FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2031674105> ?p ?o ?g. }

• W2031674105 endingPage "354" @default.
• W2031674105 startingPage "343" @default.
• W2031674105 abstract "Although smallpox has been eradicated from the human population, it is presently feared as a possible agent of bioterrorism. The smallpox virus codes for its own topoisomerase enzyme that differs from its cellular counterpart by requiring a specific DNA sequence for activation of catalysis. Here we present crystal structures of the smallpox virus topoisomerase enzyme bound both covalently and noncovalently to a specific DNA sequence. These structures reveal the basis for site-specific DNA recognition, and they explain how catalysis is likely activated by formation of a specific enzyme-DNA interface. Unexpectedly, the poxvirus enzyme uses a major groove binding α helix that is not present in the human enzyme to recognize part of the core recognition sequence and activate the enzyme for catalysis. The topoisomerase-DNA complex structures also provide a three-dimensional framework that may facilitate the rational design of therapeutic agents to treat poxvirus infections. Although smallpox has been eradicated from the human population, it is presently feared as a possible agent of bioterrorism. The smallpox virus codes for its own topoisomerase enzyme that differs from its cellular counterpart by requiring a specific DNA sequence for activation of catalysis. Here we present crystal structures of the smallpox virus topoisomerase enzyme bound both covalently and noncovalently to a specific DNA sequence. These structures reveal the basis for site-specific DNA recognition, and they explain how catalysis is likely activated by formation of a specific enzyme-DNA interface. Unexpectedly, the poxvirus enzyme uses a major groove binding α helix that is not present in the human enzyme to recognize part of the core recognition sequence and activate the enzyme for catalysis. The topoisomerase-DNA complex structures also provide a three-dimensional framework that may facilitate the rational design of therapeutic agents to treat poxvirus infections. Smallpox is caused by the variola virus, a member of the Poxviridae virus family. The virus is highly transmissible with infection typically resulting in 20%–30% mortality, making it one of the most severe infectious diseases known to humans. The efficiency with which it spreads, combined with the deadly nature of the disease, has raised fears that smallpox could be revived for use in bioterrorism (Harrison et al., 2004Harrison S.C. Alberts B. Ehrenfeld E. Enquist L. Fineberg H. McKnight S.L. Moss B. O'Donnell M. Ploegh H. Schmid S.L. et al.Discovery of antivirals against smallpox.Proc. Natl. Acad. Sci. USA. 2004; 101: 11178-11192Crossref PubMed Scopus (69) Google Scholar). Structural models of smallpox virus proteins could provide the basis for rational design of antiviral agents, but few high-resolution structures of intact proteins from variola or related viruses have so far been reported (Moss, 2001Moss B. Poxviridae: the viruses and their replication.in: Fields B.N. Virology. Lippincott-Raven, Philadelphia2001: 2637-2672Google Scholar). Poxviruses are large, double-stranded DNA viruses that carry out their replication cycles entirely in the cytoplasm of infected cells. These viruses consequently encode many of the enzymes required to replicate and transcribe their genomes. Among these is a type IB topoisomerase, which is required for efficient transcription of the viral DNA (Da Fonseca and Moss, 2003Da Fonseca F. Moss B. Poxvirus DNA topoisomerase knockout mutant exhibits decreased infectivity associated with reduced early transcription.Proc. Natl. Acad. Sci. USA. 2003; 100: 11291-11296Crossref PubMed Scopus (48) Google Scholar). Type IB topoisomerase (TopIB) enzymes introduce transient breaks in one of the two strands of duplex DNA, allowing rotation of the flanking duplexes about the uncleaved strand (Figure 1A). These enzymes play critical roles in processes such as transcription, replication, and repair by relieving the topological stress caused by underwinding or overwinding of the DNA double helix that occurs during these events (Shuman, 1998Shuman S. Vaccinia virus DNA topoisomerase: a model eukaryotic type IB enzyme.Biochim. Biophys. Acta. 1998; 1400: 321-339Crossref PubMed Scopus (89) Google Scholar, Wang, 1996Wang J.C. DNA topoisomerases.Annu. Rev. Biochem. 1996; 65: 635-692Crossref PubMed Scopus (2015) Google Scholar). The highly conserved poxvirus TopIBs are unique in several respects. They are among the smallest topoisomerases known, at only 34 kDa. Unlike the related eukaryotic cellular TopIB, which exhibits only a weak preference for certain DNA sequences (Been et al., 1984Been M.O. Burgess R.R. Champoux J.J. Nucleotide sequence preferences at rat liver and wheat germ type 1 DNA topoisomerase breakage sites in duplex SV40 DNA.Nucleic Acids Res. 1984; 12: 3097-3114Crossref PubMed Scopus (180) Google Scholar), the viral enzymes relax their substrates at specific DNA sites containing the core pentamer, 5′-(T/C)CCTT-3′ (Hwang et al., 1998Hwang Y. Wang B. Bushman F.D. Molluscum contagiosum virus topoisomerase: purification, activities and response to inhibitors.J. Virol. 1998; 72: 3401-3406Crossref PubMed Google Scholar, Shuman and Prescott, 1990Shuman S. Prescott J. Specific DNA cleavage and binding by vaccinia virus DNA topoisomerase I.J. Biol. Chem. 1990; 265: 17826-17836Abstract Full Text PDF PubMed Google Scholar). Since topoisomerase activity requires the presence of the proper recognition sequence (Hwang et al., 1999aHwang Y. Burgin A. Bushman F.D. DNA contacts stimulate catalysis by a poxvirus topoisomerase.J. Biol. Chem. 1999; 274: 9160-9168Crossref PubMed Scopus (18) Google Scholar, Shuman and Prescott, 1990Shuman S. Prescott J. Specific DNA cleavage and binding by vaccinia virus DNA topoisomerase I.J. Biol. Chem. 1990; 265: 17826-17836Abstract Full Text PDF PubMed Google Scholar, Tian et al., 2004Tian L. Claeboe C.D. Hecht S.M. Shuman S. Remote phosphate contacts trigger assembly of the active site of DNA topoisomerase IB.Structure (Camb.). 2004; 12: 31-40Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, Wittschieben and Shuman, 1997Wittschieben J. Shuman S. Mechanism of DNA transesterification by vaccinia topoisomerase: catalytic contributions of essential residues Arg-130, Gly-132, Tyr-136 and Lys-167.Nucleic Acids Res. 1997; 25: 3001-3008Crossref PubMed Scopus (66) Google Scholar), this raises important mechanistic questions about how catalysis is coupled to sequence-specific recognition in the poxvirus enzymes. Extensive biochemical studies have been carried out to explore this issue (Koster et al., 2005Koster D.A. Croquette V. Dekker C. Shuman S. Dekker N.H. Friction and torque govern the relaxation of DNA supercoils by eukaryotic topoisomerase IB.Nature. 2005; 434: 671-674Crossref PubMed Scopus (242) Google Scholar, Nagarajan et al., 2005Nagarajan R. Kwon K. Nawrot B. Stec W.J. Stivers J.T. Catalytic phosphoryl interactions of topoisomerase IB.Biochemistry. 2005; 44: 11476-11485Crossref PubMed Scopus (20) Google Scholar, Shuman, 1998Shuman S. Vaccinia virus DNA topoisomerase: a model eukaryotic type IB enzyme.Biochim. Biophys. Acta. 1998; 1400: 321-339Crossref PubMed Scopus (89) Google Scholar), and structures of the isolated domains of the vaccinia virus enzyme have been reported (Cheng et al., 1998Cheng C. Kussie P. Pavletich N. Shuman S. Conservation of structure and mechanism between eukaryotic topoisomerase I and site-specific recombinases.Cell. 1998; 92: 841-850Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar, Sharma et al., 1994Sharma A. Hanai R. Mondragon A. Crystal structure of the amino-terminal fragment of vaccinia virus DNA topoisomerase I at 1.6 A resolution.Structure. 1994; 2: 767-777Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Despite a wealth of biochemical and structural data, however, progress in understanding this system has been limited by lack of structural data for the poxvirus TopIB-DNA complex. In order to establish a framework for understanding the unique features of the poxvirus topoisomerase, we have determined the crystal structures of two variola virus topoisomerase-DNA complexes, representing the noncovalent and covalent reaction intermediates shown in Figure 1A. We first crystallized variola TopIB (vTopIB) with a 13 bp DNA duplex containing the conserved core sequence 5′-CCCTT and optimized flanking sequences (Hwang et al., 1999aHwang Y. Burgin A. Bushman F.D. DNA contacts stimulate catalysis by a poxvirus topoisomerase.J. Biol. Chem. 1999; 274: 9160-9168Crossref PubMed Scopus (18) Google Scholar). Upon cleavage of this substrate by vTopIB (Figure 1B), the trinucleotide on the 3′ side of the cleavage site was released from the complementary strand and diffused out of the active site, trapping a covalently linked topoisomerase-DNA complex (Nunes-Duby et al., 1987Nunes-Duby S.E. Matsumoto L. Landy A. Site-specific recombination intermediates trapped with suicide substrates.Cell. 1987; 50: 779-788Abstract Full Text PDF PubMed Scopus (184) Google Scholar). An essential step in obtaining well-diffracting crystals was the substitution of two nonconserved surface cysteine residues by serine to eliminate intermolecular disulfide bond formation. As described later, this C100S, C211S mutant is nearly as active as the wild-type enzyme in plasmid relaxation assays. The structure of the covalent vTopIB-DNA complex was determined at 2.9 Å using multiwavelength anomalous scattering from selenomethionine-substituted enzyme and then refined to a final resolution of 2.7 Å. Crystallographic data are summarized in Table 1, and representative electron density is shown in Figures 1C and 1D.Table 1Summary of Crystallographic DataNoncovalentCovalent (Native)Covalent (SeMet MAD)Resolution1.9 Å2.7 Å2.9 ÅSpace groupC2221C2221C2221Cell constants (Å)a = 66.2a = 68.6a = 68.5b = 133.7b = 137.0b = 137.3c = 113.0c = 113.2c = 112.8Wavelength (Å)1.03320.979170.979520.979350.96394Completeness (%)96.2 (89.1)99.7 (99.6)97.5 (98.7)97.7 (99.0)97.7 (99.0)Rmerge0.068 (0.532)0.038 (0.285)0.069 (0.335)0.071 (0.347)0.069 (0.354)Total Reflections422,113312,043311,572323,269315,893Unique Reflections36,40314,32011,74711,77311,768I/σ27.0 (1.57)28.1 (8.23)26.15 (4.88)27.24 (4.96)25.66 (4.73)Redundancy4.2 (2.1)4.3 (4.3)5.8 (5.9)6.0 (6.1)6.0 (6.1)MAD Phasing (SOLVE)Z score32.02Figure of merit0.55Number of sites found9Resolution3.0 ÅRefinementNoncovalentCovalent (Native)Rfree0.243 (0.313)0.237 (0.370)Rwork0.197 (0.273)0.191 (0.315)Number of atoms Protein26292581 DNA510509 Water39554Average B factors (Å2) Protein43.0368.98 DNA43.1670.16 Water49.9666.36Rmsd Bond lengths (Å)0.0140.011 Bond angles (°)1.7041.530Numbers in parentheses represent values in highest-resolution shell. Open table in a new tab Numbers in parentheses represent values in highest-resolution shell. Based on these results, we designed a DNA substrate to mimic the cleavage product of the 13 bp duplex, where the scissile phosphate is present as an uncleavable 3′-terminal phosphate group. Crystallization of this substrate with vTopIB led to formation of a noncovalent vTopIB-DNA complex in a nearly isomorphous crystal lattice. These crystals diffracted to 1.9 Å resolution, which allowed us to clearly visualize solvent molecules in the protein/DNA interface and in the active site. The structure was refined to conventional R and Rfree values of 0.244 and 0.197, respectively (Table 1). Electron density for the noncovalent complex is shown in Figure S1 in the Supplemental Data available with this article online. Both vTopIB-DNA complex structures have been deposited with the Protein Data Bank, with accession codes 2H7F (covalent complex) and 2H7G (noncovalent complex). The covalent and noncovalent smallpox vTopIB-DNA complex structures are similar throughout, with large differences present only in the active site of the enzyme (rmsd 0.90 Å, excluding residues 264–288). The topoisomerase is folded into two domains, as anticipated from previous work on related poxviruses (Cheng et al., 1998Cheng C. Kussie P. Pavletich N. Shuman S. Conservation of structure and mechanism between eukaryotic topoisomerase I and site-specific recombinases.Cell. 1998; 92: 841-850Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar, Cheng and Shuman, 1998Cheng C. Shuman S. A catalytic domain of eukaryotic DNA topoisomerase I.J. Biol. Chem. 1998; 273: 11589-11595Crossref PubMed Scopus (27) Google Scholar, Hwang et al., 1999bHwang Y. Park M. Fischer W.H. Bushman F. Domain structure of the type-1B topoisomerase encoded by molluscum contagiosum virus.Virology. 1999; 262: 479-491Crossref PubMed Scopus (11) Google Scholar, Sharma et al., 1994Sharma A. Hanai R. Mondragon A. Crystal structure of the amino-terminal fragment of vaccinia virus DNA topoisomerase I at 1.6 A resolution.Structure. 1994; 2: 767-777Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). The two protein domains bind on either side of the core 5′-CCCTT-3′ sequence, forming a C-shaped clamp around the DNA (Figure 2A), as originally proposed based on biochemical data (Sekiguchi and Shuman, 1994Sekiguchi J. Shuman S. Vaccinia topoisomerase binds circumferentially to DNA.J. Biol. Chem. 1994; 269: 31731-31734Abstract Full Text PDF PubMed Google Scholar). A secondary structure assignment for the full-length smallpox topoisomerase in the DNA bound structures versus those found in the isolated domains is provided in Figure S2. The amino-terminal domain (N domain) is composed of a twisted, five-stranded antiparallel β sheet (β1–β5) with two short α helices (α1 and α2). The β5 strand of this domain is bound deeply in the major groove of the core DNA sequence, where it makes extensive direct contacts with the bases. There are very few changes in secondary or tertiary structure that occur upon DNA binding, based on comparison with the isolated N domain from vaccinia TopIB (Sharma et al., 1994Sharma A. Hanai R. Mondragon A. Crystal structure of the amino-terminal fragment of vaccinia virus DNA topoisomerase I at 1.6 A resolution.Structure. 1994; 2: 767-777Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Superposition of the DNA bound variola N domain and the unbound vaccinia N domain results in an rmsd of 0.7 Å for Cα atoms. The larger catalytic domain of vTopIB is centered on the opposite, minor groove face of the core DNA sequence, and the two domains are connected by the α3 helix. The long and sharply bent α3 helix forms the side of the C-shaped clamp (Figure 2A), passing along the DNA near positions +1 and +2, where the side chains of His76 and Arg80 contact the phosphate backbone (Figures 2A and 2B). Although the topoisomerase “clamp” formed around the core recognition site appears to be open on one face, a salt bridge between Lys65 in the β4–β5 hairpin and Glu139 in the α5 helix links the two domains in the noncovalent complex to fully encircle the DNA (Figure 2B). This salt bridge is not present in the covalent complex, due to an alternative choice of hydrogen bonding partners for Lys65 and Glu139. There are no significant changes in backbone conformation in these regions, indicating that the covalent and noncovalent complexes do not differ by a domain-level opening or closing of the protein clamp around the DNA substrate. In the human TopIB-DNA (hTopIB-DNA) complex, the protein forms a more substantially closed clamp around the DNA through interactions between loops arising from N-terminal subdomain I and the catalytic domain (Figure 2C). These interacting loops were originally referred to as the “Lips” of the topoisomerase (Redinbo et al., 1998Redinbo M.R. Stewart L. Kuhn P. Champoux J.J. Hol W.G.J. Crystal structures of human topoisomerase I in covalent and noncovalent complexes with DNA.Science. 1998; 279: 1504-1513Crossref PubMed Scopus (739) Google Scholar, Stewart et al., 1998Stewart L. Redinbo M.R. Qiu X. Hol W.G.J. Champoux J.J. A model for the mechanism of human topoisomerase I.Science. 1998; 179: 1534-1541Crossref Scopus (590) Google Scholar) and were more recently designated “Lip1” and “Lip2,” respectively (Patel et al., 2006Patel A. Shuman S. Mondragon A. Crystal structure of bacterial type IB DNA topoisomerase reveals a preassembled active site in the absence of DNA.J. Biol. Chem. 2006; 281: 6030-6037Crossref PubMed Scopus (21) Google Scholar). The poxvirus enzymes do not contain sequences corresponding to the Lip1 region. As shown in Figures 2B and 2C, there is also no structural equivalent of Lip2 in the vTopIB-DNA complex. In the structure of the isolated vaccinia TopIB catalytic domain (Cheng et al., 1998Cheng C. Kussie P. Pavletich N. Shuman S. Conservation of structure and mechanism between eukaryotic topoisomerase I and site-specific recombinases.Cell. 1998; 92: 841-850Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar) and in the structure of the uncomplexed D. radiodurans TopIB (drTopIB) (Patel et al., 2006Patel A. Shuman S. Mondragon A. Crystal structure of bacterial type IB DNA topoisomerase reveals a preassembled active site in the absence of DNA.J. Biol. Chem. 2006; 281: 6030-6037Crossref PubMed Scopus (21) Google Scholar), the residues in the region corresponding to Lip2 are disordered. As shown in Figure 2B and discussed in more detail below, the corresponding region in vTopIB folds into an α helix when bound to DNA, and this helix plays a role in specific DNA recognition. Overall, the vTopIB catalytic domain is primarily α-helical (α4–α12) but contains a small, three-stranded β sheet (β6–β8) that is highly conserved among the type IB topoisomerases and the tyrosine recombinases (Patel et al., 2006Patel A. Shuman S. Mondragon A. Crystal structure of bacterial type IB DNA topoisomerase reveals a preassembled active site in the absence of DNA.J. Biol. Chem. 2006; 281: 6030-6037Crossref PubMed Scopus (21) Google Scholar, Redinbo et al., 1999aRedinbo M.R. Champoux J.J. Hol W.G. Structural insights into the function of type IB topoisomerases.Curr. Opin. Struct. Biol. 1999; 9: 29-36Crossref PubMed Scopus (54) Google Scholar, Van Duyne, 2002Van Duyne G.D. A structural view of tyrosine recombinase site-specific recombination.in: Craig N.L. Craigie R. Gellert M. Lambowitz A.M. Mobile DNA II. ASM Press, Washington, D.C.2002: 93-117Google Scholar). A large structural reorganization of this domain occurs upon DNA binding relative to the structure of the unliganded catalytic domain (Cheng et al., 1998Cheng C. Kussie P. Pavletich N. Shuman S. Conservation of structure and mechanism between eukaryotic topoisomerase I and site-specific recombinases.Cell. 1998; 92: 841-850Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar), with an rmsd of 3.5 Å for residues 81–310 (Figure S3 and Movie S1). The conformational change can be described as a 23° rotation of the segment spanning helices α4–α7 and the β sheet (Lobe1 in Figure S3), relative to the segment that includes helices α8–α12 (Lobe2). This subdomain rotation is crucial to formation of the enzyme active site, since the catalytic tyrosine (Tyr274) is located in Lobe2 and moves by 3.6 Å (Cα atom) upon formation of the complex with DNA. The catalytic domain forms an extensive interface with the DNA substrate upstream of the cleavage site (base pairs +1 to +9), including minor groove interactions near the active site and major groove interactions involving the α5 helix (Figure 2A). The DNA residues downstream of the cleavage site on the cleaved strand (positions −1 to −3) were lost in the process of trapping the covalent TopIB-DNA complex (Figure 1B); thus we cannot directly observe interactions that are present between the enzyme and the downstream sequence. However, the 5′ overhang in the DNA substrate produced as a result of cleavage interacts with a symmetry-related copy of itself in the crystal lattice, taking the place of the lost trinucleotide that would normally be present 3′ of the cleavage site. The enzyme makes a number of contacts with the resulting pseudocontinuous DNA duplex in this region via the α10a and α10b helices, strongly suggesting that the downstream DNA is contacted to at least the −2 position (data not shown). A similar conclusion was reached by modeling the DNA in the noncovalent complex as an extended DNA duplex. In the related hTopIB-DNA complex, extensive contacts are made to the downstream DNA by the coiled-coil linker (residues 636–712) and the larger N-terminal subdomains (residues 215–433), leading to a model in which these contacts control DNA rotation in the covalent intermediate (Redinbo et al., 1999aRedinbo M.R. Champoux J.J. Hol W.G. Structural insights into the function of type IB topoisomerases.Curr. Opin. Struct. Biol. 1999; 9: 29-36Crossref PubMed Scopus (54) Google Scholar, Stewart et al., 1998Stewart L. Redinbo M.R. Qiu X. Hol W.G.J. Champoux J.J. A model for the mechanism of human topoisomerase I.Science. 1998; 179: 1534-1541Crossref Scopus (590) Google Scholar). In the much smaller poxvirus enzymes, neither the coiled-coil linker nor the additional N-domain sequences are present, indicating that control of rotation in this system (Koster et al., 2005Koster D.A. Croquette V. Dekker C. Shuman S. Dekker N.H. Friction and torque govern the relaxation of DNA supercoils by eukaryotic topoisomerase IB.Nature. 2005; 434: 671-674Crossref PubMed Scopus (242) Google Scholar, Stivers et al., 1997Stivers J.T. Harris T.K. Mildvan A. Vaccinia DNA topoisomerase I: evidence supporting a free rotation mechanism for DNA supercoil relaxation.Biochemistry. 1997; 36: 5212-5222Crossref PubMed Scopus (88) Google Scholar) must involve a different mechanism. The core sequence that is recognized by the poxvirus topoisomerases is 5′-(T/C)CCTT-3′, where cleavage occurs at the phosphate following the 3′-terminal thymidine (Figure 1B). In the covalent and noncovalent vTopIB/DNA crystal structures, the β5 strand in the amino-terminal domain and the α5 helix in the catalytic domain form an extensive network of major groove contacts to this core sequence (Figure 3). The side chains of residues Tyr70 and Tyr72 from β5 lie flat along the major groove, with Tyr70 covering the Cyt+3 and Cyt+4 bases and Tyr72 stacking on both the +3 ribose ring and the Thy+2 base (Figure 3A). Both tyrosine side chains also hydrogen bond to the phosphate backbone. This intimate interface explains previous observations that these residues in the vaccinia TopIB could be crosslinked to cytosines in the core DNA substrate (Sekiguchi and Shuman, 1996Sekiguchi J. Shuman S. Identification of contacts between topoisomerase I and its target DNA by site-specific photocrosslinking.EMBO J. 1996; 15: 3448-3457Crossref PubMed Scopus (46) Google Scholar). A third direct contact from β5 involves Gln69, which makes a classic double hydrogen bonding interaction with Ade+2 (Figure 3A). Together, the major groove contacts involving the β5 strand explain the high degree of specificity for the +2 to +4 positions of the core recognition sequence. It is interesting to note that subdomain I of human TopIB shares some similarity in structure with the N domain of poxvirus TopIB, including the placement of a β strand in the major groove of the DNA target (Redinbo et al., 1998Redinbo M.R. Stewart L. Kuhn P. Champoux J.J. Hol W.G.J. Crystal structures of human topoisomerase I in covalent and noncovalent complexes with DNA.Science. 1998; 279: 1504-1513Crossref PubMed Scopus (739) Google Scholar). However, in the hTopIB-DNA complex, this β strand is shifted out of the groove by ∼3 Å relative to the position observed in the vTopIB-DNA complex, thereby preventing direct contacts to the bases. With one exception, there is little sequence similarity in this region between the poxvirus and eukaryotic cellular TopIB families. Remarkably, Tyr70 is conserved in both families of enzymes, despite playing a different role in complex formation. In the hTopIB-DNA complex, the corresponding residue (Tyr426) is both shifted out of the major groove and rotated so that it interacts only with the flanking ribose and phosphate groups (Figure 2C). The α5 helix from the vTopIB catalytic domain also forms a complex network of contacts to the major groove of the DNA substrate (Figure 3B). In this case, water molecules play a more prominent role, forming numerous bridging hydrogen bonds that are readily visualized in the high resolution noncovalent enzyme-DNA complex (data not shown). In the core recognition sequence, Tyr136 packs against the +3 sugar and hydrogen bonds to N7 of Gua+4, while Lys133 hydrogen bonds to both the N7 and O6 atoms of guanine in position +5. In the case of Lys133, it seems likely that a minor adjustment of the side chain would allow it to interact primarily with the N7 atom of an adenine base in the +5 position, explaining the more relaxed requirement for either Thy or Cyt on the opposite strand. Outside of the core recognition sequence, Lys135 from this helix hydrogen bonds to N7 of the +6 Gua base. The observation of helix α5 in the vTopIB-DNA complex was not expected. This region (residues 133–143) is disordered in the structures of the vaccinia TopIB catalytic domain and the drTopIB protein. It was logical to assume that, upon binding DNA, these residues would form an ordered loop analogous to the Lip2 segment in the human TopIB/DNA structures (Figure 2C) and that this loop would interact primarily with the sugar-phosphate backbone of the DNA (Cheng et al., 1998Cheng C. Kussie P. Pavletich N. Shuman S. Conservation of structure and mechanism between eukaryotic topoisomerase I and site-specific recombinases.Cell. 1998; 92: 841-850Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar, Patel et al., 2006Patel A. Shuman S. Mondragon A. Crystal structure of bacterial type IB DNA topoisomerase reveals a preassembled active site in the absence of DNA.J. Biol. Chem. 2006; 281: 6030-6037Crossref PubMed Scopus (21) Google Scholar). Instead, this region of poxvirus TopIB folds into an α helix and docks in the major groove where it interacts with both the bases and the backbone. The poxvirus TopIB enzyme therefore achieves its specificity for the core recognition sequence through the N domain β5 and the C domain α5 interactions with bases in the major groove. The β5 interactions specify positions +2, +3, and +4, and the α5 interactions specify positions +4 and +5. The sequence chosen for the region upstream of the core recognition site (positions +6 to +9) in these structural studies was based on identification of an optimal target for poxvirus topoisomerases (Hwang et al., 1999aHwang Y. Burgin A. Bushman F.D. DNA contacts stimulate catalysis by a poxvirus topoisomerase.J. Biol. Chem. 1999; 274: 9160-9168Crossref PubMed Scopus (18) Google Scholar). In addition to the Lys135 interaction discussed above, vTopIB makes direct contacts to bases in this region via Arg206 and Tyr209 in the α7 helix (Figure 3c). Arg206 makes a canonical bidentate hydrogen bonding interaction with Gua+9, representing the most upstream contact between enzyme and substrate that we observe. Tyr209 makes van der Waals contact with the +6 Cyt base. As with other specific protein-DNA complexes, there are numerous polar and nonpolar interactions between the vTopIB enzyme and the sugar-phosphate backbone of the DNA duplex. All of the direct vTopIB/DNA interactions observed in the noncovalent complex are summarized schematically in Figure S4. In addition to the specific interface formed by the β5 and α5 motifs discussed above, the TopIB active site may also contribute to DNA sequence specificity. The side chain of Lys167 hydrogen bonds to O2 of Thy+1 in the minor groove, an interaction that is similar to that seen in hTopIB-DNA complexes (Champoux, 2001Champoux J.J. DNA topoisomerases: structure, function, and mechanism.Annu. Rev. Biochem. 2001; 70: 369-413Crossref PubMed Scopus (2041) Google Scholar) and in the tyrosine recombinases (Van Duyne, 2002Van Duyne G.D. A structural view of tyrosine recombinase site-specific recombination.in: Craig N.L. Craigie R. Gellert M. Lambowitz A.M. Mobile DNA II. ASM Press, Washington, D.C.2002: 93-117Google Scholar). On the major groove face of the same +1 base pair, Arg80 from the α3 helix stacks its aromatic guanidino group on the C5-methyl groups of Thy+1 and Thy+2. Together, the Lys167 and Arg80 interactions may explain the preference for Thy in the +1 position. The availability of several human TopIB-DNA complex structures (Redinbo et al., 1998Redinbo M.R. Stewart L. Kuhn P. Champoux J.J. Hol W.G.J. Crystal structures of human topoisomerase I in covalent and noncovalent complexes with DNA.Science. 1998; 279: 1504-1513Crossref PubMed Scopus (739) Google Scholar, Redinbo et al., 1999bRedinbo M.R. Stewart L. Champoux J.J. Hol W.G. Structural flexibility in human topoisomerase I revealed in multiple non-isomorphous crystal structures.J. Mol. Biol. 1999; 292: 685-696Crossref PubMed Scopus (71) Google Scholar, Redinbo et al., 2000Redinbo M.R. Champoux J.J. Hol W.G. Novel insights into catalytic mechanism from a crystal structure of human topoisomerase I in complex with DNA.Biochemistry. 2000; 39: 6832-6840Crossref PubMed Scopus (125) Google Scholar, Stewart et al., 1998Stewart L. Redinbo M.R. Qiu X. Hol W.G.J. Champoux J.J. A model for the mechanism of human topoisomerase I.Science. 1998; 179: 1534-1541Crossref Scopus (590) Google Scholar) allows us to compare the protein DNA interfaces formed by the highly specific viral TopIB to the less-specific human enzyme. In vTopIB, there are nine residues that make direct interactions with DNA bases in the major groove (Figure S4). Some side chains make multiple independent contacts (e.g., Tyr70 and Tyr72; Figure 3A). In contrast, the hTopIB enzyme makes no direct contacts to bases in the maj" @default.
• W2031674105 created "2016-06-24" @default.
• W2031674105 creator A5005104278 @default.
• W2031674105 creator A5018267030 @default.
• W2031674105 creator A5052800273 @default.
• W2031674105 creator A5071741783 @default.
• W2031674105 date "2006-08-01" @default.
• W2031674105 modified "2023-09-30" @default.
• W2031674105 title "Structural Basis for Specificity in the Poxvirus Topoisomerase" @default.
• W2031674105 cites W1534825972 @default.
• W2031674105 cites W1539796472 @default.
• W2031674105 cites W1544370487 @default.
• W2031674105 cites W1560750175 @default.
• W2031674105 cites W1571281254 @default.
• W2031674105 cites W1575138663 @default.
• W2031674105 cites W1600250250 @default.
• W2031674105 cites W1742808449 @default.
• W2031674105 cites W1871475547 @default.
• W2031674105 cites W1965277349 @default.
• W2031674105 cites W1984535154 @default.
• W2031674105 cites W1985336260 @default.
• W2031674105 cites W1995017064 @default.
• W2031674105 cites W1999924817 @default.
• W2031674105 cites W2006424757 @default.
• W2031674105 cites W2006853138 @default.
• W2031674105 cites W2008883352 @default.
• W2031674105 cites W2013083986 @default.
• W2031674105 cites W2014799642 @default.
• W2031674105 cites W2024632389 @default.
• W2031674105 cites W2025395911 @default.
• W2031674105 cites W2028231353 @default.
• W2031674105 cites W2028727595 @default.
• W2031674105 cites W2031733136 @default.
• W2031674105 cites W2038692006 @default.
• W2031674105 cites W2038840577 @default.
• W2031674105 cites W2041173626 @default.
• W2031674105 cites W2045208351 @default.
• W2031674105 cites W2046545014 @default.
• W2031674105 cites W2059013391 @default.
• W2031674105 cites W2059390603 @default.
• W2031674105 cites W2060601870 @default.
• W2031674105 cites W2062634597 @default.
• W2031674105 cites W2067332276 @default.
• W2031674105 cites W2067560715 @default.
• W2031674105 cites W2072069994 @default.
• W2031674105 cites W2078248419 @default.
• W2031674105 cites W2082346169 @default.
• W2031674105 cites W2083301155 @default.
• W2031674105 cites W2087036937 @default.
• W2031674105 cites W2090247166 @default.
• W2031674105 cites W2112200653 @default.
• W2031674105 cites W2118595974 @default.
• W2031674105 cites W2132600317 @default.
• W2031674105 cites W2145846015 @default.
• W2031674105 cites W2155733781 @default.
• W2031674105 cites W2160459145 @default.
• W2031674105 cites W2161620968 @default.
• W2031674105 cites W4236530034 @default.
• W2031674105 doi "https://doi.org/10.1016/j.molcel.2006.06.015" @default.
• W2031674105 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/16885024" @default.
• W2031674105 hasPublicationYear "2006" @default.
• W2031674105 type Work @default.
• W2031674105 sameAs 2031674105 @default.
• W2031674105 citedByCount "50" @default.
• W2031674105 countsByYear W20316741052013 @default.
• W2031674105 countsByYear W20316741052014 @default.
• W2031674105 countsByYear W20316741052015 @default.
• W2031674105 countsByYear W20316741052016 @default.
• W2031674105 countsByYear W20316741052017 @default.
• W2031674105 countsByYear W20316741052019 @default.
• W2031674105 countsByYear W20316741052021 @default.
• W2031674105 countsByYear W20316741052022 @default.
• W2031674105 countsByYear W20316741052023 @default.
• W2031674105 crossrefType "journal-article" @default.
• W2031674105 hasAuthorship W2031674105A5005104278 @default.
• W2031674105 hasAuthorship W2031674105A5018267030 @default.
• W2031674105 hasAuthorship W2031674105A5052800273 @default.
• W2031674105 hasAuthorship W2031674105A5071741783 @default.
• W2031674105 hasBestOaLocation W20316741051 @default.
• W2031674105 hasConcept C104317684 @default.
• W2031674105 hasConcept C147897179 @default.
• W2031674105 hasConcept C159047783 @default.
• W2031674105 hasConcept C2779827044 @default.
• W2031674105 hasConcept C2781356689 @default.
• W2031674105 hasConcept C40767141 @default.
• W2031674105 hasConcept C54355233 @default.
• W2031674105 hasConcept C552990157 @default.
• W2031674105 hasConcept C70721500 @default.
• W2031674105 hasConcept C86803240 @default.
• W2031674105 hasConceptScore W2031674105C104317684 @default.
• W2031674105 hasConceptScore W2031674105C147897179 @default.
• W2031674105 hasConceptScore W2031674105C159047783 @default.
• W2031674105 hasConceptScore W2031674105C2779827044 @default.
• W2031674105 hasConceptScore W2031674105C2781356689 @default.
• W2031674105 hasConceptScore W2031674105C40767141 @default.
• W2031674105 hasConceptScore W2031674105C54355233 @default.
• W2031674105 hasConceptScore W2031674105C552990157 @default.
• W2031674105 hasConceptScore W2031674105C70721500 @default.

• next