b Structure of the T6 full-length Topo II with a 52 bp intact DNA. c Structure of the T6 ATPase domain with AMPPNP (left) and close-up view of the local interactive residues with AMPPNP and Mg2+ (right). d Structure of the T6 central domain with an intact DNA. e Display of the bent DNA alone in d. f Structure of T4 Topo II apo central domain (left) and zoom on the unique TOPRIM insertion domain. g Structure of the T6 central domain with a cleaved DNA and antineoplastic agent m-AMSA (left and middle). Close-up view of the interaction between m-AMSA and DNA base pairs (right).
In subunit gp39, the ATPase domain is connected with the TOPRIM domain by a short flexible linker (aa 380-390), leading to the inherent flexibility of the ATPase domain, as shown in representative 2D classes (Fig. 1i), which is also a limiting factor for acquiring high-resolution full-length structures. It is worth mentioning that this is the first cryo-EM structure of the Topo II ATPase domain bound with AMPPNP and Mg2+, whose density is well defined and interacting residues are indicated in Fig. 2c, including E54, D57, N58, D61, E62, W112, N120 and K331. Adequate particles allow us to reconstruct high-resolution ATPase domain structure using C1 symmetry and investigate each catalytic center. We found the two catalytic centers display significant difference: one contains a Mg2+ ion while the other does not, and the bound Mg2+ ion is not in a canonical way (Supplementary Fig. 7e, f). In cryo-EM structure, E54, D57, D61 and phosphate group interact with Mg2+. We also determined a crystal structure of the T6 ATPase domain at 2.8 Å (Supplementary Table 2), in which one asymmetric unit contains only one protomer. And the catalytic center shows classical Mg2+-AMPPNP interaction mode (Supplementary Fig. 7a-c). In crystal structure, E54, N58, D61 and phosphate group interact with Mg2+, but not D57. Comparison of the two structures shows that the Transducer subdomain of the cryo-EM determined ATPase domain undergoes an obvious displacement of ~ 6 Å. In addition, the distance between two Mg2+ ion is about 4 Å (Supplementary Fig. 7d, e). The differences between the two structures illustrate that the ATPase domain undergoes intrinsic cavity opening or closing in solution, which is important for trapping the T-segment DNA. Compared with previous crystal structures, T6 Topo II ATPase domain is more like eukaryotic Topo II which has a smaller central cavity than prokaryotic gyrase’s or topo IV’s ATPase domain9,44,45 (Supplementary Fig. 8). Additionally, the positions of Mg2+ in previous crystal structures are different from that of the determined T6 phage cryo-EM structure, which has an obvious displacement (on average ~4 Å change) in the ATP binding pocket (Supplementary Fig. 8b, c), suggesting a potential catalytic intermediate state, in which the Mg2+ occupies a distinct position and the ATPase domain undergoes local conformational transitions. Moreover, the asymmetric state of two catalytic centers indicates a potential asynchronous ATP usage by type IIA topoisomerase as proposed in previous reports46,47.
Compared with T6 Topo II, T4 Topo II is further split into three subunits, gp39, gp60 and gp52 (Fig. 1a). Furthermore, the C terminus of the truncated gp39 and the N terminus of the newly created gp60 contain a TOPRIM insertion (~70aa) that was not present in the original gp39 homologs29 (Fig. 1a). Previous study showed that the insertion in T4 gene 39 encoded a putative mobA homing endonuclease, but the amino acid insertion in T4 Topo II TOPRIM domain may play a role in assembly of the spilt subunits into an enzymatically active topoisomerase II48,49 (Supplementary Fig. 9a). In this work, we also obtained cryo-EM structures of the fused T4 Topo II (gp39 and gp60 are fused for stable protein expression and purification) apo and DNA-bound central domains (the ATPase domains are missing in the maps). Both structures show that the unique TOPRIM insertion resembles a pendant hanging from the outside of the TOPRIM domain without direct interaction with DNA and has no discernable influence on the structure of the central domain when compared with T6 Topo II (Fig. 2f and Supplementary Fig. 9b). Results from relaxation assay also show that this insertion has little influence on T4 Topo II enzymatic activity (Supplementary Fig. 1c, d). Since it’s difficult to purify T4 Topo II gp39 and gp60 independently, this result further strengthens the hypothesis that the unique TOPRIM insertion serves as an assembly chaperone without affecting the enzymatic activity48,50.
In addition to apo structures, we also determined a 52 bp intact G-segment DNA-bound T6 Topo II central domain and full-length structures. These structures show extensive protein-DNA interactions and the DNA is bent remarkably by ~120°, resembling other reported DNA-bound structures5,39,40 (Fig. 2b, d, e and Supplementary Figs. 6a, b). As expected, the orientation of the ATPase domain is distinct from those of T6 apo full-length structure, human Topo IIα and E. Coli gyrase39,40 (Fig. 2a, b and Supplementary Figs. 6a, b). We further added the antineoplastic agent m-AMSA to the intact DNA-bound T6 Topo II complex and the determined structure clearly shows a cleaved DNA duplex with 4 bp sticky end as well as m-AMSA intercalated into the base pairs (Fig. 2g). Furthermore, alignment of the intact DNA and cleaved DNA displays an obvious unwinding and stretching during DNA cleavage process (Supplementary Fig. 10a).
As for the functional experiments in vitro, we performed various enzymatic assays to confirm the relaxation activities of the purified Topo II, including hetero-tetramer T4 Topo II (gp39-gp60 fused), hetero-hexamer T4 Topo II (natural condition) and T6 Topo II. Both T4 Topo II exhibit similar relaxation activities, while the fused one has a higher protein yield. In addition, the inhibition assay confirmed the inhibitory effect of m-AMSA on the relaxation activity of T6 Topo II at μM level. Results from T6 Topo II relaxation assay with various divalent ions show that Mn2+ promotes relaxation activity more than Mg2+, while no relaxation activity is observed with Ca2+ or Zn2+ (Supplementary Fig. 1b-f).
Interactions between T6 Bacteriophage Topo II and DNA
Similar to other reported Topo II structures, the central domain of T4&T6 Topo II also forms a positive charged groove which is electrostatically complementary with DNA5,16, and the interactive interface between the T6 Topo II central domain and DNA spans about 6900 Å2 (Fig. 3a). In this way, T4 Topo II is highly similar to T6 Topo II, so we take T6 Topo II for detailed analysis. The WHD, TOPRIM and Tower domains together interact with DNA backbone, reshaping its conformation. The WHD domain is composed of three distinctive conserved α-helices (α1-α3) and an important β-hairpin namely Wing 151 (Fig. 3b and Supplementary Fig. 10b). The major groove of the G-segment DNA is supported by insertion of two α3 helices from two protomers, together with assistance of α1 and α2 helix (Fig. 3b). In the conserved Wing 1, tyrosine117 from catalytic center directly participates in the cleavage of DNA, forming a covalent phosphodiester bond between hydroxyl of tyrosine and the 5 prime phosphate end of DNA52, which is especially obvious in the cleaved DNA-bound central domain structure (Fig. 3c, g). The Tower domain consists of a conserved β-hairpin denoted as Wing 251,53, intercalating into the minor groove and a conserved residue isoleucine interact directly with base pair stacks, which leads to bending and conformational change of the bound DNA5 (Fig. 3e). Besides, a set of residues including Q214, R300, R301, S302, N304 in the β-sheets of the Tower domain also interact with DNA backbone, serving as a supportive scaffold for the distal region of the bent DNA (Supplementary Fig. 10c). The TOPRIM domain has an intrusion loop (Y435 ~ S444) stuck on the outside of the DNA base pair stacks, in which side chains of R438 and K440 form a “Y” shaped crotch, stabilizing the bent DNA. K556 and D490 also interact with DNA (Fig. 3d). In addition, there is a conserved metal binding pocket in this domain formed by E\(\cdots\)DxD motif (E415, D486 and D488 in T6 Topo II), which is crucial for the nucleophilic attack of tyrosine and stability of the cleaved intermediate complex52 (Fig. 3h).
a Electrostatic potential diagram of the structure T6 Topo II central domain with an intact DNA. DNA is shown as cartoon. b Cross-section diagram of the interactions between the intact DNA and the T6 Topo II central domain, mainly TOPRIM, Tower and WHD domains. c Close-up view of the catalytic center “RY”. d Close-up view of interactions between the TOPRIM and DNA. e Isoleucine on the Wing 2 of the Tower domain intercalates between base pairs, bending the G-segment DNA. f Interactions between the cleaved DNA and the T6 Topo II central domain. g Close-up view of the catalytic center “RY”, a covalent phosphodiester bond is formed between “–OH” of tyrosine and 5’- PO43-. Positions of the antineoplastic agent m-AMSA are indicated. h Close-up view of the metal-binding center with conversed residues shown as stick. Mg2+ ion is colored in green.
Previous study reported a crystal structure of a T-segment DNA-bound ParE ATPase domain of topoisomerase IV from Streptococcus pneumoniae45, but none has been reported for other Topo II by cryo-EM, thus trapping of the T-segment DNA and the exact role of ATPase domain have long been unsolved problems. Unexpectedly, we found a novel 2D class of T6 Topo II-DNA complex, which shows direct attachment between the nearly recumbent ATPase domain and one of the DNA overhangs, as well as the highly flexible linker (Supplementary Fig. 3c). Furthermore, 3D initial model shows that one linker is stretched and the ATPase domain grabs the DNA overhang (Supplementary Fig. 10d). This conformation is unique because the ATPase domain traps the stretched G-segment DNA terminus, distinct from previous conventional DNA binding mode, suggesting a potential for trapping a T-segment DNA by the ATPase domain. However, higher-resolution structures of this conformation need to be further determined, which is vital to exactly reveal the intrinsic mechanism.
Conformational changes are directly coupled to G-segment DNA cleavage
During relaxation catalysis, Topo II experiences numerous conformational changes (G-segment DNA binding, cleavage, opening and T-segment DNA transport)1,10,11, which is also the case for phage Topo II. Firstly, comparing full-length T6 apo Topo II with DNA-bound state, the ATPase domain went through major rotating and lateral movement (Fig. 4a). Superimposition of the two structures (alignment based on two central domains) shows that the DNA-bound T6 Topo II ATPase domain has a greater tilt angle relative to the central domain (apo: ~100° vs DNA-bound: ~125°). Meanwhile, a 22 ~ 50 Å lateral movement and ~25° anticlockwise rotation can be detected (Supplementary Fig. 11a). In fact, the wobbling of the ATPase domain should be constant as shown in 2D classes as a result of highly flexible linkers (Fig. 1i and Supplementary Fig. 3c). Furthermore, in apo state, the linkers seem to lie flat, forming a lid above the DNA binding groove, while in DNA-bound state, the linkers lift up and extend outward (Supplementary Fig. 11b).
a Superimposition of the T6 Topo II apo (gray) and DNA-bound (yellow) full-length structure. The ATPase domain shows obvious lateral and rotating movement. b Superimposition of the T6 Topo II apo (gray) and T4 central-open (green) structure. c Superimposition of the T6 apo (gray) and DNA-bound (yellow) central domain structure. d Superimposition of the DNA-bound (yellow) and the cleaved DNA, m-AMSA-bound (blue) central domain structure. e–g Close-up view of the catalytic center of the T6 Topo II in apo, pre-cleavage and cleaved states. For each conformation, the catalytic tyrosine, the metal binding residues, and the distances between the two catalytic tyrosine, as well as distances between metal binding motif and catalytic tyrosine (Taking D486 as an example, the distance is between the side chain “O” of Y117 and the side chain “O” of D486, cause these radical groups directly participate in the catalytic reaction) are shown to illustrate structural changes in the central domain of T6 Topo II during the apo-to-pre-cleavage and pre-cleavage-to-cleaved conformational transitions.
Secondly, the central domain experiences obvious domain displacements17. When binding a G-segment DNA, the overall structure of the central domain contracts to encompass the DNA. In detail, the Tower domain has an upward movement of ~10 Å, the TOPRIM domain approaches the bound DNA by ~12 Å, and the WHD domain also draws near by ~ 5 Å, by which the catalytic tyrosine moves by 5.4 Å (Fig. 4c and Supplementary Fig. 10b). As a result, the two heterodimers contract to form a steady pre-cleavage state Topo II-DNA complex. When the G-segment DNA is cleaved (m-AMSA is added to stabilize the cleavage complex), the Tower domain undergoes downward movement by ~10 Å. Meanwhile, the TOPRIM domain continues to move unidirectionally by ~ 5 Å and slightly tilts ~10° in the vertical direction. In addition, the upper region of the Coiled-coil domain opens slightly through the bottom dimeric interface as a rotating axis (Fig. 4d). These changes lead to a widened gap between two heterodimers, preparing for opening of the cleaved G-segment DNA and transport of T-segment DNA13. Indeed, in the central-open state structure, the Tower, WHD domain and the upper region of the Coiled-coil domain open up to a greater degree, which is ~14 Å, ~8 Å and ~10 Å respectively compared to the apo state (Fig. 4b). A modeled T-segment DNA can get through the open G-segment channel as expected (Supplementary Fig. 11c).
The superimposition of T6 apo and DNA bound central domain structures also show that key residues in the catalytic center shifted remarkably (Fig. 4e–g and Supplementary Fig. 11d). During the mentioned progress above, the distance between two catalytic tyrosine residues subsequently decreases and finally increases (apo: 31.3 Å, Pre-cleavage: 24.7 Å, Cleaved: 16.0 Å, central-open: 30.9 Å). Accordingly, the metal-binding motif gets closer to the catalytic Y117 and then becomes farther on the whole (taking D486 as an example, the distance is between the side chain “O” of Y117 and the side chain “O” of D486: apo: 4.8 Å, Pre-cleavage: 4.2 Å, cleaved: 11.2 Å, the TOPRIM domain is missing in the central-open structure). These subtle changes in key residues and overall conformational changes together provide explicit information for understanding reaction mechanisms of phage Topo II.
Indications for asynchronous G-segment DNA cleavage
Previous studies demonstrated a single-stranded DNA cleavage mechanism by both eukaryotic Topo II and prokaryotic Topo IV or gyrase, which indicates that DNA cleavage by Topo II proceeds through two asymmetric single-stranded cleavages14,15,30. However, this asymmetric or asynchronous single-strand DNA cleavage has been lacking of direct structural evidence. In this work, we obtained a high-resolution T6 Topo II structure bound with an intact DNA at 2.8 Å resolution with C1 symmetry, enabling us to investigate two catalytic centers independently. Unexpectedly, the well-defined density map displayed obvious difference in two catalytic centers. Although overall dimer architecture exhibits C2 symmetry, one subunit has an obvious Mg2+ binding while the other does not, giving the direct structural evidence for asymmetric state in each catalytic center, indicating the G-segment DNA cleavage is completed by two asynchronous single-stranded DNA cleavage (Fig. 5a, b). The DNA cleavage assay by T6 Topo II also displayed single-stranded cleavage, proved by the presence of nicked open circular plasmids when ATP is not included, and this single-stranded cleavage activity is affected by replacement with different divalent metal ions, especially in the presence of Ca2+, consistence with previous report5. In the presence of Mg2+ and Mn2+, single-stranded cleavage activity can be detected, although ATP-independent relaxation activity can also be detected, which is similar to a recent report (Fig. 5c)54. Together, these structural and functional evidences show directly the asymmetric form of catalytic centers, indicating an asynchronous G-segment DNA cleavage by the T6 Topo II, which may be a more universal mechanism for DNA cleavage by type IIA topoisomerase.
a, b Close-up observation of the two catalytic centers of the T6 intact DNA-bound central domain structure. Distances between key atoms are displayed. Comparison of these distances shows obviously different forms of the two catalytic centers, suggesting asynchronous DNA cleavage (contour level: 0.8. For clarity, the residue E415 and D488 are omitted). c cleavage assay of the T6 Topo II without ATP in the presence of different divalent ions. Single-stranded cleavage activity is affected by replacement with different divalent metal ions, especially in the presence of Ca2+. All enzymatic assays were performed at least twice independently.
Discussion
Since its first identification in 1970s, T4 bacteriophage topoisomerase II has been found playing a vital role in the initiation of T4 bacteriophage DNA replication in vivo and illegitimate recombination in vitro19,55. In addition, T4 Topo II consists of three subunits (gp39, gp60 and gp52), distinct from its T2/T6 homologs and other type IIA topoisomerases11,29. Although extensive functional studies have been conducted on T4 Topo II, structures of T-even type bacteriophage Topo II have not been reported, limiting our understanding of these specific Topo II encoded by viruses. In this work, we took T4 and T6 Topo IIs as examples, determined their apo and DNA-bound cryo-EM structures in different conformations and a crystal structure, as well as characterized their relaxation activities in the presence of ATP, and cleavage activities in the absence of ATP, providing detailed information and new insights for the correspondence between their structures and functions.
In total, four high-resolution structures, two moderate resolution and three marginal resolution structures were determined by cryo-EM, including T4 apo central domain, T4 DNA-bound central domain, T6 ATPase domain bound with AMPPNP, T6 apo central domain, T6 DNA-bound central domain and T6 central domain with DNA and m-ASMA, in addition to T6 apo full-length, T6 DNA-bound full-length and T4 central-open structures. Clearly, these structures display that overall architectures of T4 and T6 Topo II are more similar to eukaryotic ones rather than prokaryotic ones, at the same time T4 and T6 Topo II structure comparison shows that the unique T4 TOPRIM insertion serves as an assembly bracket for the split proteins into an intact one without affecting its central domain structure and DNA-binding property, which suggests its function remains unaffected, proved by additional enzymatic assays. In addition, the first cryo-EM structure of T6 ATPase domain displays a similar dimeric conformation but a novel binding mode of Mg2+ in its catalytic center compared with its crystal structure and other homologous ATPase domains. This finding may suggest a distinct catalytic intermediate of ATP hydrolysis, in which the cavity of the ATPase domain slightly opens. Moreover, the asymmetric dimeric Mg2+ binding indicates the potential mechanism of asynchronous ATP energy usage by type IIA topoisomerase46. However, relatively low resolution limits our further investigation, more cryo-EM studies on the ATPase domain are needed to verify the hypothesis. Anyway, we think this question is usually ignored and should be addressed in future studies, which may explain the energy transfer mechanism in the course of DNA relaxation, unknotting or decatenation processes by type IIA topoisomerase.
As evidenced by other reported structures, Topo II generally undergoes various conformational changes during relaxation process1,11. Here, we displayed more comprehensive and detailed changes of this process, taking T6 Topo II as an example. Briefly, apo state T6 Topo II central domain has a relatively loose conformation characterized by a ~ 31 Å distance between two catalytic tyrosine. Upon binding of an intact DNA duplex, the central domain apparently contracts to accommodate the DNA, with a ~ 25 Å distance between two catalytic tyrosine. Agent like m-AMSA promotes cleavage of the DNA duplex, leading to formation of the covalent phosphodiester bond between tyrosine and DNA, as well as a contraction of ~ 16 Å between two catalytic tyrosine. Then, the cleaved G-segment DNA splits so that a second T-segment DNA can pass through, which is characterized by a ~ 31 Å distance between two catalytic tyrosine. After passage of the T-segment DNA, the cleaved G-segment DNA can be resealed and released, thus the enzyme is reset for a next catalytic cycle1,34. Another interesting thing is that we find the two catalytic centers are asymmetric in T6 Topo II DNA-bound central domain structure determined at 2.8 Å. This difference indicates asynchronous cleavage reaction in each catalytic center. However, it should be noted that short linear DNA is not easy to be cleaved by Topo II compared with supercoiled plasmid DNA in natural condition56. Therefore, the high-resolution structure of Topo II bound with a singly-nicked DNA which represents the direct intermediate of single-stranded cleavage by Topo II is difficult to obtain. The T4 topo II-DNA bound structure shows two Mg2+ ions at each catalytic center, but their distances are slightly different from each other by ~ 1.3 Å (Supplementary Figs. 12a, b). The T6 Topo II-cleaved DNA bound structure shows one Mg2+ ion binding at each catalytic center in a similar way. However, detailed analysis is limited by the relatively low resolution of the two structures. Further studies are still needed. Moreover, DNA cleavage assay further provided evidence on the existence of single-stranded cleaved DNA intermediate (nicked open circular plasmid). Based on the above, we proposed a detailed mechanism governing relaxation assay of T6 Topo II (Fig. 6), and we think this model should also be applicable to other Topo II.
Four states of the central domain are displayed. In apo state, the central domain has a relatively loose conformation. In the pre-cleavage state, upon binding of an intact DNA duplex, the central domain apparently contracts to accommodate the DNA. During the cleavage process, DNA cleavage is completed by two asymmetric single-stranded cleavages. In the Open state, the doubly cleaved G-segment DNA splits so that a second T-segment DNA can pass through the breach. The dashed boxes represent changes in DNA at each step.
The exact role of ATPase domain in Topo II has been a longstanding question except for its basic ATPase activity8,54,57. In this work, the novel 2D class and 3D initial model of the new T6 Topo II conformation show direct interaction between the ATPase domain and one of the DNA overhangs. We speculate the ATPase domain opens its C-terminal interface by stretching its flexible linker, grabbing the DNA overhang, just like trapping a T-segment DNA. Thus, we think it’s reasonable the ATPase domain can open and grab another DNA duplex as T-segment DNA. However, this initial model is difficult to promote due to the preferred orientation problem and limited particles. Further studies are still needed.
From evolutionary perspective, previous phylogenetic analysis suggested Type IIA family forms three subgroups, in which Topo II from T4 bacteriophage superfamily stands as an independent subgroup, distinguishing from Topo IIA of eukaryotes and related viruses, as well as bacterial gyrase, Topo IV and archaeal gyrase58. This can be further proved by our result of phylogenetic analysis (Supplementary Figs. 13, 14). However, structural analysis suggested a situation similar to eukaryotic and their viral Topo II. In other words, despite of low amino acid sequence similarity with host bacterial gyrase or Topo IV, T-even type phage Topo II contains the two most fundamental domains (ATPase and central domain) without additional C-terminal domains like eukaryotic Topo II or prokaryotic gyrase and Topo IV, which have been proved to play an important role in the cell cycle, including mediating functional interactions with chromatin, serving as a scaffold to recruit mitotic regulators to centromeres (eukaryotic Topo II)59, as well as determining the preference for different topological states of DNA substate (gyrase or Topo IV)43,60 (Supplementary Figs. 5, 6a, b and 13). This potentially corresponds to the relatively simpler replication manner of virus compared with prokaryotic or eukaryotic cells and indicates a possibility of viral origin of both eukaryotic and bacterial type IIA topoisomerases58,61,62. On the other hand, there is another possibility that viral Topo II encodes unidentified proteins to serve as the corresponding CTDs63. But for now, this is just a hypothesis and needs further studies.
As mentioned before, out of seven types of E. coli infecting phages, T-even type phages encode their own Topo II and cause cellular death through cell lysis upon infection, while T-odd type phages don’t encode their own Topo II and basically establish a latent or chronic infection20,22 (except T5 phage). This distinction indicates the importance of Topo II in T-even type phages during the immediate lytic cycle, which requires topoisomerase II to relax potential DNA supercoils or to resolve DNA knots of the rapidly replicating genome, consistent with the previous idea that T4 Topo II appears to have an essential role in the initiation of T4 bacteriophage DNA replication19.
In conclusion, we determined atomic resolution cryo-EM structures of T4 and T6 Topo II in different conformations as well as one crystal structure, revealing distinct architectures of Topo II encoded by T-even type bacteriophages, which are important model organisms. These various structures provide abundant details for understanding the connection between protein structure and function. Beyond that, novel findings on position of Mg2+ within the ATPase domain, the new initial model of the full-length Topo II with distinct orientation of the ATPase domain and the asynchronous DNA cleavage model together provide further insights into mechanisms of topoisomerase II.
Methods
Recombinant protein overexpression and purification
The codon-optimized sequences of T4 bacteriophage topoisomerase II large subunit (gp39, NP_049621.1) and small subunit (gp60, NP_049618.1) were fused and inserted into pET-28a (+) vector containing a C-terminal 6 × His-tag for facilitating structural analysis. And the codon-optimized sequence of T6 bacteriophage topoisomerase II large subunit (gp39, YP_010067157.1) was inserted into pET-28a (+) vector containing a C-terminal 6 × His-tag. T4 Topo II medium subunit (gp52, NP_049875.1) was constructed in the same way with a C-terminal 10 × His-tag. In addition, T4 gp39 and gp60 were inserted into pRSFDuet-1 vector (for the co-expression of two proteins) and overexpressed independently. These recombinant proteins were overexpressed in E. coli BL21 (DE3), inducing with 0.5 mM Isopropyl β-D-1-Thiogalactopyranoside (IPTG) at 18 °C overnight. The cell pellet of T4 gp39-gp60 fused protein, gp52 and T6 gp39 was suspended using Lysis Buffer containing 50 mM MES (pH 6.0), 300 mM NaCl, 2 mM TCEP and 10% glycerol, then sonicated for 45 min on ice. The lysate was centrifuged at 30,700 g for 40 min at 4 °C. The supernatant was incubated with Ni-NTA at 4 °C for 1 h in the column, then washed with Lysis Buffer plus 30 mM imidazole to remove unbound proteins and eluted with Lysis Buffer plus 500 mM imidazole. The eluted protein was concentrated and loaded on a Superdex 200 column (Cytiva) using SEC Buffer (same as Lysis Buffer but with 5 mM MgCl2 and without glycerol) before concentrating and storing at −80 °C. Purified T4 gp39-gp60 fused protein and T6 gp39 were incubated with T4 gp52, respectively, followed by a second size-exclusion chromatography in SEC Buffer (T4 gp52 and T6 gp52 differ by only two amino acids, P250S and N253D, which almost has no effect on T6 Topo II structure and function. So, we incubated T6 gp39 and T4 gp52 to constitute a chimeric T6 Topo II that is nearly the same as the natural product). The resulted full-length T4 and T6 Topo II were concentrated and stored at −80 °C for further use.
T6 Topo II ATPase domain (1 ~ 385) from large subunit (gp39, YP_010067157.1) was inserted into pET-28a (+) vector containing a C-terminal 6×His-tag. The recombinant protein was overexpressed and purified as the same as above, except changing the Lysis buffer to 50 mM Hepes (pH 7.2), 300 mM NaCl, 2 mM TCEP, 10% Glycerol, 5 mM MgCl2 (SEC buffer: 50 mM Hepes (pH 7.2), 150 mM NaCl, 2 mM TCEP, 5 mM MgCl2). Purified protein was concentrated and stored at −80 °C for crystal screening.
Nucleic acid preparation
DNA oligonucleotides were ordered from GenScript, China. Briefly, two 52 bp complementary DNA oligonucleotides were dissolved in DNase-free water at a molar ratio of 1: 132, and then annealed by incubating at 95 °C for 2 min and decreasing the temperature by 1 °C per minute to 4 °C. Native T4 DNA contains glycosylated hydroxymethyl cytosine, but for simplicity, they are not included in the DNA substrate used here.
Cryo-EM sample preparation
For apo state sample, purified full-length T4 and T6 Topo II were incubated at 0.8 mg/mL for 30 min on ice after adding non-hydrolysable ATP homolog, AMPPNP, to the final concentration of 1 mM. For DNA-binding sample, purified proteins were incubated with the 52 bp DNA duplex for 30 min on ice at a molar ratio of 1:2 before adding AMPPNP. For cleaved-DNA sample, m-AMSA was further added to the DNA-binding sample to the final concentration of 1 mM and incubated for 30 min at 30 °C. Then BS3 was added to the protein complex to the final concentration of 0.5 mM and incubated on ice for 30 min before centrifuging at 13,000 rpm for 30 min to remove potential aggregates (Supplementary Fig. 2, BS3 is a protein crosslinker used in this study to stabilize protein complexes and improve particle dispersibility, without affecting native protein structure or protein-DNA interaction64,65). 4 μL of the well-prepared protein complex was applied to the amorphous alloy film35 (R1.2/1.3, Au, 300 mesh) before glow-discharging for 30 s with hydrogen and oxygen. Finally, the grids were plunge-frozen for 3 ~ 6 s in liquid ethane with 100% chamber humidity and 4 °C using Vitrobot Mark IV (FEI) and stored in liquid nitrogen for screening and data collection.
Cryo-EM data collection
Cryo-EM micrographs were collected on FEI Titan Krios (300 kV) and Talos Arctica (200 kV) equipped with GIF-quantum energy filter (Gatan) and Gatan K2-summit detector using Serial EM software (http://bio3d.colorado.edu/SerialEM/) in super-resolution counting mode with a defocus range of −1.5 to −2.5 μm and a pixel size of 1.04 Å (300 kV) or 1.0 Å (200 kV). Each image was recorded with a total dose of 60 e-/Å2, fractioned into 32 frames. In total, 3934 micrographs of apo T4 Topo II were collected with a pixel size of 1.0 Å at 200 kV. Other images were all recorded with a pixel size of 1.04 Å at 300 kV, including 3076 micrographs of T4 Topo II bound with DNA, 2765, 3022 and 3183 micrographs of apo T6 Topo II, 3142 and 3432 micrographs of T6 Topo II bound with intact DNA as well as 3031 micrographs of T6 Topo II bound with cleaved DNA and m-AMSA.
Cryo-EM image processing
Basically, CryoSPARC v4.3.0 was used to process all micrographs and processing of each dataset followed a similar procedure66. All movie stacks of each dataset were subjected to motion correction using MotionCor267. The resulting mrc files were imported to CryoSPARC for contrast Transfer Function (CTF) estimation and micrographs with resolution better than 4 ~ 5 Å (depending on image quality of each dataset) were selected for subsequent auto picking. For simplicity, 400 micrographs were randomly separated for Blob Picker to obtain an initial set of particles, which were extracted and subjected to 2D classification to generate templates for the Template Picker of all selected micrographs. Then, all picked particles were extracted and subjected to several rounds of 2D classification to remove any potential junk particles and contaminations. The selected particles were used to do Ab-initio Reconstruction and Heterogeneous Refinement. The particles of the best 3D class were re-extracted and subjected to further Homogeneous Refinement and Non-uniform Refinement.
In detail, 3934 micrographs were used to select 2,320,720 particles, in which 634,311 good particles were selected to build four classes of ab-initio models and do Heterogenous Refinement. Finally, 278,632 good particles were used to obtain the best density map of T4 Topo II apo central domain structure at 3.62 Å using C2 symmetry. 3076 micrographs were used to select 2,758,338 particles, in which 729,866 good particles were selected to build four classes of ab-initio models and do Heterogenous Refinement. Finally, 269,648 good particles were used to obtain the best density map of T4 Topo II central domain bound with DNA structure at 3.20 Å using C1 symmetry. 3183 micrographs were used to select 1,553,757 particles, in which 327,205 good particles were selected to build three classes of ab-initio models and do Heterogenous Refinement. Finally, 125,156 good particles were used to obtain the best density map of T6 Topo II apo central domain structure at 3.93 Å using C2 symmetry. 3142 micrographs were used to select 2,505,974 particles, in which 1,122,496 good particles were selected to build four classes of ab-initio models and do Heterogenous Refinement. Finally, 713.922 good particles were used to obtain the best density map of T6 Topo II central domain bound with DNA structure at 2.81 Å using C1 symmetry. 3031 micrographs were used to select 2,601,779 particles, in which 989,991 good particles were selected to build four classes of ab-initio models and do Heterogenous Refinement. Finally, 459,464 good particles were used to obtain the best density map of T6 Topo II central domain bound with DNA and m-AMSA structure at 3.16 Å using C1 symmetry. 2765 micrographs were used to select 3,886,893 particles, in which 415,580 good particles were selected to build three classes of ab-initio models and do Heterogenous Refinement. Finally, 148,327 good particles were used to obtain the best density map of T6 Topo II ATPase domain bound with AMPPNP structure at 3.40 Å using C1 symmetry. 8970 micrographs were used to select 6,722,951 particles, in which 315,759 good particles were selected to build ten classes of ab-initio models and do Heterogenous Refinement. Finally, 39,189 good particles were used to obtain the best density map of T6 Topo II central-open structure at 6.12 Å using C2 symmetry. And full-length initial model was used as template for the reconstruction of T6 Topo II apo full-length structure. In total, 68,911 good particles were used to obtain the best density map at 6.80 Å using C1 symmetry. 3432 micrographs were used to select 2,380,539 particles, in which 39,324 good particles were selected to build five classes of ab-initio models and do Heterogenous Refinement. Finally, 11,206 good particles were used to obtain the best density map of T6 Topo II full-length structure bound with DNA at 6.73 Å using C1 symmetry. In addition, 11,206 good particles were used to obtain an initial model of T6 Topo II DNA-bound full-length structure with novel state (Supplementary Fig. 4a-i).
Crystallization, diffraction data collection and structural solution of T6 ATPase domain
Initial crystal screening was performed by sitting drop on the 96-well plate using crystal screen kit (Hampton Research). Purified T6 ATPase domain protein was concentrated to the final concentration of 5-15 mg/mL, then incubated with 1 mM AMPPNP (final concentration) on ice for 30 min. Then protein was mixed with well solutions at a volume ratio of 1:1 by mosquito (SPT Labtech). Best crystal was grown under the condition of 0.1 M succinic acid (pH 7.0) and 12% PEG3350. Crystals were transferred for three minutes to the reservoir solution plus 10% glycerol before looped and flash-frozen in liquid nitrogen. Diffraction data were collected at the rotating-anode X-ray source MicroMax 007/Satun 944 HG/Varimax HF at a wavelength of 1.5418 Å (Institute of Biophysics, Chinese Academy of Sciences, CAS). Collected data was processed using HKL200068 and molecular replacement (MR) was carried out using CCP4 v7.169 with AlphaFold2 predicted model38. The final MR solution contains a monomer in one asymmetric unit. Model building was carried out in COOT v0.9.736, followed by PHENIX v1.20.137 for further refinement and validation.
Model building and Refinement of cryo-EM structures
T4 Topo II gp39-gp60, gp52, and T6 Topo II gp39 were predicted as homodimers using AlphaFold v2.3.238, respectively. Predicated models were used as initial models for manual model building in UCSF chimera v1.17.170 and COOT36. Then real-space refinement and comprehensive validation in PHENIX37 were used for further optimization.
Relaxation assay
Relaxation activity of T6 Topo II was measured by using an increasing concentration of T6 Topo II incubating with 200 ng (~6 nM) supercoiled pUC19 plasmid (Solarbio) in a reaction mixture of 20 μL containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 6 mM MgCl2, 1 mM DTT, 100 μg/mL BSA and 1 mM ATP. The mixture was incubated at 37°C for 30 min. Then, 2 μL 10× DNA loading buffer was added and the final reaction mixture was loaded on a native agarose gel and electrophoresed with TAE buffer (40 mM Tris-Acetate, 1 mM EDTA) for 2 hours on ice at 60 V before being stained with StarStain Red Plus (GenStar) and imaged using UV transillumination (Gel DocTM EZ Imager, BIO-RAD). Reaction activity of T6 Topo II with different divalent ion (Mn2+, Ca2+, and Zn2+) was measured by the same method except for changing the divalent ion in the reaction buffer. At least two independent experiments were performed.
Relaxation activity of T4 Topo II was measured using the same method except for changing the reaction buffer to 50 mM Tris-HCl (pH 7.8), 40 mM KCl, 25 mM MgCl2, 0.5 mM DTT, 0.5 mM Na3EDTA, 30 μg/mL BSA and 1 mM ATP (referring to previous report19). At least two independent experiments were performed.
Cleavage assay
Cleavage activity was performed by similar method as relaxation assay except for omitting ATP in the reaction buffer (concentration of Mn2+ was 1 mM due to its high activity). The mixture was incubated at 37 °C for 1 h, then 1 μL 10% SDS was added to stop the reaction and Protease K (Sangon) was added to a finial concentration of 1 mg/mL to digest the protein at 37 °C for 1 h. The reaction mixture was electrophoresed and imaged as the same as the above except for 6 h electrophoresis at 60 V. At least two independent experiments were performed.
Inhibition Assay with m-AMSA
Inhibition assay was performed on the basis of relaxation assay. Briefly, a decreasing concentration of m-AMSA was prepared by diluting in DMSO and added to the reaction mixture containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 6 mM MgCl2, 1 mM DTT, 100 μg/mL BSA, 1 mM ATP, 60 nM protein and 200 ng (~ 6 nM) pUC19 plasmid. The mixture was incubated at 37 °C for 30 min. The final reaction mixture was electrophoresed and imaged as the same as relaxation assay. At least two independent experiments were performed.
Figure preparation
SEC figures were generated by GraphPad Prism v10.2.0. All structural model and cryo-EM density maps were generated using UCSF ChimeraX v1.771 and PyMOL v2.5.472. Multiple sequence alignment was obtained using Clustal X73 and ESPript74. Phylogenetic analysis was obtained by iTOL v675.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
Data availability
All structures and corresponding cryo-EM density maps of T4 & T6 bacteriophage Topo II have been deposited to the Protein Data Bank (PDB) and Electron Microscopy Data Bank (EMDB) under the accession codes 8YO3 and EMD-39434 for T4 Topo II apo central domain; 8YO4 and EMD-39435 for T4 Topo II central domain bound with DNA; 8YO5 and EMD-39436 for T6 Topo II apo central domain; 8YLU and EMD-39391 for T6 Topo II central domain bound with DNA; 8YO7 and EMD-39437 for T6 Topo II central domain bound with DNA and m-AMSA; 8YO1 and EMD-39433 for T6 Topo II ATPase domain; 8YO9 and EMD-39438 for T4 Topo II central-open structure; 8YOD and EMD-39444 for T6 Topo II apo full-length structure; 8YON and EMD-39454 for T6 Topo II full-length bound with DNA. Crystal structure of T6 Topo II ATPase domain has been deposited to the PDB under the accession code 9IMJ. Structures used for structural comparisons/analyzes have the following accession codes from the PDB: 6zy5, 6zy7, 6rkw, 8kgm, 4i3h, 1ei1, 1zxm, 5j5p. Source data are provided with this paper.
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Acknowledgements
We thank Jianguo zhang, Boling Zhu, Jitong Yan, Longlong zhang, Lihong Chen and Xiaojun Huang in the Center for Biological imaging (CBI) at Institute of Biophysics (IBP) for their assistance in cryo-EM data collection. We thank Yan Wu for his research assistant service. We thank Nan Wang, Jie Deng and Wangjun Fu for their assistance in cryo-EM data processing. We thank Ya Wang, Yi Han, Xianjin Ou for their technical assistance in crystal screening and data collection. All research described in this article is supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB37030200), NSFC General Program grant (32270179) and Research on the Application of Space Protein Molecular Assembly (YYWT-0901-EXP-10).
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Y.X. and R.X. conceived the study and designed the experiments; Y.X., R.X., and Y.Y. performed protein purification and cryo-EM sample preparation; Y.X. and R.X. performed cryo-EM data collection and processing. Y.X., R.X., and Y.Y. conducted biochemical experiments; Y.X., R.X., and J.C. refined and built the atomic models. Y.X., R.X., Y.Y., and J.C. wrote the manuscript together with X.L. and Y.C. Z.R and Y.C. supervised the project.
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Xin, Y., Xian, R., Yang, Y. et al. Structural and functional insights into the T-even type bacteriophage topoisomerase II. Nat Commun 15, 8719 (2024). https://doi.org/10.1038/s41467-024-53037-3
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DOI: https://doi.org/10.1038/s41467-024-53037-3
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