An Open Conformation of the Thermus thermophilusGyrase B ATP-binding Domain

Lamour, Valérie; Hoermann, Laurence; Jeltsch, Jean‐Marc; Oudet, P; Moras, Dino

doi:10.1074/jbc.m111740200

Cited by 94 publications

(102 citation statements)

References 41 publications

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“…The crystal structures of the bacterial 24-kDa N-terminal domain of GyrB (domain 1) in complex with numerous coumarins have been solved. However, this domain alone is monomeric and cannot bind ATP (17) The crystal structures of the full dimeric ATP binding domain (domain 1 and domain 2), also called the 43-kDa domain, in complex with ADPPNP and more recently with novobiocin (8,13,32,34,48,54) have been determined. Both structures show a dimeric organization of this enzyme.…”

Section: Resultsmentioning

confidence: 99%

“…Most of the residues implicated in quinolone resistance and residing in the ATP binding region of S. pneumoniae are conserved among the three protein sequences. The crystal structures of the E. coli GyrB N-terminal 43-kDa domain in complex with ADPPNP and of the T. thermophilus 43-kDa domain in complex with novobiocin (8,32) show that the ATP binding site can adopt different conformations of the active site, closed and open, respectively. These conformations are defined by the position adopted by the loop closing the active site (from residues 99 to 119 in the E. coli sequence) and stabilized by the dimeric contacts between the subunits.…”

Section: Resultsmentioning

confidence: 99%

“…The His103Tyr mutation responsible for the resistance was located outside the ParE QRDR at a position equivalent to His99 in the 43-kDa N-terminal domain of Escherichia coli GyrB. The crystal structures of the 43-kDa domain of GyrB have shown that this residue is involved in the intramolecular and intermolecular interactions of the two GyrB subunits, resulting in stabilization of the dimer in the presence of ATP or novobiocin (8,32).…”

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ATP-Bound Conformation of Topoisomerase IV: a Possible Target for Quinolones in Streptococcus pneumoniae

et al. 2003

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Topoisomerase IV, a C 2 E 2 tetramer, is involved in the topological changes of DNA during replication. This enzyme is the target of antibacterial compounds, such as the coumarins, which target the ATP binding site in the ParE subunit, and the quinolones, which bind, outside the active site, to the quinolone resistance-determining region (QRDR). After site-directed and random mutagenesis, we found some mutations in the ATP binding site of ParE near the dimeric interface and outside the QRDR that conferred quinolone resistance to Streptococcus pneumoniae, a bacterial pathogen. Modeling of the N-terminal, 43-kDa ParE domain of S. pneumoniae revealed that the most frequent mutations affected conserved residues, among them His43 and His103, which are involved in the hydrogen bond network supporting ATP hydrolysis, and Met31, at the dimeric interface. All mutants showed a particular phenotype of resistance to fluoroquinolones and an increase in susceptibility to novobiocin. All mutations in ParE resulted in resistance only when associated with a mutation in the QRDR of the GyrA subunit. Our models of the closed and open conformations of the active site indicate that quinolones preferentially target topoisomerase IV of S. pneumoniae in its ATP-bound closed conformation.Topoisomerase IV and DNA gyrase are bacterial type II topoisomerases, which modify DNA topology during the replication process by unlinking DNA and facilitating chromosome segregation (59). Topoisomerase IV forms a C 2 E 2 tetramer involved in segregation of the chromosome at cell division (1, 30, 59). The ParC subunit contains the site of topoisomerization catalyzing the double-stranded DNA break (30, 53), while the ParE subunit catalyzes the hydrolysis of ATP, providing the free energy necessary for these reactions (2, 6, 11). The ParE and ParC subunits share extensive sequence homology with, respectively, the GyrB and GyrA subunits of the DNA gyrase A 2 B 2 tetramer, which catalyzes negative DNA supercoiling during the initiation and elongation processes of DNA replication (16,53).Both topoisomerases are the targets of antibacterial molecules, such as the quinolones and the coumarins. The coumarins inhibit supercoiling and enzyme turnover by preventing the binding and hydrolysis of ATP (45). The quinolones form a ternary complex with the topoisomerases in the presence of DNA, resulting in lethal double-stranded DNA breaks (11). Enzymatic studies and binding assays have also shown that the quinolones can form a complex with the topoisomerases before binding DNA (15, 29); however, some binding data indicate the occurrence of a specific and higher level of binding to the enzyme-DNA complex rather than to the enzyme alone (43,56). These families of molecules, in particular, the fluoroquinolones (FQs), are under continuous development as resistance to antibiotics in pathogenic bacteria has dramatically increased during the last decade (20,22,49).

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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confidence: 99%

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ATP-Bound Conformation of Topoisomerase IV: a Possible Target for Quinolones in Streptococcus pneumoniae

et al. 2003

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“…The second domain, called the transducer domain, forms the walls of the clamp and connects it to the enzyme core. Furthermore, communication between the ATP-binding domain and the central domain of the enzyme, responsible for DNA cleavage/ligation, has been suggested to go through the transducer domain (4,(12)(13)(14)(15). Upon clamp closure, the transducer domain of each protomer approaches one another, creating a very tight cavity that is actually too small to hold a T-segment (10).…”

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Hindering the Strand Passage Reaction of Human Topoisomerase IIα without Disturbing DNA Cleavage, ATP Hydrolysis, or the Operation of the N-terminal Clamp

Oestergaard

Giangiacomo²,

Bjergbæk³

et al. 2004

Journal of Biological Chemistry

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DNA topoisomerase II is an essential enzyme that releases a topological strain in DNA by introduction of transient breaks in one DNA helix through which another helix is passed. While changing DNA topology, ATP is required to drive the enzyme through a series of conformational changes dependent on interdomain communication. We have characterized a human topoisomerase II␣ enzyme with a two-amino acid insertion at position 351 in the transducer domain. The mutation specifically abolishes the DNA strand passage event of the enzyme, probably because of a sterical hindrance of T-segment transport. Thus, the enzyme fails to decatenate and relax DNA, even though it is fully capable of ATP hydrolysis, closure of the N-terminal clamp, and DNA cleavage. The cleavage activity is increased, suggesting that the transducer domain has a role in regulating DNA cleavage. Furthermore, the enzyme has retained a tendency to increase DNA cleavage upon nucleotide binding and also responds to DNA with elevated ATP hydrolysis. However, the DNA-mediated increase in ATP hydrolysis is lower than that obtained with the wild-type enzyme but similar to that of a cleavage-deficient topoisomerase II␣ enzyme. Our results strongly suggest that the strand passage event is required for efficient DNA stimulation of topoisomerase II-mediated ATP hydrolysis, whereas the stimulation occurs independent of the DNA cleavage reaction per se. A comparison of the strand passage deficient-enzyme described here and the cleavage-deficient enzyme may have applications in other studies where a clear distinction between strand passage and topoisomerase II-mediated DNA cleavage is desirable.Type II DNA topoisomerases are highly complex enzymes that are able to change the topological conformation of DNA by introducing a transient double strand break in one DNA helix, the G-segment, whereas another intact helix, the T-segment, is transported coordinately through the gated DNA (1). The process depends on ATP, which drives the enzyme through a series of conformational changes (2-5).The homodimeric eukaryotic topoisomerase II enzyme contains two highly conserved catalytic entities, which also share homology with the bacterial DNA topoisomerase II counterpart, DNA gyrase. The ATPase activity is found in the Nterminal region, whereas the DNA cleavage and ligation activities are held by the central region (6). The extreme C termini of the type II topoisomerases are divergent and dispensable for catalytic activity in vitro (7-9).The structural data reveal that the N-terminal region of topoisomerase II forms an ATP-operated clamp, which closes upon ATP binding (10, 11). The dimeric N-terminal clamp contains two domains in each protomer (10). The most Nterminal domain is responsible for dimer interactions during clamp closure and also holds the ATP-binding site. The second domain, called the transducer domain, forms the walls of the clamp and connects it to the enzyme core. Furthermore, communication between the ATP-binding domain and the central domain of the enzyme, respon...

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“…La caractérisation structurale de ces complexes nucléoprotéiques représente un vrai défi scientifique et technique, de par la taille (~320 kDa) et la flexibilité de cet édifice moléculaire. Les études structurales, jusqu'à présent cantonnées aux domaines catalytiques isolés des Top2 en présence d'oligonucléotides et d'inhibiteurs [6][7][8], fournissent des informations très partielles sur les conformations adoptées par ces protéines modulaires au cours du cycle catalytique, et ne permettent pas d'étudier le positionnement des longues séquences d'ADN sur l'enzyme. Nous avons entrepris l'analyse structurale par cryo-microscopie électronique d'une ADN gyrase en complexe avec un ADN long de 155pb et un inhibiteur poison de la famille des quinolones, la ciprofloxacine (CFX) [9].…”

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L’architecture moléculaire complète de l’ADN gyrase révélée par cryo-microscopie électronique

et al. 2014

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An Open Conformation of the Thermus thermophilusGyrase B ATP-binding Domain

Cited by 94 publications

References 41 publications

ATP-Bound Conformation of Topoisomerase IV: a Possible Target for Quinolones in Streptococcus pneumoniae

ATP-Bound Conformation of Topoisomerase IV: a Possible Target for Quinolones in Streptococcus pneumoniae

Hindering the Strand Passage Reaction of Human Topoisomerase IIα without Disturbing DNA Cleavage, ATP Hydrolysis, or the Operation of the N-terminal Clamp

L’architecture moléculaire complète de l’ADN gyrase révélée par cryo-microscopie électronique

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