Structure of the topoisomerase VI-B subunit: implications for type II topoisomerase mechanism and evolution
2002; Springer Nature; Volume: 22; Issue: 1 Linguagem: Inglês
10.1093/emboj/cdg008
ISSN1460-2075
Autores Tópico(s)Neuroblastoma Research and Treatments
ResumoArticle2 January 2003free access Structure of the topoisomerase VI-B subunit: implications for type II topoisomerase mechanism and evolution Kevin D. Corbett Kevin D. Corbett Department of Molecular and Cellular Biology, University of California, Berkeley, 327 Hildebrand Hall 3206, Berkeley, CA, 94720 USA Search for more papers by this author James M. Berger Corresponding Author James M. Berger Department of Molecular and Cellular Biology, University of California, Berkeley, 327 Hildebrand Hall 3206, Berkeley, CA, 94720 USA Search for more papers by this author Kevin D. Corbett Kevin D. Corbett Department of Molecular and Cellular Biology, University of California, Berkeley, 327 Hildebrand Hall 3206, Berkeley, CA, 94720 USA Search for more papers by this author James M. Berger Corresponding Author James M. Berger Department of Molecular and Cellular Biology, University of California, Berkeley, 327 Hildebrand Hall 3206, Berkeley, CA, 94720 USA Search for more papers by this author Author Information Kevin D. Corbett1 and James M. Berger 1 1Department of Molecular and Cellular Biology, University of California, Berkeley, 327 Hildebrand Hall 3206, Berkeley, CA, 94720 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2003)22:151-163https://doi.org/10.1093/emboj/cdg008 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Type IIA and type IIB topoisomerases each possess the ability to pass one DNA duplex through another in an ATP-dependent manner. The role of ATP in the strand passage reaction is poorly understood, particularly for the type IIB (topoisomerase VI) family. We have solved the structure of the ATP-binding subunit of topoisomerase VI (topoVI-B) in two states: an unliganded monomer and a nucleotide-bound dimer. We find that topoVI-B is highly structurally homologous to the entire 40–43 kDa ATPase region of type IIA topoisomerases and MutL proteins. Nucleotide binding to topoVI-B leads to dimerization of the protein and causes dramatic conformational changes within each protomer. Our data demonstrate that type IIA and type IIB topoisomerases have descended from a common ancestor and reveal how ATP turnover generates structural signals in the reactions of both type II topoisomerase families. When combined with the structure of the A subunit to create a picture of the intact topoisomerase VI holoenzyme, the ATP-driven motions of topoVI-B reveal a simple mechanism for strand passage by the type IIB topoisomerases. Introduction DNA topoisomerases are a broad group of enzymes with the ability to manipulate the topological state of DNA. Through a complicated and still incompletely understood series of DNA breakage and rejoining steps, these enzymes can perform various reactions on DNA including decatenation, unknotting and modulation of superhelicity. DNA topoisomerases fall into two general classes, type I and type II, which are distinguished by their ability to cleave one or both strands of a DNA duplex, respectively (for reviews, see Wang, 1996, 2002; Champoux, 2001). Type II topoisomerases carry out strand passage by first generating a transient double-strand (ds) DNA break in a 'gate' or G-segment through nucleophilic attack on the DNA backbone and the formation of 5′-phosphotyrosyl enzyme–DNA linkages. The broken G-segment ends are then separated, a second duplex (the 'transfer', or T-segment) passed through the break, and the broken G-segment duplex resealed. The type II topoisomerase reaction is coupled to ATP binding and hydrolysis, which coordinates the sequential opening and closing of enzyme 'gates' that drive G-segment separation and T-segment transport (Roca and Wang, 1992, 1994). Type II topoisomerases can be divided into two sub-classes: type IIA and type IIB (Bergerat et al., 1997; Nichols et al., 1999). The type IIA enzymes are the predominant form and are found in some bacteriophages, viruses and archaea, and in all bacteria and eukaryotes. All type IIA topoisomerases are related to each other at the amino acid sequence level, though their oligomeric organization sometimes differs. For example, microbial type IIA topoisomerases are A2B2 heterotetramers, while the eukaryotic enzymes are homodimers of a B-A subunit fusion protein (Figure 1A). Catalytic functions within the type IIA enzymes are arrayed in a modular fashion, with the ATPase site located in the B region and the DNA binding/cleavage activity formed from domains in the B and A regions (Figure 1A). Figure 1.TopoVI-B′ monomer. (A) Domain organization of type II topoisomerases. Key functional modules of type IIA and IIB topoisomerases are labeled as follows: GHKL (yellow), CAP (green) and toprim (red). A conserved 'transducer' domain (Trans.) is shown in orange and the H2TH domain of topoVI-B′ is shown in pink. The split of the A and B regions in prokaryotic type IIA enzymes into discrete subunits is indicated by the double hash mark. The active-site tyrosines (yellow circles) are labeled as 'Y' and conserved acidic residues within the toprim fold are shown as blue circles. While the GHKL and toprim folds of type IIB topoisomerases are identifiable by sequence homology, the type IIB CAP region (striped green) has no sequence homology to the equivalent fold in type IIA proteins. Furthermore, the N- to C-terminal orientation of the CAP and toprim modules is swapped between the two type II topoisomerase families. (B) Structure-based sequence alignment of topoVI-B′ with the ATPase regions of E.coli GyrB and MutL. The secondary structural elements of topoVI-B′ are shown below the sequences. The three domains are colored as in (A). Residue numbering is that of topoVI-B′ followed by that of GyrB in parentheses. Highlighted in blue are the GHKL motifs identified by Bergerat et al. (1997). Highlighted in red is the invariant lysine (427 in topoVI-B′, 337 in GyrB) that contacts the γ-phosphate of bound nucleotide and highlighted in green is a highly conserved asparagine (375 in topoVI-B′, 271 in GyrB) that serves as an anchoring element between the GHKL and transducer domains. (C) Structure of the monomer (apo) form of topoVI-B′. Domains are colored as in (A). Secondary structural elements are labeled as in (B). (B) was generated by ALSCRIPT (Barton, 1993) and (C) with RIBBONS (Carson, 1991). Download figure Download PowerPoint Type IIB topoisomerases comprise a much smaller group than the type IIA enzymes, with topoisomerase VI as the sole representative of the family (Champoux, 2001). Topoisomerase VI enzymes are A2B2 heterotetramers found principally in archaea, though recent evidence has shown that they also exist in plants (Hartung and Puchta, 2001; Hartung et al., 2002; Sugimoto-Shirasu et al., 2002; Yin et al., 2002). The topoisomerase VI A subunit (topoVI-A) shares sparse sequence similarity with the type IIA topoisomerases, principally in a region required for DNA cleavage known as a 'toprim' domain (Aravind et al., 1998; Nichols et al., 1999). However, topoVI-A is not homologous to regions of type IIA enzymes outside the toprim domain, and instead shares general homology with Spo11, a protein responsible for creating dsDNA breaks to initiate meiotic recombination (Keeney et al., 1997; Celerin et al., 2000; Martini and Keeney, 2002). Indeed, the similarity between topoVI-A and Spo11 helped prove that the A subunit of type IIB topoisomerases contains the DNA binding and cleavage activities, and further, allowed identification of the active site tyrosine responsible for DNA cleavage (Bergerat et al., 1997). The only region of the topoisomerase VI B subunit (topoVI-B) held in common with type IIA enzymes at the amino acid sequence level is an ∼200 amino acid domain at its N-terminus that is homologous to the GHKL-type ATPases, a large family that also includes MutL, Hsp90 and CheA proteins (Bergerat et al., 1997; Dutta and Inouye, 2000). Since only sporadic sequence motifs are shared between the two type II topoisomerase families (Figure 1A), it has remained unclear whether type IIA and IIB enzymes are remote cousins of each other or represent distinct type II topoisomerase sub-classes. Structural studies of type IIB topoisomerases to date have only deepened this question. The structure of topoVI-A from Methanococcus jannaschii revealed that, in addition to the conserved toprim domain, the A subunit contains a helix–turn–helix fold of the catabolite activator protein (CAP) family of DNA-binding proteins, and that the active site tyrosine responsible for DNA cleavage resides on this domain (Nichols et al., 1999). In type IIA topoisomerases, the active site tyrosine is also located on a CAP fold (Berger et al., 1996), though on a different secondary structural element than in topoVI-A (Nichols et al., 1999). In order to maintain proper active site architecture, the positioning of the toprim and CAP domains is different in type IIB and type IIA topoisomerases, resulting in globally different subunit structures in the two enzyme families. The presence of the CAP and toprim domains in topoVI-A indicates that the DNA cleavage mechanisms of type IIA and type IIB topoisomerases are chemically similar. However, the major structural differences between the two families' DNA binding and cleavage subunits have left their evolutionary kinship open to debate. Ancestry aside, the physical mechanism by which type IIB topoisomerases pass one DNA duplex through another has likewise remained unresolved. The architecture of topoVI-A initially indicated that the G-segment would lie across the A subunit dimer interface, and that the two halves of the A subunit dimer would separate during G-segment opening to allow T-segment transport (Nichols et al., 1999). This model, combined with the observation that the GHKL domains of type IIA topoisomerases and MutL proteins dimerize upon nucleotide binding (Wigley et al., 1991; Ali et al., 1995; Ban et al., 1999), suggested that the B subunits might act as a nucleotide-dependent gate to 'bridge' the cleaved G-segment while the A subunit dimer was separated. The model further predicted that since strand passage is ATP dependent, the B subunits would physically transmit nucleotide-dependent conformational signals to the A subunits that would guide their separation to allow T-segment exit. It has not been demonstrated, however, whether the GHKL folds of the topoVI-B subunits self-associate in response to nucleotide binding, nor has it been shown how these subunits communicate with the A subunits. To address these mechanistic questions and to clarify the ancestral relationship of the type IIA and type IIB topoisomerases, we determined the three-dimensional structure of a truncated form of topoVI-B, both free and complexed to the ATP analog adenosine [β,γ-imido]triphosphate (AMP-PNP). Unexpectedly, our data reveal that topoVI-B is structurally homologous to the entire 40–43 kDa ATPase region of type IIA topoisomerases and MutL proteins. TopoVI-B contains not only an ATP-binding GHKL module, but also a domain that is known to be important in nucleotide hydrolysis and the transduction of structural signals from the ATP-binding site to the DNA breakage/reunion regions of these enzymes. Our data show that topoVI-B dimerizes in response to ATP binding and undergoes significant interdomain rearrangements centered about the active site pocket, which together explain how ATP stimulates G-segment cleavage and opening by the topoVI-A subunits. This work demonstrates that the type IIA and IIB topoisomerases are directly related evolutionarily, and that the nucleotide-dependent switching elements of GHKL ATPases are conserved. Results Apo structure During purification of the 530 amino acid full-length Sulfolobus shibatae topoVI-B subunit, a contaminating species was observed to co-purify with the full-length product. Mass spectrometry identified this protein as a proteolyzed fragment of topoVI-B that ended at amino acid 470. This truncation (topoVI-B′) was subsequently cloned, purified and crystallized without nucleotide in the space group P21212, with one protein monomer per asymmetric unit. The structure was solved to 2.0 Å resolution using multi-wavelength anomalous dispersion methods (MAD) with selenomethione-labeled protein. All residues in the final model were traceable directly from the solvent-flattened, experimentally determined electron density maps. The final model includes amino acids 10–96 and 98–470, and was refined to an R-factor of 21.4% and an Rfree of 23.9% (Table I). Table 1. Data collection, refinement and stereochemistry Data collection Native 1a Se-met peak 1 Se-met remote 1 Se-met (native) 2a Se-met peak 2 Se-met remote 2 Resolution (Å) 20 − 2.0 20 − 2.5 20 − 2.7 20 − 2.3 30 − 2.8 30 − 2.9 Wavelength (Å) 1.127 0.9793 1.078 1.100 0.9796 1.100 Space group P21212 P21212 I/σ (last shell) 16.7 (2.7) 13.1 (4.5) 13.3 (5.6) 10.0 (1.8) 16.2 (5.6) 16.7 (6.7) Rsymb (last shell) (%) 0.058 (0.405) 0.075 (0.211) 0.073 (0.171) 0.111 (0.473) 0.085 (0.234) 0.079 (0.177) Completeness (last shell) % 95.8 (90.6) 96.7 (94.9) 96.6 (93.8) 96.1 (88.9) 98.5 (87.3) 98.9 (93.4) No. of reflections 284 447 243 169 191 859 2 006 747 2 043 065 1 861 489 Unique 37 738 19 710 15 640 149 698 84 924 76 768 No. of sites – 5 5 – 30 30 Refinement Crystal form 1a Crystal form 2a Resolution (Å) 20 − 2.0 20 − 2.3 No. of reflections 34 608 137 434 Working 31 478 124 899 Free (% total) 3130 (8.3) 12 535 (8.4) Rworkc (last shell) (%) 21.4 (26.0) 21.4 (25.7) Rfreec (last shell) (%) 23.9 (30.2) 26.3 (30.7) Structure and stereochemistry Crystal form 1 Crystal form 2 No. of atoms 3932 23 098 Protein 3684 22 053 Water 246 853 Nucleotide 0 186 Ions 2 6 R.m.s.d. bond lengths (Å) 0.011 0.013 R.m.s.d. bond angles (°) 1.313 1.423 a Crystal form 1, unliganded monomer; crystal form 2, AMP-PNP-bound dimer. b Rsym = ΣΣj|Ij − |/ΣIj, where Ij is the intensity measurement for reflection j and is the mean intensity for multiply recorded reflections. c Rwork, free = Σ‖Fobs| − |Fcalc‖/|Fobs|, where the working and free R-factors are calculated using the working and free reflection sets, respectively. The free reflections were held aside throughout refinement. The topoVI-B′ protomer contains three domains (Figure 1C). The N-terminal domain (amino acids 10–229) consists of an eight-stranded mixed β-sheet backed on one side by five α-helices. The fold, as anticipated, belongs to the GHKL family of ATPases, which includes MutL, Hsp90, CheA-type histidine kinases and type IIA topoisomerases (Bergerat et al., 1997; Dutta and Inouye, 2000). The structure of this domain is particularly similar to the GHKL domain of Escherichia coli gyrase B (GyrB) (Wigley et al., 1991), a type IIA topoisomerase. The Cα r.m.s.d. between the GHKL regions of topoVI-B′ and GyrB is 1.17 Å for 112 core residues (Figure 2A), despite 50 amino acids long and extends from the body of the protein by several turns. Unexpectedly, this domain is structurally related to the C-terminal domain of both the 43 kDa GyrB and 40 kDa MutL ATPase fragments (Wigley et al., 1991; Ban and Yang, 1998), and is an example of an unusual left-handed βαβ crossover seen in proteins such as EF-G, ribosomal protein S5 and RNase P (Ramakrishnan and White, 1992; Ævarsson et al., 1994; Czworkowski et al., 1994; Murzin, 1995; Stams et al., 1998). In gyrase, this domain has been proposed to mediate intersubunit communication by structurally transducing signals from the ATP binding and hydrolysis site of the GHKL domain to the DNA binding and cleavage domains of the gyrase holoenzyme. The architecture of the topoVI-B′ 'transducer' domain is slightly more diverged from the equivalent region of GyrB than is the GHKL domain, but is still a very close structural match. The Cα r.m.s.d. for core secondary structural elements is 1.54 Å over 54 amino acids (Figure 2C), a structural similarity that is all the more remarkable given that <11% sequence identity exists between these two domains. The only point at which the structure of the transducer domain of topoVI-B′ differs significantly from that seen in GyrB is at its C-terminal-most α-helix. In GyrB, this helix bends back sharply towards a molecular two-fold axis to interact with a dimer-related helix, while in the topoVI-B′ monomer structure, this helix is straight. It does not appear that the sharp bend observed in the GyrB C-terminal helix could be accommodated by topoVI-B′. The GyrB C-terminal helix is anchored to the core of the transducer domain by a number of hydrophobic residues after the bend, whereas the topoVI-B′ helix becomes highly polar immediately upon its protrusion from the transducer domain core. Nucleotide-bound structure To understand the effects of ATP binding on topoVI-B, we co-crystallized topoVI-B′ with the non-hydrolyzable ATP analog AMP-PNP. These crystals belong to space group P21212 and contain six protein chains per asymmetric unit. The AMP-PNP form was solved to 2.3 Å resolution using MAD with selenomethionine-labeled protein. The final model contains 97% of all residues in the six protein chains (see Materials and methods), and was refined to an R-factor of 21.4% and an Rfree of 26.4%. The six protomers in the asymmetric unit superimpose closely, with a Cα r.m.s.d. of ∼0.5 Å for any given pair. The GHKL domains of type IIA topoisomerases and MutL proteins are known to dimerize in response to ATP binding. In the strand passage reaction of type IIA topoisomerases, this step represents the closure of the 'entry' or 'N-gate' of the enzyme (Osheroff, 1986; Roca and Wang, 1992; Ali et al., 1995). Strikingly, we observe that topoVI-B′ also dimerizes when bound to AMP-PNP, creating an interprotomer interaction that is very similar to that seen in the GyrB and MutL ATPase fragment structures (Figure 3; Wigley et al., 1991; Ban et al., 1999). This interaction is mediated almost entirely between the lateral sides of the GHKL domains of each monomer. Figure 3.Comparison of topoVI-B′, GyrB and MutL dimer structures. (A) Structure of the AMP-PNP bound topoVI-B′ dimer. Coloring is as in Figure 1C. Two views are shown: front (top) and top-down (bottom). Bound nucleotides and Mg2+ ions are shown in blue. (B) and (C) AMP-PNP-bound dimer structures of GyrB (Wigley et al., 1991) and MutL (Ban et al., 1999), respectively. The GHKL and transducer domains are colored as in (A), and the views are equivalent for all three proteins. Download figure Download PowerPoint The topoVI-B′ dimerization interaction also shares a second prominent structural feature with the GyrB and MutL dimer structures, in which the first 10–15 N-terminal amino acids of one protomer extend outward and latch onto the top of the GHKL domain of the opposite protomer. Each of these N-terminal 'straps' buries ∼570 Å2 of surface area. Additionally, 1630 Å2 of each GHKL domain, 530 Å2 of each H2TH domain and 550 Å2 of each transducer domain are buried upon dimerization. Mutational analysis of the GyrB N-terminal strap has identified residues that are critical for dimerization, including a conserved isoleucine (Ile10 in GyrB; Brino et al., 1997, 1999, 2000), which binds in a hydrophobic pocket and anchors the strap to the opposite protomer (Wigley et al., 1991). In topoVI-B′, Phe7 appears to play an analogous role, binding in the same hydrophobic pocket as Ile10 of GyrB. Interestingly, another strap residue shown to be important in GyrB, Tyr5, appears to have no functional counterpart in topoVI-B′. It remains to be seen whether the absence of this tyrosine is important to function in topoVI-B′. The conservation of dimerization geometry and the N-terminal strap in topoVI-B′ further emphasizes its mechanistic similarity to type IIA topoisomerases. The H2TH domains of topoVI-B′ also contribute to the dimer interface, packing against the GHKL domain of the dimer-related subunit. In the monomer form, this domain partially occludes the GHKL domain dimer interface, and it moves ∼9 Å upon dimerization to open up this surface. The role of the H2TH domains in the mechanism of topoVI is unknown, and topoVI-B does not share any obvious activities with Fpg/endoVIII glycosylases other than as a general DNA-binding protein (Melamede et al., 1994). One may speculate that this domain could assist with binding of DNA to the enzyme or, given its proximity to the ATP-binding pockets, could affect nucleotide turnover. In the structure of a nucleotide-bound dimer form of the 43 kDa GyrB ATPase fragment, the inner sides of the transducer domains, together with their kinked, C-terminal-most helices and the GHKL dimer interface, enclose a hole ∼20 Å in diameter (Figure 3B; Wigley et al., 1991). This hole has been proposed to accommodate a captured T-segment prior to the strand passage reaction (Wigley et al., 1991; Tingey and Maxwell, 1996). In the topoVI-B′ dimer structure, the space between the transducer domains is only ∼16 Å at its widest, and the C-terminal helices do not bend and cross over one another as seen in gyrase. As a result, there is no hole formed by the topoVI-B′ dimer that is analogous to that observed for the GyrB ATPase fragment. Because non-crystallographic symmetry in this crystal form affords three independent views of the topoVI-B′ dimer, and given that the polar character of its C-terminal transducer helices appears to preclude their bending back toward the two-fold axis of the dimer, it seems unlikely that the lack of the hole in topoVI-B′ is due to crystal packing artifacts. Rather, these features suggest that despite the overall architectural and catalytic similarity of topoVI-B′ to GyrB, the C-terminal end of topoVI-B has been adapted to interact with the altered DNA binding and cleavage machinery used by the topoVI-A subunit. Intra- and interdomain rearrangements mediated by nucleotide binding In addition to dimerization upon ATP binding, other structural changes are evident upon comparison of the AMP-PNP-bound and unbound topoVI-B′ structures. At the ATP-binding site, a number of side chain and main chain elements undergo local positional changes to become liganding groups for the bound nucleotide (Figure 4A). Asn42 binds a Mg2+ ion, which is also coordinated by the three phosphate groups of AMP-PNP and two water molecules; the two water molecules are in turn held in place by Glu38 and Glu41. Gly97 and Lys98, contained in a loop that is disordered in the unbound state, ligand the ribose and β-phosphate, respectively, while Asp76, Gly80 and Thr170 help position the adenine moiety. Finally, the glycine-rich P-loop (amino acids 107–111) moves by up to 6.4 Å from its position in the unbound state to form an extensive hydrogen bond network between four backbone amide nitrogens and the γ-phosphate of AMP-PNP (Figure 4A). Most of the residues involved in nucleotide contacts are part of the GHKL signature sequence motifs conserved in the type IIA topoisomerases and MutL proteins (Figure 1B), and are structurally analogous to nucleotide-binding residues in GyrB (Figure 4), illustrating the high degree of conservation in the nucleotide-binding pocket of the GHKL domains of these proteins. Figure 4.Comparison of topoVI-B′ and GyrB ATP-binding sites. (A) Stereo view of AMP-PNP coordination by topoVI-B′. Bound AMP-PNP is shown in magenta, protein side chains in gray, and the protein backbone and specific main chain bonds in white. Hydrogen bonds are represented as dotted lines. Carbon atoms are shown in green, nitrogen in blue (main-chain nitrogen atoms enlarged for visualization), oxygen in red, phosphorus in pink and bound Mg2+ in black. Amino acids are labeled with their one-letter code and residue number. (B) Stereo view of AMP-PNP coordination by GyrB. The view and the color scheme are essentially identical to that in (A). Residue Y5(B) resides on the N-terminal strap of the opposite GyrB protomer and has no analog in topoVI-B′. Download figure Download PowerPoint The local structure of the transducer domain is also affected by nucleotide binding. The most dramatic difference in this region is that the loop immediately N-terminal to the C-terminal-most helix of this domain moves translationally by 5–6 Å, concomitantly flipping Lys427 by 180° and inserting it into the ATP-binding pocket of the GHKL domain (Figure 5A). This configurational change positions the Nζ of Lys427 within hydrogen bonding distance of a non-bridging oxygen of the γ-phosphate of AMP-PNP. Rearrangements of this loop region, together with a conserved lysine contacting an oxygen of the γ-phosphate, have also been observed in GyrB and MutL (Wigley et al., 1991; Ban and Yang, 1998; Ban et al., 1999; Lamour et al., 2002). Figure 5.Nucleotide binding induces structural changes in topoVI-B′. (A) Least-squares superposition of the topoVI-B′ ATP-binding site in both nucleotide states, highlighting γ-phosphate interactions. The apo form of the protein is colored white, while the AMP-PNP-bound form is colored as in Figure 1C, with the GHKL domain yellow and the transducer domain orange. The 'switch' lysine (K427) is observed to flip positions and hydrogen bond the γ-phosphate of bound AMP-PNP. (B) Superposition of topoVI-B′ monomers in both nucleotide-bound and unbound states. Only the GHKL domains were included for superposing the two states. Two views are shown, rotated 90° relative to each other. The apo form is colored gray and the AMP-PNP-bound form is colored as in Figure 1C. The 15 Å motion of the distal end of the C-terminal α-helix is depicted with an arrow (right panel), as is the 9 Å motion of the H2TH domain (left panel). (C) Model for topoVI-B′ transducer motions in a dimerized state. A 'closed' AMP-PNP-bound GHKL/transducer domain orientation (left) is shown next to a modeled 'open' configuration (right). The 'open' model was created by combining the GHKL/transducer domain orientations seen in the topoVI-B′ apo structure with the GHKL/H2TH dimer geometry of the AMP-PNP-bound structure. The space between transducer domains increases dramatically in this configuration; the distances shown are between Cα atoms of Glu366 (upper arrows) and Lys466 (lower arrows). Download figure Download PowerPoint The motion of the loop containing Lys427 has significant structural consequences for the relative positioning of domains in topoVI-B′. Upon nucleotide binding and dimerization, the transducer domain rotates ∼11° relative to its position in the apo structure, moving the distal end of the C-terminal helix by ∼15 Å (Figure 5B). Each of the three copies of the dimer in the asymmetric unit displays an identical orientation of all three domains, indicating that
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