Toprim Domain Research Articles

Structure of the E. Coli Gyrase DNA Binding and Cleavage Core Reveals A Unique Domain

DNA topoisomerases are essential enzymes that sustain chromosome supercoiling homeostasis in all forms of life. DNA gyrase, a heterotetrameric type IIA topoisomerase, has the unique ability to introduce negative supercoils into DNA, helping maintain bacterial genomes in a compact, underwound state. Though all gyrase orthologs use a set of homologous domains and a central “two-gate” mechanism for passing one DNA segment through another, they also exhibit critical family-specific differences. For example, the metal- and DNA-binding TOPRIM domain of gyrases found in many gamma- and beta-proteobacteria contains a 170-amino acid insertion of unknown function. We have solved the crystal structure of the E.coli gyrase DNA binding and cleavage core, visualizing this insertion for the first time. Biochemical analyses of a structure-guided deletion mutant lacking this region reveal that it may help coordinate the activities of gyrase's distal ATPase and DNA binding gates.

Deciphering the Distinct Role for the Metal Coordination Motif in the Catalytic Activity of Mycobacterium smegmatis Topoisomerase I

Mycobacterium smegmatis topoisomerase I (MstopoI) is distinct from typical type IA topoisomerases. The enzyme binds to both single- and double-stranded DNA with high affinity, making specific contacts. The enzyme comprises conserved regions similar to type IA topoisomerases from Escherichia coli and other eubacteria but lacks the typically found zinc fingers in the carboxy-terminal domain. The enzyme can perform DNA cleavage in the absence of Mg2+, but religation needs exogenously added Mg2+. One molecule of Mg2+ tightly bound to the enzyme has no role in DNA cleavage but is needed only for the religation reaction. The toprim (topoisomerase–primase) domain in MstopoI comprising the Mg2+ binding pocket, conserved in both type IA and type II topoisomerases, was subjected to mutagenesis to understand the role of Mg2+ in different steps of the reaction. The residues D108, D110, and E112 of the enzyme, which form the acidic triad in the DXDXE motif, were changed to alanines. D108A mutation resulted in an enzyme that is Mg2+ dependent for DNA cleavage unlike MstopoI and exhibited enhanced DNA cleavage property and reduced religation activity. The mutant was toxic for cell growth, most likely due to the imbalance in cleavage–religation equilibrium. In contrast, the E112A mutant behaved like wild-type enzyme, cleaving DNA in a Mg2+-independent fashion, albeit to a reduced extent. Intra- and intermolecular religation assays indicated specific roles for D108 and E112 residues during the reaction. Together, these results indicate that the D108 residue has a major role during cleavage and religation, while E112 is important for enhancing the efficiency of cleavage. Thus, although architecturally and mechanistically similar to topoisomerase I from E. coli, the metal coordination pattern of the mycobacterial enzyme is distinct, opening up avenues to exploit the enzyme to develop inhibitors.

A Unique 45-Amino-Acid Region in the Toprim Domain of Plasmodium falciparum Gyrase B Is Essential for Its Activity

DNA gyrase is the only topoisomerase that can introduce negative supercoils into the DNA at the cost of ATP hydrolysis. Some but not all the steps of the topoisomerization reaction are understood clearly for both eukaryotic topoII and DNA gyrase. This study is an attempt to understand whether the B subunit of DNA gyrase binds to DNA directly, which may be central to the stimulation of its ATPase activity essential for gyrase function. We have dissected the Plasmodium falciparum gyrase B (PfGyrB) subunit to identify a 45-amino-acid region in the toprim domain that is responsible for its intrinsic DNA binding activity, DNA-stimulated ATPase activity, and DNA cleavage. We find that DNA has to enter through the ATP-operated clamp of PfGyrB to gain access to the DNA binding region. Furthermore, the rate of ATP hydrolysis of PfGyrB increases significantly with increasing DNA length, suggesting a possible communication between the ATPase domain and the DNA binding region that can account for its optimal ATPase activity. These results not only highlight the mechanism of GyrB action in the deadly human parasite P. falciparum but also provide meaningful insights into the current mechanistic model of DNA transport by gyrase during the topoisomerization reaction.

Crystal structure of DNA gyrase B′ domain sheds lights on the mechanism for T-segment navigation

DNA gyrase is an indispensible marvelous molecular machine in manipulating the DNA topology for the prokaryotes. In the ‘two-gate’ mechanism of DNA topoisomerase, T-segment navigation from N- to DNA-gate is a critical step, but the structural basis supporting this scheme is unclear. The crystal structure of DNA gyrase B′ subfragment from Mycobacterium tuberculosis reveals an intrinsic homodimer. The two subunits, each consisting of a Tail and a Toprim domain, are tightly packed one another to form a ‘crab-like’ organization never observed previously from yeast topo II. Structural comparisons show two orientational alterations of the Tail domain, which may be dominated by a 43-residue peptide at the B′ module C-terminus. A highly conserved pentapeptide mediates large-scale intrasubunit conformational change as a hinge point. Mutational studies highlight the significant roles of a negatively charge cluster on a groove at dimer interface. On the basis of structural analysis and mutation experiments, a sluice-like model for T-segment transport is proposed.

Probing the Differential Interactions of Quinazolinedione PD 0305970 and Quinolones with Gyrase and Topoisomerase IV

Quinazoline-2,4-diones, such as PD 0305970, are new DNA gyrase and topoisomerase IV (topo IV) inhibitors with potent activity against gram-positive pathogens, including quinolone-resistant isolates. The mechanistic basis of dione activity vis-à-vis quinolones is not understood. We present evidence for Streptococcus pneumoniae gyrase and topo IV that PD 0305970 and quinolones interact differently with the enzyme breakage-reunion and Toprim domains, DNA, and Mg2+-four components that are juxtaposed in the topoisomerase cleavage complex to effect DNA scission. First, PD 0305970 targets primarily gyrase in Streptococcus pneumoniae. However, unlike quinolones, which select predominantly for gyrA (or topo IV parC) mutations in the breakage-reunion domain, unusually the dione selected for novel mutants with alterations that map to a region of the Toprim domain of GyrB (R456H and E474A or E474D) or ParE (D435H and E475A). This "dione resistance-determining region" overlaps the GyrB quinolone resistance-determining region and the region that binds essential Mg2+ ions, each function involving conserved EGDSA and PLRGK motifs. Second, dione-resistant gyrase and topo IV were inhibited by ciprofloxacin, whereas quinolone-resistant enzymes (GyrA S81F and ParC S79F) remained susceptible to PD 0305970. Third, dione-promoted DNA cleavage by gyrase occurred at a distinct repertoire of sites, implying that structural differences with quinolones are sensed at the DNA level. Fourth, unlike the situation with quinolones, the Mg2+ chelator EDTA did not reverse dione-induced gyrase cleavage nor did the dione promote Mg2+-dependent DNA unwinding. It appears that PD 0305970 interacts uniquely to stabilize the cleavage complex of gyrase/topo IV perhaps via an altered orientation directed by the bidentate 3-amino-2,4-dione moiety.

Structural insight into the quinolone–DNA cleavage complex of type IIA topoisomerases

Type II topoisomerases alter DNA topology by forming a covalent DNA-cleavage complex that allows DNA transport through a double-stranded DNA break. We present the structures of cleavage complexes formed by the Streptococcus pneumoniae ParC breakage-reunion and ParE TOPRIM domains of topoisomerase IV stabilized by moxifloxacin and clinafloxacin, two antipneumococcal fluoroquinolones. These structures reveal two drug molecules intercalated at the highly bent DNA gate and help explain antibacterial quinolone action and resistance.

Asp-to-Asn Substitution at the First Position of the DxD TOPRIM Motif of Recombinant Bacterial Topoisomerase I Is Extremely Lethal to E. coli

The TOPRIM domain found in many nucleotidyl transferases contains a DxD motif involved in magnesium ion coordination for catalysis. Medium- to high-copy-number plasmid clones of Yersinia pestis topoisomerase I (YpTOP) with Asp-to-Asn substitution at the first aspartate residue (D117N) of this motif could not be generated in Escherichia coli without second-site mutation even when expression was under the control of the tightly regulated BAD promoter and suppressed by 2% glucose in the medium. Arabinose induction of a single-copy YpTOP-D117N mutant gene integrated into the chromosome resulted in ∼ 10 5-fold of cell killing in 2.5 h. Attempt to induce expression of the corresponding E. coli topoisomerase I mutant (EcTOP-D111N) encoded on a high-copy-number plasmid resulted in either loss of viability or reversion of the clone to wild type. High-copy-number plasmid clones of YpTOP-D119N and EcTOP-D113N with the Asn substitution at the second Asp of the TOPRIM motif could be stably maintained, but overexpression also decreased cell viability significantly. The Asp-to-Asn substitutions at these TOPRIM residues can selectively decrease Mg 2+ binding affinity with minimal disruption of the active-site geometry, leading to trapping of the covalent complex with cleaved DNA and causing bacterial cell death. The extreme sensitivity of the first TOPRIM position suggested that this might be a useful site for binding of small molecules that could act as topoisomerase poisons.

Mutagenesis in the α3α4 GyrA Helix and in the Toprim Domain of GyrB Refines the Contribution of Mycobacterium tuberculosis DNA Gyrase to Intrinsic Resistance to Quinolones

The replacement of M74 in GyrA, A83 in GyrA, and R447 in GyrB of Mycobacterium tuberculosis gyrase by their Escherichia coli homologs resulted in active enzymes as quinolone susceptible as the E. coli gyrase. This demonstrates that the primary structure of gyrase determines intrinsic quinolone resistance and was supported by a three-dimensional model of N-terminal GyrA.

Crystal structure and putative function of small Toprim domain‐containing protein from Bacillus stearothermophilus

The crystal structure of the Midwest Center for Structural Genomics target APC35832, a 14.7-kDa cytosolic protein from Bacillus stearothermophilus, has been determined at 1.3 A resolution by the single anomalous diffraction method from a mercury soaked crystal. The APC35832 protein is a representative of large group of bacterial and archeal proteins entirely consisting of the Toprim (topoisomerase-primase) domain. This domain is found in the catalytic centers of many enzymes catalyzing phosphodiester bond formation or cleavage, but the function of small Toprim domain proteins remains unknown. Consistent with the sequence analysis, the APC35832 structure shows a conserved Toprim fold, with a central 4-stranded parallel beta-sheet surrounded by four alpha-helixes. Comparison of the APC35832 structure with its closest structural homolog, the catalytic core of bacteriophage T7 primase, revealed structural conservation of a metal binding site and isothermal titration calorimetry indicates that APC35832 binds Mg2+ with a sub-millimolar dissociation constant (K(d)). The APC35832-Mg2+ complex structure was determined at 1.65 A and reveals the role of conserved acidic residues in Mg2+ ion coordination. The structural similarities to other Toprim domain containing proteins and potential function and substrates of APC35832 are discussed in this article.

SSB Protein Limits RecOR Binding onto Single-stranded DNA

The RecO and RecR proteins form a complex that promotes the nucleation of RecA protein filaments onto SSB protein-coated single-stranded DNA (ssDNA). However, even when RecO and RecR proteins are provided at optimal concentrations, the loading of RecA protein is surprisingly slow, typically proceeding with a lag of 10 min or more. The rate-limiting step in RecOR-promoted RecA nucleation is the binding of RecOR protein to ssDNA, which is inhibited by SSB protein despite the documented interaction between RecO and SSB. Full activity of RecOR is seen only when RecOR is preincubated with ssDNA prior to the addition of SSB. The slow binding of RecOR to SSB-coated ssDNA involves the C terminus of SSB. When an SSB variant that lacks the C-terminal 8 amino acids is used, the capacity of RecOR to facilitate RecA loading onto the ssDNA is largely abolished. The results are used in an expanded model for RecOR action.

Identification of the RecR Toprim Domain as the Binding Site for both RecF and RecO: A ROLE OF RecR IN RecFOR ASSEMBLY AT DOUBLE-STRANDED DNA-SINGLE-STRANDED DNA JUNCTIONS

The RecR protein forms complexes with RecF or RecO that direct the specific loading of RecA onto gapped DNA. However, the binding sites of RecF and RecO on RecR have yet to be identified. In this study, a Thermus thermophilus RecR dimer model was constructed by NMR analysis and homology modeling. NMR titration analysis suggested that the hairpin region of the helix-hairpin-helix motif in the cavity of the RecR dimer is a binding site for double-stranded DNA (dsDNA) and that the acidic cluster region of the Toprim domain is a RecO binding site. Mutations of Glu-84, Asp-88, and Glu-144 residues comprising that acidic cluster were generated. The E144A and E84A mutations decreased the binding affinity for RecO, but the D88A did not. Interestingly, the binding ability to RecF was abolished by E144A, suggesting that the region surrounding the RecR Glu-144 residue could be a binding site not only for RecO but also for RecF. Furthermore, RecR and RecF formed a 4:2 heterohexamer in solution that was unaffected by adding RecO, indicating a preference by RecR for RecF over RecO. The RecFR complex is considered to be involved in the recognition of the dsDNA-ssDNA junction, whereas RecO binds single-stranded DNA (ssDNA) and ssDNA-binding protein. Thus, the RecR Toprim domain may contribute to the RecO interaction with RecFR complexes at the dsDNA-ssDNA junction site during recombinational DNA repair mediated by the RecFOR.

Bacterial type IA DNA topoisomerase as a target for novel antibacterial agents

At least one type IA DNA topoisomerases can be found in every bacterium, making it a logical target for antibacterial agents that can convert the enzyme into poison by trapping its covalent complex with DNA. We isolated a mutant of recombinant Yersinia pestis topoisomerase I by screening for its ability to induce the SOS response in E. coli. Overexpression of this mutant topoisomerase I results in bacterial cell death. From sequence analysis and site-directed mutagenesis, it was determined that a single amino acid substitution in the TOPRIM domain changing a strictly conserved glycine residue to serine in either the Y. pestis or Escherichia coli topoisomerase I can result in a mutant enzyme that has the SOS inducing and cell killing properties. Analysis of the purified mutant enzymes showed that they have no relaxation activity but retain the ability to cleave DNA and form a covalent complex. These results demonstrate that perturbation of the active site region of bacterial topoisomerase I can result in stabilization of the covalent intermediate, with the in vivo consequence of bacterial cell death. Small molecules that induce similar perturbation in the enzyme-DNA complex should be candidates as leads for novel antibacterial agents. Second site mutations that can enhance the cell killing property of topoisomerase I are being studied. The SOS-inducing mutant topoisomerase I is useful as a model to study the mechanism of cell killing initiated by accumulation of topoisomerase I-mediated DNA cleavage.

Crosstalk between Primase Subunits Can Act to Regulate Primer Synthesis in trans

The coordination of primase function within the replisome is an essential but poorly understood feature of lagging strand synthesis. By using crystallography and small-angle X-ray scattering (SAXS), we show that functional elements of bacterial primase transition between two dominant conformations: an extended form that uncouples a regulatory domain from its associated RNA polymerase core and a compact state that sequesters the regulatory region from the site of primer synthesis. FRET studies and priming assays reveal that the regulatory domain of one primase subunit productively associates with nucleic acid that is bound to the polymerase domain of a second protomer in trans. This intersubunit interaction allows primase to select initiation sites on template DNA and implicates the regulatory domain as a "molecular brake" that restricts primer length. Our data suggest that the replisome may cooperatively use multiple primases and this conformational switch to control initiation frequency, processivity, and ultimately, Okazaki fragment synthesis.

Bacterial Cell Killing Mediated by Topoisomerase I DNA Cleavage Activity

DNA topoisomerases are important clinical targets for antibacterial and anticancer therapy. At least one type IA DNA topoisomerase can be found in every bacterium, making it a logical target for antibacterial agents that can convert the enzyme into poison by trapping its covalent complex with DNA. However, it has not been possible previously to observe the consequence of having such a stabilized covalent complex of bacterial topoisomerase I in vivo. We isolated a mutant of recombinant Yersinia pestis topoisomerase I that forms a stabilized covalent complex with DNA by screening for the ability to induce the SOS response in Escherichia coli. Overexpression of this mutant topoisomerase I resulted in bacterial cell death. From sequence analysis and site-directed mutagenesis, it was determined that a single amino acid substitution in the TOPRIM domain changing a strictly conserved glycine residue to serine in either the Y. pestis or E. coli topoisomerase I can result in a mutant enzyme that has the SOS-inducing and cell-killing properties. Analysis of the purified mutant enzymes showed that they have no relaxation activity but retain the ability to cleave DNA and form a covalent complex. These results demonstrate that perturbation of the active site region of bacterial topoisomerase I can result in stabilization of the covalent intermediate, with the in vivo consequence of bacterial cell death. Small molecules that induce similar perturbation in the enzyme-DNA complex should be candidates as leads for novel antibacterial agents.

The Effects of Metal Ions on the Structure and Stability of the DNA Gyrase B Protein

The effects of mono- and divalent metal ions on the DNA gyrase B subunit, on its 43 kDa and 47 kDa domains, and on two mutants in the Toprim domain (D498A and D500C) were investigated by means of circular dichroism and protein melting experiments. Both types of metal ion, with the notable exception of Mn2+, did not affect the conformational properties of the enzyme subunit at room temperature, but were able to produce selective and differential effects on protein stability. In particular, monovalent (K+) ions increased the stability of the gyrase B structure, whereas destabilising effects were most prominent using Mn2+ as the metal ion. Ca2+ and Mg2+ produced comparable changes in the gyrase B melting profile. Additionally, we found that monovalent (K+) ions were more effective in the 43 kDa N-terminal domain where ATP binding occurs, whereas divalent ions caused large modifications in the conformational stability of the 47 kDa C-terminal domain. Our results on gyrase B mutants indicate that D498 interacts with Mn2+, whereas it has little effect on the binding of the other ions tested. A D500C mutation, in contrast, effectively impairs Mg2+ affinity, suggesting effective contacts between this ion and D500 in the wild-type enzyme. Hence, the sites of metal ion complexation within the Toprim domain are modulated by the nature of the ion species. These results suggest a double role played by metal ions in the catalytic steps involving DNA gyrase B. One has to do with direct involvement of cations complexed to the Toprim domain in the DNA cutting-rejoining process, the other, until now overlooked, is connected to the dramatic changes in protein flexibility produced by ion binding, which reduces the energy required for the huge conformational changes essential for the catalytic cycle to occur.

The 5S rRNA maturase, ribonuclease M5, is a Toprim domain family member

The maturation of 5S ribosomal RNA in low G+C Gram-positive bacteria is catalyzed by a highly conserved, ∼190 residue, enzyme, called ribonuclease M5 (RNase M5). Sequence alignment had predicted that the N-terminal half of RNase M5 would consist of a Toprim domain, a protein fold found in type IA and type II topoisomerases, DnaG-like primases, OLD family nucleases and RecR proteins [L. Aravind, D. D. Leipe and E. V. Koonin (1998) Nucleic Acids Res., 26, 4205–4213]. Here, we present structural modelling data and a mutational analysis of RNase M5 that confirms this hypothesis. The N-terminal half of RNase M5 can be fitted to the Toprim domain of the DnaG catalytic core. Mutation of amino acid residues highly conserved among RNase M5 enzymes and members of the Toprim domain family showed that alteration of residues critical for topoisomerase and primase activity also had a dramatic effect on the cleavage of 5S rRNA precursor by RNase M5 both in vivo and in vitro. This suggests that the mechanisms of double-stranded RNA cleavage by RNase M5 and double-stranded DNA cleavage by members of the topoisomerase family are related.

Acidic Residues in the Nucleotide-binding Site of the Bacteriophage T7 DNA Primase

DNA primases catalyze the synthesis of oligoribonucleotides to initiate lagging strand DNA synthesis during DNA replication. Like other prokaryotic homologs, the primase domain of the gene 4 helicase-primase of bacteriophage T7 contains a zinc motif and a catalytic core. Upon recognition of the sequence, 5'-GTC-3' by the zinc motif, the catalytic site condenses the cognate nucleotides to produce a primer. The TOPRIM domain in the catalytic site contains several charged residues presumably involved in catalysis. Each of eight acidic residues in this region was replaced with alanine, and the properties of the altered primases were examined. Six of the eight residues (Glu-157, Glu-159, Asp-161, Asp-207, Asp-209, and Asp-237) are essential in that altered gene 4 proteins containing these mutations cannot complement T7 phage lacking gene 4 for T7 growth. These six altered gene 4 proteins can neither synthesize primers de novo nor extend an oligoribonucleotide. Despite the inability to catalyze phosphodiester bond formation, the altered proteins recognize the sequence 5'-GTC-3' in the template and deliver preformed primer to T7 DNA polymerase. The alterations in the TOPRIM domain result in the loss of binding affinity for ATP as measured by surface plasmon resonance assay together with ATP-agarose affinity chromatography.

The Bacterial Helicase-Primase Interaction: A Common Structural/Functional Module

The lack of a high-resolution structure for the bacterial helicase-primase complex and the fragmented structural information for the individual proteins have been hindering our detailed understanding of this crucial binary protein interaction. Two new structures for the helicase-interacting domain of the bacterial primases from Escherichia coli and Bacillus stearothermophilus have recently been solved and both revealed a unique and surprising structural similarity to the amino-terminal domain of the helicase itself. In this minireview, the current data are discussed and important new structural and functional aspects of the helicase-primase interaction are highlighted. An attractive structural model with direct biological significance for the function of this complex and also for the development of new antibacterial compounds is examined.

Ring-shaped architecture of RecR: implications for its role in homologous recombinational DNA repair.

RecR, together with RecF and RecO, facilitates RecA loading in the RecF pathway of homologous recombinational DNA repair in procaryotes. The human Rad52 protein is a functional counterpart of RecFOR. We present here the crystal structure of RecR from Deinococcus radiodurans (DR RecR). A monomer of DR RecR has a two-domain structure: the N-terminal domain with a helix-hairpin-helix (HhH) motif and the C-terminal domain with a Cys4 zinc-finger motif, a Toprim domain and a Walker B motif. Four such monomers form a ring-shaped tetramer of 222 symmetry with a central hole of 30-35 angstroms diameter. In the crystal, two tetramers are concatenated, implying that the RecR tetramer is capable of opening and closing. We also show that DR RecR binds to both dsDNA and ssDNA, and that its HhH motif is essential for DNA binding.

Crystallization and preliminary X-ray crystallographic analysis of the C-terminal domain of ParC protein from Bacillus stearothermophilus.

Type IIA topoisomerases are multidomain enzymes composed of four major domains: the ATPase domain, the TOPRIM domain, the DNA-cleavage/religation domain and the C-terminal domain (CTD). Although crystal structures of the first three domains are available, the three-dimensional structure of the less-conserved CTD has yet to be determined. In order to provide a three-dimensional structure of this structurally uncharacterized region, the 36 kDa CTD of ParC protein, the DNA-cleavage/religation subunit of topoisomerase IV, from Bacillus stearothermophilus has been cloned, purified and crystallized. The crystals belonged to the trigonal space group P3(1) (or P3(2)), with unit-cell parameters a = b = 83.5, c = 45.1 A. The asymmetric unit contains one molecule and the solvent content is 51.2%. A 98.9% complete native data set has been collected from a frozen crystal to 2.0 A resolution with an overall R(merge) of 6.5%.