RESUMO
DNA topoisomerases are nature's tools for resolving the unique problems of DNA entanglement that occur owing to unwinding and rewinding of the DNA helix during replication, transcription, recombination, repair, and chromatin remodeling. These enzymes perform topological transformations by providing a transient DNA break, formed by a covalent adduct with the enzyme, through which strand passage can occur. The active site tyrosine is responsible for initiating two transesterifications to cleave and then religate the DNA backbone. The cleavage reaction intermediate is exploited by cytotoxic agents, which have important applications as antibiotics and anticancer drugs. The reactions mediated by these enzymes can also be regulated by their binding partners; one example is a DNA helicase capable of modulating the directionality of strand passage, enabling important functions like reannealing denatured DNA and resolving recombination intermediates. In this review, we cover recent advances in mechanistic insights into topoisomerases and their various cellular functions.
Assuntos
Replicação do DNA , DNA Topoisomerases Tipo II , DNA Topoisomerases Tipo I , Antineoplásicos/farmacologia , Domínio Catalítico , DNA/genética , DNA/metabolismo , DNA Topoisomerases Tipo I/química , DNA Topoisomerases Tipo I/genética , DNA Topoisomerases Tipo I/metabolismo , DNA Topoisomerases Tipo II/química , DNA Topoisomerases Tipo II/genética , DNA Topoisomerases Tipo II/metabolismo , Humanos , Estrutura Terciária de ProteínaRESUMO
The ability to catalyze reversible DNA cleavage and religation is central to topoisomerases' role in regulating DNA topology. In type IIA topoisomerases (Top2), the formation of its DNA cleavage-religation center is driven by DNA-binding-induced structural rearrangements. These changes optimally position key catalytic modules, such as the active site tyrosine of the WHD domain and metal ion(s) chelated by the TOPRIM domain, around the scissile phosphodiester bond to perform reversible transesterification. To understand this assembly process in detail, we report the catalytic core structures of human Top2α and Top2ß in an on-pathway conformational state. This state features an in trans formation of an interface between the Tower and opposing TOPRIM domain, revealing a groove for accommodating incoming G-segment DNA. Structural superimposition further unveils how subsequent DNA-binding-induced disengagement of the TOPRIM and Tower domains allows a firm grasp of the bound DNA for cleavage/religation. Notably, we identified a previously undocumented protein-DNA interaction, formed between an arginine-capped C-terminus of an α-helix in the TOPRIM domain and the DNA backbone, significantly contributing to Top2 function. This work uncovers a previously unrecognized role of the Tower domain, highlighting its involvement in anchoring and releasing the TOPRIM domain, thus priming Top2 for DNA binding and cleavage.
RESUMO
Formation of programmed DNA double-strand breaks is essential for initiating meiotic recombination. Genetic studies on Arabidopsis thaliana and Mus musculus have revealed that assembly of a type IIB topoisomerase VI (Topo VI)-like complex, composed of SPO11 and MTOPVIB, is a prerequisite for generating DNA breaks. However, it remains enigmatic if MTOPVIB resembles its Topo VI subunit B (VIB) ortholog in possessing robust ATPase activity, ability to undergo ATP-dependent dimerization, and activation of SPO11-mediated DNA cleavage. Here, we successfully prepared highly pure A. thaliana MTOPVIB and MTOPVIB-SPO11 complex. Contrary to expectations, our findings highlight that MTOPVIB differs from orthologous Topo VIB by lacking ATP-binding activity and independently forming dimers without ATP. Most significantly, our study reveals that while MTOPVIB lacks the capability to stimulate SPO11-mediated DNA cleavage, it functions as a bona fide DNA-binding protein and plays a substantial role in facilitating the dsDNA binding capacity of the MOTOVIB-SPO11 complex. Thus, we illustrate mechanistic divergence between the MTOPVIB-SPO11 complex and classical type IIB topoisomerases.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , DNA Topoisomerases Tipo II , Trifosfato de Adenosina/metabolismo , Arabidopsis/genética , Arabidopsis/enzimologia , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas Arqueais , Quebras de DNA de Cadeia Dupla , DNA Topoisomerases/metabolismo , DNA Topoisomerases/genética , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Endodesoxirribonucleases/metabolismo , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/química , Evolução Molecular , Meiose , Multimerização ProteicaRESUMO
Deoxypodophyllotoxin contains a core of four fused rings (A to D) with three consecutive chiral centers, the last being created by the attachment of a peripheral trimethoxyphenyl ring (E) to ring C. Previous studies have suggested that the iron(II)- and 2-oxoglutarate-dependent (Fe/2OG) oxygenase, deoxypodophyllotoxin synthase (DPS), catalyzes the oxidative coupling of ring B and ring E to form ring C and complete the tetracyclic core. Despite recent efforts to deploy DPS in the preparation of deoxypodophyllotoxin analogs, the mechanism underlying the regio- and stereoselectivity of this cyclization event has not been elucidated. Herein, we report 1) two structures of DPS in complex with 2OG and (±)-yatein, 2) in vitro analysis of enzymatic reactivity with substrate analogs, and 3) model reactions addressing DPS's catalytic mechanism. The results disfavor a prior proposal of on-pathway benzylic hydroxylation. Rather, the DPS-catalyzed cyclization likely proceeds by hydrogen atom abstraction from C7', oxidation of the benzylic radical to a carbocation, Friedel-Crafts-like ring closure, and rearomatization of ring B by C6 deprotonation. This mechanism adds to the known pathways for transformation of the carbon-centered radical in Fe/2OG enzymes and suggests what types of substrate modification are likely tolerable in DPS-catalyzed production of deoxypodophyllotoxin analogs.
Assuntos
Berberidaceae/enzimologia , Medicamentos de Ervas Chinesas/química , Ligases/química , Proteínas de Plantas/química , Podofilotoxina/análogos & derivados , Oxirredução , Podofilotoxina/químicaRESUMO
The propagation of the hepatitis C virus (HCV) is regulated in part by the phosphorylation of its nonstructural protein NS5A that undergoes sequential phosphorylation on several highly conserved serine residues and switches from a hypo- to a hyperphosphorylated state. Previous studies have shown that NS5A sequential phosphorylation requires NS3 encoded on the same NS3-NS4A-NS4B-NS5A polyprotein. Subtle mutations in NS3 without affecting its protease activity could affect NS5A phosphorylation. Given the ATPase domain in the NS3 COOH terminus, we tested whether NS3 participates in NS5A phosphorylation similarly to the nucleoside diphosphate kinase-like activity of the rotavirus NSP2 nucleoside triphosphatase (NTPase). Mutations in the NS3 ATP-binding motifs blunted NS5A hyperphosphorylation and phosphorylation at serines 225, 232, and 235, whereas a mutation in the RNA-binding domain did not. The phosphorylation events were not rescued with wild-type NS3 provided in trans. When provided with an NS3 ATPase-compatible ATP analog, N6-benzyl-ATP-γ-S, thiophosphorylated NS5A was detected in the cells expressing the wild-type NS3-NS5B polyprotein. The thiophosphorylation level was lower in the cells expressing NS3-NS5B with a mutation in the NS3 ATP-binding domain. In vitro assays with a synthetic peptide and purified wild-type NS3 followed by dot blotting and mass spectrometry found weak NS5A phosphorylation at serines 222 and 225 that was sensitive to an inhibitor of casein kinase Iα but not helicase. When casein kinase Iα was included in the assay, much stronger phosphorylation was observed at serines 225, 232, and 235. We concluded that NS5A sequential phosphorylation requires the ATP-binding domain of the NS3 helicase and that casein kinase Iα is a potent NS5A kinase. IMPORTANCE For more than 20 years, NS3 was known to participate in NS5A sequential phosphorylation. In the present study, we show for the first time that the ATP-binding domain of NS3 is involved in NS5A phosphorylation. In vitro assays showed that casein kinase Iα is a very potent kinase responsible for NS5A phosphorylation at serines 225, 232, and 235. Our data suggest that ATP binding by NS3 probably results in conformational changes that recruit casein kinase Iα to phosphorylate NS5A, initially at S225 and subsequently at S232 and S235. Our discovery reveals intricate requirements of the structural integrity of NS3 for NS5A hyperphosphorylation and HCV replication.
Assuntos
Hepacivirus , Hepatite C , RNA Polimerase Dependente de RNA , Proteínas não Estruturais Virais , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Caseína Quinase Ialfa/metabolismo , Hepacivirus/enzimologia , Hepacivirus/genética , Hepatite C/virologia , Humanos , Fosforilação , Poliproteínas/metabolismo , Domínios Proteicos/genética , RNA Polimerase Dependente de RNA/metabolismo , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismoRESUMO
Mechanisms of enzymatic epoxidation via oxygen atom transfer (OAT) to an olefin moiety is mainly derived from the studies on thiolate-heme containing epoxidases, such as cytochrome P450 epoxidases. The molecular basis of epoxidation catalyzed by nonheme-iron enzymes is much less explored. Herein, we present a detailed study on epoxidation catalyzed by the nonheme iron(II)- and 2-oxoglutarate-dependent (Fe/2OG) oxygenase, AsqJ. The native substrate and analogues with different para substituents ranging from electron-donating groups (e.g., methoxy) to electron-withdrawing groups (e.g., trifluoromethyl) were used to probe the mechanism. The results derived from transient-state enzyme kinetics, Mössbauer spectroscopy, reaction product analysis, X-ray crystallography, density functional theory calculations, and molecular dynamic simulations collectively revealed the following mechanistic insights: (1) The rapid O2 addition to the AsqJ Fe(II) center occurs with the iron-bound 2OG adopting an online-binding mode in which the C1 carboxylate group of 2OG is trans to the proximal histidine (His134) of the 2-His-1-carboxylate facial triad, instead of assuming the offline-binding mode with the C1 carboxylate group trans to the distal histidine (His211); (2) The decay rate constant of the ferryl intermediate is not strongly affected by the nature of the para substituents of the substrate during the OAT step, a reactivity behavior that is drastically different from nonheme Fe(IV)-oxo synthetic model complexes; (3) The OAT step most likely proceeds through a stepwise process with the initial formation of a C(benzylic)-O bond to generate an Fe-alkoxide species, which is observed in the AsqJ crystal structure. The subsequent C3-O bond formation completes the epoxide installation.
Assuntos
Aspergillus nidulans/metabolismo , Compostos de Epóxi/metabolismo , Proteínas Fúngicas/metabolismo , Ácidos Cetoglutáricos/metabolismo , Oxigênio/metabolismo , Oxigenases/metabolismo , Aspergillus nidulans/química , Aspergillus nidulans/enzimologia , Cristalografia por Raios X , Compostos de Epóxi/química , Proteínas Fúngicas/química , Ferro/química , Ferro/metabolismo , Modelos Moleculares , Oxigênio/química , Oxigenases/químicaRESUMO
Many cancer type-specific anticancer agents have been developed and significant advances have been made toward precision medicine in cancer treatment. However, traditional or nonspecific anticancer drugs are still important for the treatment of many cancer patients whose cancers either do not respond to or have developed resistance to cancer-specific anticancer agents. DNA topoisomerases, especially type IIA topoisomerases, are proved therapeutic targets of anticancer and antibacterial drugs. Clinically successful topoisomerase-targeting anticancer drugs act through topoisomerase poisoning, which leads to replication fork arrest and double-strand break formation. Unfortunately, this unique mode of action is associated with the development of secondary cancers and cardiotoxicity. Structures of topoisomerase-drug-DNA ternary complexes have revealed the exact binding sites and mechanisms of topoisomerase poisons. Recent advances in the field have suggested a possibility of designing isoform-specific human topoisomerase II poisons, which may be developed as safer anticancer drugs. It may also be possible to design catalytic inhibitors of topoisomerases by targeting certain inactive conformations of these enzymes. Furthermore, identification of various new bacterial topoisomerase inhibitors and regulatory proteins may inspire the discovery of novel human topoisomerase inhibitors. Thus, topoisomerases remain as important therapeutic targets of anticancer agents.
Assuntos
Antineoplásicos/química , DNA Topoisomerases Tipo II/química , DNA de Neoplasias/química , Terapia de Alvo Molecular/métodos , Neoplasias/tratamento farmacológico , Inibidores da Topoisomerase/química , Antibacterianos/química , Antibacterianos/farmacologia , Antineoplásicos/farmacologia , Domínio Catalítico , DNA/química , DNA/genética , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , DNA Topoisomerases Tipo II/genética , DNA Topoisomerases Tipo II/metabolismo , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Desenho de Fármacos , Expressão Gênica , Humanos , Simulação de Acoplamento Molecular , Neoplasias/enzimologia , Neoplasias/genética , Neoplasias/patologia , Estrutura Secundária de Proteína , Relação Estrutura-Atividade , Inibidores da Topoisomerase/farmacologiaRESUMO
Human type II topoisomerase (Top2) isoforms, hTop2α and hTop2ß, are targeted by some of the most successful anticancer drugs. These drugs induce Top2-mediated DNA cleavage to trigger cell-death pathways. The potency of these drugs correlates positively with their efficacy in stabilizing the enzyme-mediated DNA breaks. Structural analysis of hTop2α and hTop2ß revealed the presence of methionine residues in the drug-binding pocket, we therefore tested whether a tighter Top2-drug association may be accomplished by introducing a methionine-reactive Pt2+ into a drug to further stabilize the DNA break. Herein, we synthesized an organoplatinum compound, etoplatin-N2ß, by replacing the methionine-juxtaposing group of the drug etoposide with a cis-dichlorodiammineplatinum(II) moiety. Compared to etoposide, etoplatin-N2ß more potently inhibits both human Top2s. While the DNA breaks arrested by etoposide can be rejoined, those captured by etoplatin-N2ß are practically irreversible. Crystallographic analyses of hTop2ß complexed with DNA and etoplatin-N2ß demonstrate coordinate bond formation between Pt2+ and a flanking methionine. Notably, this stable coordinate tether can be loosened by disrupting the structural integrity of drug-binding pocket, suggesting that Pt2+ coordination chemistry may allow for the development of potent inhibitors with protein conformation-dependent reversibility. This approach may be exploited to achieve isoform-specific targeting of human Top2s.
Assuntos
Antineoplásicos/química , Quebras de DNA , Proteínas de Ligação a DNA/antagonistas & inibidores , Compostos Organoplatínicos/química , Podofilotoxina/análogos & derivados , Inibidores da Topoisomerase II/química , Antígenos de Neoplasias/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , DNA/química , DNA Topoisomerases Tipo II/química , Proteínas de Ligação a DNA/química , Células HL-60 , Humanos , Metionina/química , Compostos Organoplatínicos/farmacologia , Podofilotoxina/química , Podofilotoxina/farmacologia , Proteínas de Ligação a Poli-ADP-Ribose , Conformação Proteica , Inibidores da Topoisomerase II/farmacologiaRESUMO
Polyamines are organic polycations essential for cell growth and differentiation; their aberrant accumulation is often associated with diseases, including many types of cancer. To maintain polyamine homeostasis, the catalytic activity and protein abundance of ornithine decarboxylase (ODC), the committed enzyme for polyamine biosynthesis, are reciprocally controlled by the regulatory proteins antizyme isoform 1 (Az1) and antizyme inhibitor (AzIN). Az1 suppresses polyamine production by inhibiting the assembly of the functional ODC homodimer and, most uniquely, by targeting ODC for ubiquitin-independent proteolytic destruction by the 26S proteasome. In contrast, AzIN positively regulates polyamine levels by competing with ODC for Az1 binding. The structural basis of the Az1-mediated regulation of polyamine homeostasis has remained elusive. Here we report crystal structures of human Az1 complexed with either ODC or AzIN. Structural analysis revealed that Az1 sterically blocks ODC homodimerization. Moreover, Az1 binding triggers ODC degradation by inducing the exposure of a cryptic proteasome-interacting surface of ODC, which illustrates how a substrate protein may be primed upon association with Az1 for ubiquitin-independent proteasome recognition. Dynamic and functional analyses further indicated that the Az1-induced binding and degradation of ODC by proteasome can be decoupled, with the intrinsically disordered C-terminal tail fragment of ODC being required only for degradation but not binding. Finally, the AzIN-Az1 structure suggests how AzIN may effectively compete with ODC for Az1 to restore polyamine production. Taken together, our findings offer structural insights into the Az-mediated regulation of polyamine homeostasis and proteasomal degradation.
Assuntos
Proteínas de Transporte/química , Homeostase , Ornitina Descarboxilase/química , Poliaminas/química , Proteínas/química , Sequência de Aminoácidos , Biocatálise , Proteínas de Transporte/metabolismo , Cristalografia por Raios X , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Humanos , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Ornitina Descarboxilase/metabolismo , Poliaminas/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas/metabolismo , Proteólise , Homologia de Sequência de AminoácidosRESUMO
AsqJ, an iron(II)- and 2-oxoglutarate-dependent enzyme found in viridicatin-type alkaloid biosynthetic pathways, catalyzes sequential desaturation and epoxidation to produce cyclopenins. Crystal structures of AsqJ bound to cyclopeptin and its C3â epimer are reported. Meanwhile, a detailed mechanistic study was carried out to decipher the desaturation mechanism. These findings suggest that a pathway involving hydrogen atom abstraction at the C10 position of the substrate by a short-lived FeIV -oxo species and the subsequent formation of a carbocation or a hydroxylated intermediate is preferred during AsqJ-catalyzed desaturation.
Assuntos
Compostos de Epóxi/metabolismo , Proteínas Fúngicas/metabolismo , Peptídeos/metabolismo , Aspergillus nidulans/enzimologia , Biocatálise , Domínio Catalítico , Sistema Enzimático do Citocromo P-450/metabolismo , Compostos de Epóxi/química , Compostos Férricos/química , Proteínas Fúngicas/química , Ácidos Cetoglutáricos/química , Ácidos Cetoglutáricos/metabolismo , Simulação de Dinâmica Molecular , Peptídeos/química , Teoria Quântica , EstereoisomerismoRESUMO
The mer operon confers bacterial resistance to inorganic mercury (Hg(2+)) and organomercurials by encoding proteins involved in sensing, transport and detoxification of these cytotoxic agents. Expression of the mer operon is under tight control by the dual-function transcriptional regulator MerR. The metal-free, apo MerR binds to the mer operator/promoter region as a repressor to block transcription initiation, but is converted into an activator upon Hg(2+)-binding. To understand how MerR interacts with Hg(2+) and how Hg(2+)-binding modulates MerR function, we report here the crystal structures of apo and Hg(2+)-bound MerR from Bacillus megaterium, corresponding respectively to the repressor and activator conformation of MerR. To our knowledge, the apo-MerR structure represents the first visualization of a MerR family member in its intact and inducer-free form. And the Hg(2+)-MerR structure offers the first view of a triligated Hg(2+)-thiolate center in a metalloprotein, confirming that MerR binds Hg(2+) via trigonal planar coordination geometry. Structural comparison revealed the conformational transition of MerR is coupled to the assembly/disassembly of a buried Hg(2+) binding site, thereby providing a structural basis for the Hg(2+)-mediated functional switching of MerR. The pronounced Hg(2+)-induced repositioning of the MerR DNA-binding domains suggests a plausible mechanism for the transcriptional regulation of the mer operon.
Assuntos
Proteínas de Bactérias/química , Proteínas de Ligação a DNA/química , Mercúrio/química , Proteínas Repressoras/química , Transativadores/química , Bacillus megaterium/genética , Sítios de Ligação , Modelos Moleculares , Óperon , Ligação Proteica , Conformação ProteicaRESUMO
The ß-propeller is a highly symmetrical structure with 4-10 repeats of a four-stranded antiparallel ß-sheet motif. Although ß-propeller proteins with different blade numbers all adopt disc-like shapes, they are involved in a diverse set of functions, and defects in this family of proteins have been associated with human diseases. However, it has remained ambiguous how variations in blade number could alter the function of ß-propellers. In addition to the regularly arranged ß-propeller topology, a recently discovered ß-pinwheel propeller has been found. Here, we review the structural and functional diversity of ß-propeller proteins, including ß-pinwheels, as well as recent advances in the typical and atypical propeller structures.
Assuntos
Estrutura Secundária de Proteína/fisiologia , Sequência de Aminoácidos , Animais , Sequência Conservada , Enzimas/química , Enzimas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ligação Proteica , Estrutura Terciária de ProteínaRESUMO
Human cytosolic NADP(+)-dependent malic enzyme (c-NADP-ME) is neither a cooperative nor an allosteric enzyme, whereas mitochondrial NAD(P)(+)-dependent malic enzyme (m-NAD(P)-ME) is allosterically activated by fumarate. This study examines the molecular basis for the different allosteric properties and quaternary structural stability of m-NAD(P)-ME and c-NADP-ME. Multiple residues corresponding to the fumarate-binding site were mutated in human c-NADP-ME to correspond to those found in human m-NAD(P)-ME. Additionally, the crystal structure of the apo (ligand-free) human c-NADP-ME conformation was determined. Kinetic studies indicated no significant difference between the wild-type and mutant enzymes in Km,NADP, Km,malate, and kcat. A chimeric enzyme, [51-105]_c-NADP-ME, was designed to include the putative fumarate-binding site of m-NAD(P)-ME at the dimer interface of c-NADP-ME; however, this chimera remained nonallosteric. In addition to fumarate activation, the quaternary structural stability of c-NADP-ME and m-NAD(P)-ME is quite different; c-NADP-ME is a stable tetramer, whereas m-NAD(P)-ME exists in equilibrium between a dimer and a tetramer. The quaternary structures for the S57K/N59E/E73K/S102D and S57K/N59E/E73K/S102D/H74K/D78P/D80E/D87G mutants of c-NADP-ME are tetrameric, whereas the K57S/E59N/K73E/D102S m-NAD(P)-ME quadruple mutant is primarily monomeric with some dimer formation. These results strongly suggest that the structural features near the fumarate-binding site and the dimer interface are highly related to the quaternary structural stability of c-NADP-ME and m-NAD(P)-ME. In this study, we attempt to delineate the structural features governing the fumarate-induced allosteric activation of malic enzyme.
RESUMO
The jadomycins are a family of secondary metabolites produced by S. venezuelae ISP5230. Specific jadomycins have been shown to possess a variety of anticancer, antifungal, and antibacterial properties, with different molecular mechanisms of action. Herein we demonstrate qualitative and quantitative direct binding between the validated anticancer target human topoisomerase IIß and jadomycin DS using WaterLOGSY NMR spectroscopy. Additionally, we report for the first time, that jadomycin DS also binds a variety of other proteins, likely in a non-specific manner. Such interactions may rationalize the potential polypharmacology of jadomycin DS.
Assuntos
DNA Topoisomerases Tipo II/química , Proteínas de Ligação a DNA/química , Isoquinolinas/química , Sítios de Ligação , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , Espectroscopia de Ressonância Magnética , Conformação Molecular , Estereoisomerismo , Água/químicaRESUMO
Type II topoisomerases (Top2s) alter DNA topology via the formation of an enzyme-DNA adduct termed cleavage complex, which harbors a transient double-strand break in one DNA to allow the passage of another. Agents targeting human Top2s are clinically active anticancer drugs whose trapping of Top2-mediated DNA breakage effectively induces genome fragmentation and cell death. To understand the structural basis of this drug action, we previously determined the structure of human Top2 ß-isoform forming a cleavage complex with the drug etoposide and DNA, and described the insertion of drug into DNA cleavage site and drug-induced decoupling of catalytic groups. By developing a post-crystallization drug replacement procedure that simplifies structural characterization of drug-stabilized cleavage complexes, we have extended the analysis toward other structurally distinct drugs, m-AMSA and mitoxantrone. Besides the expected drug intercalation, a switch in ribose puckering in the 3'-nucleotide of the cleavage site was robustly observed in the new structures, representing a new mechanism for trapping the Top2 cleavage complex. Analysis of drug-binding modes and the conformational landscapes of the drug-binding pockets provide rationalization of the drugs' structural-activity relationships and explain why Top2 mutants exhibit differential effects toward each drug. Drug design guidelines were proposed to facilitate the development of isoform-specific Top2-targeting anticancer agents.
Assuntos
Antígenos de Neoplasias/química , Antineoplásicos/química , DNA Topoisomerases Tipo II/química , Proteínas de Ligação a DNA/química , Inibidores da Topoisomerase II/química , Amsacrina/química , Antineoplásicos/farmacologia , Desenho de Fármacos , Guias como Assunto , Humanos , Mitoxantrona/química , Modelos Moleculares , Proteínas de Ligação a Poli-ADP-Ribose , Relação Estrutura-Atividade , Inibidores da Topoisomerase II/farmacologiaRESUMO
TarI is a ribitol-5-phosphate cytidylyltransferase that catalyzes the formation of CDP-ribitol, which is involved in the biosynthesis of wall teichoic acids, from CTP and ribitol 5-phosphate. TarI from Bacillus subtilis (BsTarI) was purified and crystallized using the sitting-drop vapour-diffusion method. The crystals diffracted to a resolution of 1.78â Å and belonged to the monoclinic space group C2, with unit-cell parameters a = 103.74, b = 60.97, c = 91.80â Å, ß = 113.48°. The initial structural model indicated that the crystals of BsTarI contained a dimer in the asymmetric unit.
Assuntos
Bacillus subtilis/enzimologia , Nucleotidiltransferases/química , Cristalização , Cristalografia por Raios X , Expressão Gênica , Modelos Moleculares , Nucleotidiltransferases/genética , Nucleotidiltransferases/isolamento & purificação , Estrutura Terciária de ProteínaRESUMO
The RecQ proteins are a highly conserved group of DNA helicases which play crucial roles in the maintenance of genome stability. DrRecQ from the radioresistant bacterium Deinococcus radiodurans is a special member of the RecQ family because it contains three Helicase-and-RNase-D-C-terminal (HRDC) domains at the C-terminus. The helicase catalytic core is essential for ATPase and DNA-unwinding activities. In this work, the helicase catalytic core of DrRecQ was expressed in Escherichia coli, purified and crystallized. Crystals were obtained using the sitting-drop vapour diffusion method and X-ray diffraction data were collected to 2.9â Å resolution. The crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 84.75, b = 95.61, c = 183.83â Å.
Assuntos
Deinococcus/enzimologia , RecQ Helicases/química , Domínio Catalítico , Cristalização , Expressão Gênica , RecQ Helicases/genética , RecQ Helicases/isolamento & purificação , Difração de Raios XRESUMO
DNA gyrase is the only topoisomerase capable of introducing (-) supercoils into relaxed DNA. The C-terminal domain of the gyrase A subunit (GyrA-CTD) and the presence of a gyrase-specific 'GyrA-box' motif within this domain are essential for this unique (-) supercoiling activity by allowing gyrase to wrap DNA around itself. Here we report the crystal structure of Xanthomonas campestris GyrA-CTD and provide the first view of a canonical GyrA-box motif. This structure resembles the GyrA-box-disordered Escherichia coli GyrA-CTD, both adopting a non-planar beta-pinwheel fold composed of six seemingly spirally arranged beta-sheet blades. Interestingly, structural analysis revealed that the non-planar architecture mainly stems from the tilted packing seen between blades 1 and 2, with the packing geometry likely being defined by a conserved and unusual beta-strand-bearing proline. Consequently, the GyrA-box-containing blade 1 is placed at an angled spatial position relative to the other DNA-binding blades, and an abrupt bend is introduced into the otherwise flat DNA-binding surface. Mutagenesis studies support that the proline-induced structural twist contributes directly to gyrase's (-) supercoiling activity. To our knowledge, this is the first demonstration that a beta-strand-bearing proline may impact protein function. Potential relevance of beta-strand-bearing proline to disease phenylketonuria is also noted.
Assuntos
DNA Girase/química , Proteínas de Ligação a DNA/química , Prolina/química , Xanthomonas campestris/enzimologia , Sequência de Aminoácidos , Cristalografia por Raios X , DNA Girase/genética , DNA Girase/metabolismo , DNA Topoisomerases Tipo II/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese , Prolina/análise , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismoRESUMO
Nature has developed complexity-generating reactions within natural product biosynthetic pathways. However, direct utilization of these pathways to prepare compound libraries remains challenging due to limited substrate scopes, involvement of multiple-step reactions, and moderate robustness of these sophisticated enzymatic transformations. Synthetic chemistry, on the other hand, offers an alternative approach to prepare natural product analogs. However, owing to complex and diverse functional groups appended on the targeted molecules, dedicated design and development of synthetic strategies are typically required. Herein, by leveraging the power of chemo-enzymatic synthesis, we report an approach to bridge the gap between biological and synthetic strategies in the preparation of quinolone alkaloid analogs. Leading by in silico analysis, the predicted substrate analogs were chemically synthesized. The AsqJ-catalyzed asymmetric epoxidation of these substrate analogues was followed by an Lewis Acid-triggered ring contraction to complete the viridicatin formation. We evaluated the robustness of this method in gram-scale reactions. Lastly, through chemoenzymatic cascades, a library of quinolone alkaloids is effectively prepared.