RESUMEN
There are three key components at the core of the mpox virus (MPXV) DNA polymerase holoenzyme: DNA polymerase F8, processivity factors A22, and the Uracil-DNA glycosylase E4. The holoenzyme is recognized as a vital antiviral target because MPXV replicates in the cytoplasm of host cells. Nucleotide analogs such as cidofovir and cytarabine (Ara-C) have shown potential in curbing MPXV replication and they also display promise against other poxviruses. However, the mechanism behind their inhibitory effects remains unclear. Here, we present the cryo-EM structure of the DNA polymerase holoenzyme F8/A22/E4 bound with its competitive inhibitor Ara-C-derived cytarabine triphosphate (Ara-CTP) at an overall resolution of 3.0 Å and reveal its inhibition mechanism. Ara-CTP functions as a direct chain terminator in proximity to the deoxycytidine triphosphate (dCTP)-binding site. The extra hydrogen bond formed with Asn665 makes it more potent in binding than dCTP. Asn665 is conserved among eukaryotic B-family polymerases.
Asunto(s)
Microscopía por Crioelectrón , ADN Polimerasa Dirigida por ADN , Monkeypox virus , Proteínas Virales , Antivirales/química , Antivirales/farmacología , Sitios de Unión , Citidina Trifosfato/química , ADN Polimerasa Dirigida por ADN/química , Holoenzimas/química , Modelos Moleculares , Unión Proteica , Proteínas Virales/química , Monkeypox virus/enzimologíaRESUMEN
The novel coronavirus SARS-CoV-2 is the causative agent of the COVID-19 outbreak that is affecting the entire planet. As the pandemic is still spreading worldwide, with multiple mutations of the virus, it is of interest and of help to employ computational methods for identifying potential inhibitors of the enzymes responsible for viral replication. Attractive antiviral nucleotide analogue RNA-dependent RNA polymerase (RdRp) chain terminator inhibitors are investigated with this purpose. This study, based on molecular dynamics (MD) simulations, addresses the important aspects of the incorporation of an endogenously synthesized nucleoside triphosphate, ddhCTP, in comparison with the natural nucleobase cytidine triphosphate (CTP) in RdRp. The ddhCTP species is the product of the viperin antiviral protein as part of the innate immune response. The absence of the ribose 3'-OH in ddhCTP could have important implications in its inhibitory mechanism of RdRp. We built an in silico model of the RNA strand embedded in RdRp using experimental methods, starting from the cryo-electron microscopy structure and exploiting the information obtained by spectrometry on the RNA sequence. We determined that the model was stable during the MD simulation time. The obtained results provide deeper insights into the incorporation of nucleoside triphosphates, whose molecular mechanism by the RdRp active site still remains elusive.
Asunto(s)
COVID-19 , Citidina Trifosfato , ARN Polimerasa Dependiente del ARN , SARS-CoV-2 , Humanos , Antivirales/farmacología , Antivirales/química , Microscopía por Crioelectrón , Citidina Trifosfato/química , Simulación de Dinámica Molecular , Nucleósidos , Nucleótidos , Ribosa , ARN Viral , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , ARN Polimerasa Dependiente del ARN/química , ARN Polimerasa Dependiente del ARN/metabolismo , SARS-CoV-2/química , SARS-CoV-2/metabolismoRESUMEN
Cytidine-5'-triphosphate (CTP) synthase (CTPS) is the class I glutamine-dependent amidotransferase (GAT) that catalyzes the last step in the de novo biosynthesis of CTP. Glutamine hydrolysis is catalyzed in the GAT domain and the liberated ammonia is transferred via an intramolecular tunnel to the synthase domain where the ATP-dependent amination of UTP occurs to form CTP. CTPS is unique among the glutamine-dependent amidotransferases, requiring an allosteric effector (GTP) to activate the GAT domain for efficient glutamine hydrolysis. Recently, the first cryo-electron microscopy structure of Drosophila CTPS was solved with bound ATP, UTP, and, notably, GTP, as well as the covalent adduct with 6-diazo-5-oxo-l-norleucine. This structural information, along with the numerous site-directed mutagenesis, kinetics, and structural studies conducted over the past 50 years, provide more detailed insights into the elaborate conformational changes that accompany GTP binding at the GAT domain and their contribution to catalysis. Interactions between GTP and the L2 loop, the L4 loop from an adjacent protomer, the L11 lid, and the L13 loop (or unique flexible "wing" region), induce conformational changes that promote the hydrolysis of glutamine at the GAT domain; however, direct experimental evidence on the specific mechanism by which these conformational changes facilitate catalysis at the GAT domain is still lacking. Significantly, the conformational changes induced by GTP binding also affect the assembly and maintenance of the NH3 tunnel. Hence, in addition to promoting glutamine hydrolysis, the allosteric effector plays an important role in coordinating the reactions catalyzed by the GAT and synthase domains of CTPS.
Asunto(s)
Glutaminasa , Glutamina , Adenosina Trifosfato/metabolismo , Regulación Alostérica , Ligasas de Carbono-Nitrógeno , Microscopía por Crioelectrón , Citidina Trifosfato/química , Glutaminasa/química , Glutaminasa/metabolismo , Glutamina/metabolismo , Guanosina Trifosfato/química , Óxido Nítrico Sintasa/metabolismo , Uridina Trifosfato/química , Uridina Trifosfato/metabolismoRESUMEN
The ParABS system is supposed to be responsible for plasmid partitioning and chromosome segregation in bacteria. ParABS ensures a high degree of fidelity in inheritance by dividing the genetic material equally between daughter cells during cell division. However, the molecular mechanisms underlying the assembly of the partition complex, representing the core of the ParABS system, are still far from being understood. Here we demonstrate that the partition complex is formed via liquid-liquid phase separation. Assembly of the partition complex is initiated by the formation of oligomeric ParB species, which in turn are regulated by CTP-binding. Phase diagrams and in vivo analysis show how the partition complex can further be spatially regulated by parS. By investigating the phylogenetic variation in phase separation and its regulation by CTP, we find a high degree of evolutionary conservation among distantly related prokaryotes. These results advance the understanding of partition complex formation and regulation in general, by confirming and extending recently proposed models.
Asunto(s)
Citidina Trifosfato/química , Citidina Trifosfato/metabolismo , ADN Primasa/química , ADN Primasa/metabolismo , Bacterias/metabolismo , Fenómenos Fisiológicos Bacterianos , Proteínas Bacterianas/metabolismo , División Celular , Segregación Cromosómica , Cromosomas Bacterianos , Corynebacterium glutamicum/metabolismo , ADN Primasa/genética , ADN Primasa/aislamiento & purificación , ADN Bacteriano , Transición de Fase , FilogeniaRESUMEN
Despite the development of non-radioactive DNA/RNA labelling methods, radiolabelled nucleic acids are commonly used in studies focused on the determination of RNA fate. Nucleic acid fragments with radioactive nucleotide analoguesincorporated into the body or at the 5' or 3' terminus of the molecule can serve as probes in hybridization-based analyses of in vivo degradation and processing of transcripts. Radiolabelled oligoribonucleotides are utilized as substrates in biochemical assays of various RNA metabolic enzymes, such as exo- and endoribonucleases, nucleotidyltransferases or helicases. In some applications, the placement of the label is not a concern, while in other cases it is required that the radioactive mark is located at the 5'- or 3'-end of the molecule. An unsurpassed method for 5'-end RNA labelling employs T4 polynucleotide kinase (PNK) and [γ-32P]ATP. In the case of 3'-end labelling, several different possibilities exist. However, they require the use of costly radionucleotide analogues. Previously, we characterized an untypical nucleotidyltransferase named CutA, which preferentially incorporates cytidines at the 3'-end of RNA substrates. Here, we demonstrate that this unusual feature can be used for the development of a novel, efficient, reproducible and economical method of RNA 3'-end labelling by CutA-mediated cytidine tailing. The labelling efficiency is comparable to that achieved with the most common method applied to date, i.e. [5'-32P]pCp ligation to the RNA 3'-terminus catalysed by T4 RNA ligase I. We show the utility of RNA substrates labelled using our new method in exemplary biochemical assays assessing directionality of two well-known eukaryotic exoribonucleases, namely Dis3 and Xrn1.
Asunto(s)
Nucleotidiltransferasas/química , ARN/química , Coloración y Etiquetado/métodos , Citidina Trifosfato/química , Técnicas In Vitro , Marcaje Isotópico/métodos , Nucleótidos/química , Radioisótopos de Fósforo , ARN/genética , ARN Ligasa (ATP)/química , Coloración y Etiquetado/normas , Especificidad por Sustrato , Uridina Trifosfato/químicaRESUMEN
The naturally occurring nucleotide 3'-deoxy-3',4'-didehydro-cytidine-5'-triphosphate (ddhCTP) was recently found to exert potent and broad-spectrum antiviral activity. However, nucleoside 5'-triphosphates in general are not cell-permeable, which precludes the direct use of ddhCTP as a therapeutic. To harness the therapeutic potential of this endogenous antiviral nucleotide, we synthesized phosphoramidate prodrug HLB-0532247 (1) and found it to result in dramatically elevated levels of ddhCTP in cells. We compared 1 and 3'-deoxy-3',4'-didehydro-cytidine (ddhC) and found that 1 more effectively reduces titers of Zika and West Nile viruses in cell culture with minimal nonspecific toxicity to host cells. We conclude that 1 is a promising antiviral agent based on a novel strategy of facilitating elevated levels of the endogenous ddhCTP antiviral nucleotide.
Asunto(s)
Antivirales/farmacología , Citidina Trifosfato/farmacología , Virus del Nilo Occidental/efectos de los fármacos , Virus Zika/efectos de los fármacos , Animales , Antivirales/química , Línea Celular , Chlorocebus aethiops , Citidina Trifosfato/química , Relación Dosis-Respuesta a Droga , Humanos , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Relación Estructura-ActividadRESUMEN
Flavins play a central role in metabolism as molecules that catalyze a wide range of redox reactions in living organisms. Several variations in flavin biosynthesis exist among the domains of life, and their analysis has revealed many new structural and mechanistic insights till date. The cytidine triphosphate (CTP)-dependent riboflavin kinase in archaea is one such example. Unlike most kinases that use adenosine triphosphate, archaeal riboflavin kinases utilize CTP to phosphorylate riboflavin and produce flavin mononucleotide. In this study, we present the characterization of a new mesophilic archaeal CTP-utilizing riboflavin kinase homologue from Methanococcus maripaludis (MmpRibK), which is linked closely in sequence to the previously characterized thermophilic Methanocaldococcus jannaschii homologue. We reconstitute the activity of MmpRibK, determine its kinetic parameters and molecular factors that contribute to its unique properties, and finally establish the residues that improve its thermostability using computation and a series of experiments. Our work advances the molecular understanding of flavin biosynthesis in archaea by the characterization of the first mesophilic CTP-dependent riboflavin kinase. Finally, it validates the role of salt bridges and rigidifying amino acid residues in imparting thermostability to this unique structural fold that characterizes archaeal riboflavin kinase enzymes, with implications in enzyme engineering and biotechnological applications.
Asunto(s)
Citidina Trifosfato/química , Methanococcus/enzimología , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Ingeniería de Proteínas , Temperatura , Citidina Trifosfato/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , FilogeniaRESUMEN
NS5B is the RNA-dependent RNA polymerase that catalyzes the replication of the hepatitis C virus genome. It is a major target for antiviral drugs including nucleoside analogs, such as the prodrugs mericitabine and sofosbuvir, which get metabolized to 2'-fluoro-2'C-methylcytidine-5'-triphosphate and 2'-fluoro-2'C-methyluridine-5'-triphosphate, respectively. These analogs act as chain terminators after they are incorporated during RNA synthesis. Recently, it has been shown that NS5B can efficiently remove chain terminators by a nucleotide-mediated excision reaction that rescues RNA synthesis. In this study, we use transient-state kinetics to understand the efficiency of inhibition for five nucleoside analogs. We show that CTP analogs are readily incorporated into a growing primer by NS5B but are also efficiently excised. In contrast, although UMP analogs are more slowly incorporated, the excision of UMP is slow and inefficient, and modifications to the 2'-carbon of the UTP ribose ring further decreased rates of excision to an undetectable level. Taken together, these data suggest that the clinical effectiveness of sofosbuvir is largely a function of being intractable to nucleotide-mediated excision compared with similar nucleoside analogs.
Asunto(s)
Citidina Trifosfato , Hepacivirus/enzimología , ARN Viral/química , ARN Polimerasa Dependiente del ARN , Proteínas no Estructurales Virales/química , Citidina Trifosfato/análogos & derivados , Citidina Trifosfato/química , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , ARN Polimerasa Dependiente del ARN/químicaRESUMEN
Viperin is a radical S-adenosylmethionine (SAM) enzyme that inhibits viral replication by converting cytidine triphosphate (CTP) into 3'-deoxy-3',4'-didehydro-CTP and by additional undefined mechanisms operating through its N- and C-terminal domains. Here, we describe crystal structures of viperin bound to a SAM analogue and CTP or uridine triphosphate (UTP) and report kinetic parameters for viperin-catalyzed reactions with CTP or UTP as substrates. Viperin orients the C4' hydrogen atom of CTP and UTP similarly for abstraction by a 5'-deoxyadenosyl radical, but the uracil moiety introduces unfavorable interactions that prevent tight binding of UTP. Consistently, kcat is similar for CTP and UTP whereas the Km for UTP is much greater. The structures also show that nucleotide binding results in ordering of the C-terminal tail and reveal that this region contains a P-loop that binds the γ-phosphate of the bound nucleotide. Collectively, the results explain the selectivity for CTP and reveal a structural role for the C-terminal tail in binding CTP and UTP.
Asunto(s)
Citidina Trifosfato/química , Proteínas/química , Proteínas/metabolismo , S-Adenosilhomocisteína/química , Uridina Trifosfato/química , Animales , Cristalografía por Rayos X , Citidina Trifosfato/metabolismo , Cinética , Ratones , Modelos Moleculares , Estructura Molecular , Mutación , Proteínas/genética , S-Adenosilhomocisteína/metabolismo , Especificidad por Sustrato , Uridina Trifosfato/metabolismoRESUMEN
3'-Deoxynucleotides are an important class of drugs because they interfere with the metabolism of nucleotides, and their incorporation into DNA or RNA terminates cell division and viral replication. These compounds are generally produced by multi-step chemical synthesis, and an enzyme with the ability to catalyse the removal of the 3'-deoxy group from different nucleotides has yet to be described. Here, using a combination of HPLC, HRMS and NMR spectroscopy, we demonstrate that a thermostable fungal radical S-adenosylmethionine (SAM) enzyme, with similarity to the vertebrate antiviral enzyme viperin (RSAD2), can catalyse the transformation of CTP, UTP and 5-bromo-UTP to their 3'-deoxy-3',4'-didehydro (ddh) analogues. We show that, unlike the fungal enzyme, human viperin only catalyses the transformation of CTP to ddhCTP. Using electron paramagnetic resonance spectroscopy and molecular docking and dynamics simulations in combination with mutagenesis studies, we provide insight into the origin of the unprecedented substrate promiscuity of the enzyme and the mechanism of dehydration of a nucleotide. Our findings highlight the evolution of substrate specificity in a member of the radical-SAM enzymes. We predict that our work will help in using a new class of the radical-SAM enzymes for the biocatalytic synthesis of 3'-deoxy nucleotide/nucleoside analogues.
Asunto(s)
Citidina Trifosfato/química , Proteínas Fúngicas/química , Proteínas/química , S-Adenosilmetionina/química , Sordariales/química , Sitios de Unión , Biocatálisis , Cristalografía por Rayos X , Citidina Trifosfato/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Humanos , Cinética , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH , Filogenia , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas/genética , Proteínas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , S-Adenosilmetionina/metabolismo , Sordariales/clasificación , Sordariales/enzimología , Homología Estructural de Proteína , Especificidad por Sustrato , Termodinámica , Uridina Trifosfato/análogos & derivados , Uridina Trifosfato/química , Uridina Trifosfato/metabolismoRESUMEN
Three sets of 7-deazaadenine and cytosine nucleosides and nucleoside triphosphates bearing either unsubstituted ferrocene, octamethylferrocene and ferrocenecarboxamide linked through an alkyne tether to position 7 or 5, respectively, were designed and synthesized. The modified dNFcX TPs were good substrates for KOD XL DNA polymerase in primer extension and were used for enzymatic synthesis of redox-labelled DNA probes. Square-wave voltammetry showed that the octamethylferrocene oxidation potential was shifted to lower values, whilst the ferrocenecarboxamide was shifted to higher potentials, as compared to ferrocene. Tailed PEX products containing different ratios of Fc-labelled A (dAFc ) and FcPa-labelled C (dCFcPa ) were synthesized and hybridized with capture oligonucleotides immobilized on gold electrodes to study the electrochemistry of the redox-labelled DNA. Clearly distinguishable, fully orthogonal and ratiometric peaks were observed for the dAFc and dCFcPa bases in DNA, demonstrating their potential for use in redox coding of nucleobases and for the direct electrochemical measurement of the relative ratio of nucleobases in an unknown sequence of DNA.
Asunto(s)
ADN/química , Compuestos Ferrosos/química , Metalocenos/química , Nucleótidos/química , Coloración y Etiquetado/métodos , Citidina Trifosfato/química , ADN/metabolismo , Sondas de ADN/síntesis química , Sondas de ADN/química , ADN Polimerasa Dirigida por ADN/metabolismo , Técnicas Electroquímicas , Oxidación-Reducción , Especificidad por SustratoRESUMEN
ParABS systems facilitate chromosome segregation and plasmid partitioning in bacteria and archaea. ParB protein binds centromeric parS DNA sequences and spreads to flanking DNA. We show that ParB is an enzyme that hydrolyzes cytidine triphosphate (CTP) to cytidine diphosphate (CDP). parS DNA stimulates cooperative CTP binding by ParB and CTP hydrolysis. A nucleotide cocrystal structure elucidates the catalytic center of the dimerization-dependent ParB CTPase. Single-molecule imaging and biochemical assays recapitulate features of ParB spreading from parS in the presence but not absence of CTP. These findings suggest that centromeres assemble by self-loading of ParB DNA sliding clamps at parS ParB CTPase is not related to known nucleotide hydrolases and might be a promising target for developing new classes of antibiotics.
Asunto(s)
Bacillus subtilis/enzimología , Proteínas Bacterianas/química , Centrómero/enzimología , Citidina Trifosfato/química , Pirofosfatasas/química , Bacillus subtilis/genética , Proteínas Bacterianas/genética , Secuencias Hélice-Giro-Hélice , Hidrólisis , Secuencias Invertidas Repetidas , Dominios Proteicos , Multimerización de Proteína , Pirofosfatasas/genéticaRESUMEN
Several pathogenic bacteria utilize sialic acid, including host-derived N-acetylneuraminic acid (Neu5Ac), in at least two ways: they use it as a nutrient source and as a host-evasion strategy by coating themselves with Neu5Ac. Given the significant role of sialic acid in pathogenesis and host-gut colonization by various pathogenic bacteria, including Neisseria meningitidis, Haemophilus influenzae, Pasteurella multocida and Vibrio cholerae, several enzymes of the sialic acid catabolic, biosynthetic and incorporation pathways are considered to be potential drug targets. In this work, findings on the structural and functional characterization of CMP-N-acetylneuraminate synthetase (CMAS), a key enzyme in the incorporation pathway, from Vibrio cholerae are reported. CMAS catalyzes the synthesis of CMP-sialic acid by utilizing CTP and sialic acid. Crystal structures of the apo and the CDP-bound forms of the enzyme were determined, which allowed the identification of the metal cofactor Mg2+ in the active site interacting with CDP and the invariant Asp215 residue. While open and closed structural forms of the enzyme from eukaryotic and other bacterial species have already been characterized, a partially closed structure of V. cholerae CMAS (VcCMAS) observed upon CDP binding, representing an intermediate state, is reported here. The kinetic data suggest that VcCMAS is capable of activating the two most common sialic acid derivatives, Neu5Ac and Neu5Gc. Amino-acid sequence and structural comparison of the active site of VcCMAS with those of eukaryotic and other bacterial counterparts reveal a diverse hydrophobic pocket that interacts with the C5 substituents of sialic acid. Analyses of the thermodynamic signatures obtained from the binding of the nucleotide (CTP) and the product (CMP-sialic acid) to VcCMAS provide fundamental information on the energetics of the binding process.
Asunto(s)
Proteínas Bacterianas/química , N-Acilneuraminato Citidililtransferasa/química , Vibrio cholerae/enzimología , Secuencia de Aminoácidos , Proteínas Bacterianas/farmacología , Proteínas Bacterianas/fisiología , Sitios de Unión , Dominio Catalítico , Cristalización , Cristalografía por Rayos X/métodos , Citidina Difosfato/química , Citidina Difosfato/metabolismo , Ácido N-Acetilneuramínico Citidina Monofosfato/química , Ácido N-Acetilneuramínico Citidina Monofosfato/metabolismo , Citidina Trifosfato/química , Citidina Trifosfato/metabolismo , N-Acilneuraminato Citidililtransferasa/farmacología , N-Acilneuraminato Citidililtransferasa/fisiología , Dominios y Motivos de Interacción de Proteínas , Estructura Cuaternaria de Proteína , Ácidos Siálicos/metabolismoRESUMEN
The leading cause of bacterial gastroenteritis, Campylobacter jejuni, is a Gram-negative pathogen that contains a unique O-methyl phosphoramidate (MeOPN) on its capsular polysaccharide. Previously, MeOPN has been linked to the evasion of host immune responses and serum resistance. Despite the involvement of MeOPN in pathogenicity, the complete biosynthesis of this modification is unknown; however, the first four enzymatic steps have been elucidated. The second enzyme in this pathway, Cj1416, is a CTP/phosphoglutamine cytididylyltransferase that catalyzes the displacement of pyrophosphate from MgCTP by l-glutamine phosphate to form CDP-l-glutamine. Initially, Cj1416 was predicted to use phosphoramidate to form cytidine diphosphoramidate, but no activity was detected with MgATP as a substrate. However, in the presence of MnCTP, Cj1416 can directly catalyze the formation of cytidine diphosphoramidate from phosphoramidate and MnCTP. Here we characterize the manganese-induced promiscuity of Cj1416. In the presence of Mn2+, Cj1416 catalyzes the formation of 12 different reaction products using l-glutamine phosphate, phosphoramidate, methyl phosphate, methyl phosphonate, phosphate, arsenate, ethanolamine phosphate, glycerol-1-phosphate, glycerol-2-phosphate, serinol phosphate, l-serine phosphate, or 3-phospho-d-glycerate as the nucleophile to displace pyrophosphate from CTP.
Asunto(s)
Proteínas Bacterianas/metabolismo , Campylobacter jejuni/enzimología , Glutamina/metabolismo , Manganeso/metabolismo , Nucleotidiltransferasas/metabolismo , Amidas/química , Amidas/metabolismo , Biocatálisis , Citidina Trifosfato/química , Citidina Trifosfato/metabolismo , Modelos Químicos , Estructura Molecular , Ácidos Fosfóricos/química , Ácidos Fosfóricos/metabolismo , Especificidad por SustratoRESUMEN
Discrimination of single nucleotides by a nanopore remains a challenge because of the minor difference among the four types of single nucleotides. Here, the blockade currents induced by the translocation of single nucleotides through a 1.8 nm diameter silicon nitride nanopore have been measured. It is found that the single nucleotides are driven through the nanopore by an electroosmotic flow instead of electrophoretic force when a bias voltage is applied. The blockade currents for the four types of single nucleotides are unique and differentiable, following the order of the nucleotide volume. Also, the dwell time for each single nucleotide can last for several hundred microseconds with the advantage of the electroosmotic flow, which is helpful for single nucleotide identification. The dwell-time distributions are found to obey the first-passage time distribution from the 1D Fokker-Planck equation, from which the velocity and diffusion constant of each nucleotide can be deduced. Interestingly, the larger nucleotide is found to translocate faster than the smaller one inside the nanopore because the larger nucleotide has a larger surface area, which may produce larger drag force induced by the electroosmotic flow, which is validated by molecular dynamics simulations.
Asunto(s)
Nanoporos , Nucleótidos/química , Adenosina Trifosfato/química , Citidina Trifosfato/química , Citidina Trifosfato/metabolismo , Electroósmosis , Guanosina Trifosfato/química , Simulación de Dinámica Molecular , Tiamina/químicaRESUMEN
Viral infections continue to represent major challenges to public health, and an enhanced mechanistic understanding of the processes that contribute to viral life cycles is necessary for the development of new therapeutic strategies 1 . Viperin, a member of the radical S-adenosyl-L-methionine (SAM) superfamily of enzymes, is an interferon-inducible protein implicated in the inhibition of replication of a broad range of RNA and DNA viruses, including dengue virus, West Nile virus, hepatitis C virus, influenza A virus, rabies virus 2 and HIV3,4. Viperin has been suggested to elicit these broad antiviral activities through interactions with a large number of functionally unrelated host and viral proteins3,4. Here we demonstrate that viperin catalyses the conversion of cytidine triphosphate (CTP) to 3'-deoxy-3',4'-didehydro-CTP (ddhCTP), a previously undescribed biologically relevant molecule, via a SAM-dependent radical mechanism. We show that mammalian cells expressing viperin and macrophages stimulated with IFNα produce substantial quantities of ddhCTP. We also establish that ddhCTP acts as a chain terminator for the RNA-dependent RNA polymerases from multiple members of the Flavivirus genus, and show that ddhCTP directly inhibits replication of Zika virus in vivo. These findings suggest a partially unifying mechanism for the broad antiviral effects of viperin that is based on the intrinsic enzymatic properties of the protein and involves the generation of a naturally occurring replication-chain terminator encoded by mammalian genomes.
Asunto(s)
Antivirales/metabolismo , Citidina Trifosfato/metabolismo , Genoma Humano/genética , Proteínas/genética , Proteínas/metabolismo , Terminación de la Transcripción Genética , Animales , Antivirales/química , Chlorocebus aethiops , Citidina Trifosfato/biosíntesis , Citidina Trifosfato/química , Células HEK293 , Humanos , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , ARN Polimerasa Dependiente del ARN/metabolismo , Ribonucleótidos , Especificidad por Sustrato , Células Vero , Virus Zika/enzimología , Virus Zika/metabolismoRESUMEN
Potentiometric and NMR spectroscopic studies of the nucleotide (NucP)/polyamine (PA) system (where NucPâ¯=â¯CDP, CTP, PAâ¯=â¯putrescine or spermidine) revealed the formation of molecular complexes (NucP)(Hx+y)(PA) (where Hx+yâ¯=â¯number of protons; x - from NucP and y - from PA). Their thermodynamic parameters were determined and the modes of their interactions were proposed. The main reaction centers were found to be the protonated amine groups of polyamine (positive centers) and phosphate groups of nucleotide (negative centers). The pH ranges in which the complex occurs correspond to those of amine protonation and -PO3x- group deprotonation, which unambiguously confirms the dipole-dipole type of interaction. In the pH range of total deprotonation of NHx+ groups from the polyamine, the molecular complexes disappear. The equilibrium and spectroscopic studies of the ternary systems Cu(II)/NucP/PA evidenced the formation of Cu(NucP)Hx+y(PA) type coordination compounds and Cu(NucP)â¯(PA)(Hx) type molecular complexes with polyamine in the outer coordination sphere. The main sites of metal ion bonding in the latter species are the phosphate groups of the nucleotide, while in the coordination compounds - besides the phosphate groups - also the donor nitrogen atoms from the polyamines. In this paper we have also quantitatively calculated the effect of metal ions on the formation of the molecular complexes.
Asunto(s)
Aminas Biogénicas/química , Cobre/química , Citidina Difosfato/química , Citidina Trifosfato/química , Nucleótidos/química , Putrescina/química , Espermidina/químicaRESUMEN
The divergence of archaea, bacteria and eukaryotes was a fundamental step in evolution. One marker of this event is a major difference in membrane lipid chemistry between these kingdoms. Whereas the membranes of bacteria and eukaryotes primarily consist of straight fatty acids ester-bonded to glycerol-3-phosphate, archaeal phospholipids consist of isoprenoid chains ether-bonded to glycerol-1-phosphate. Notably, the mechanisms underlying the biosynthesis of these lipids remain elusive. Here, we report the structure of the CDP-archaeol synthase (CarS) of Aeropyrum pernix (ApCarS) in the CTP- and Mg2+-bound state at a resolution of 2.4 Å. The enzyme comprises a transmembrane domain with five helices and cytoplasmic loops that together form a large charged cavity providing a binding site for CTP. Identification of the binding location of CTP and Mg2+ enabled modeling of the specific lipophilic substrate-binding site, which was supported by site-directed mutagenesis, substrate-binding affinity analyses, and enzyme assays. We propose that archaeol binds within two hydrophobic membrane-embedded grooves formed by the flexible transmembrane helix 5 (TM5), together with TM1 and TM4. Collectively, structural comparisons and analyses, combined with functional studies, not only elucidated the mechanism governing the biosynthesis of phospholipids with ether-bonded isoprenoid chains by CTP transferase, but also provided insights into the evolution of this enzyme superfamily from archaea to bacteria and eukaryotes.
Asunto(s)
Aeropyrum/enzimología , Proteínas Arqueales/química , Transferasas/química , Proteínas Arqueales/metabolismo , Sitios de Unión , Citidina Trifosfato/química , Lípidos de la Membrana/biosíntesis , Metales/química , Modelos Moleculares , Thermotoga maritima/enzimología , Transferasas/metabolismoRESUMEN
Noncanonical RNA nucleotidyltransferases (NTases), including poly(A), poly(U) polymerases (PAPs/PUPs), and C/U-adding enzymes, modify 3'-ends of different transcripts affecting their functionality and stability. They contain PAP/OAS1 substrate-binding domain (SBD) with inserted NTase domain. Aspergillus nidulans CutA (AnCutA), synthesizes C/U-rich 3'-terminal extensions in vivo. Here, using high-throughput sequencing of the 3'-RACE products for tails generated by CutA proteins in vitro in the presence of all four NTPs, we show that even upon physiological ATP excess synthesized tails indeed contain an unprecedented number of cytidines interrupted by uridines and stretches of adenosines, and that the majority end with two cytidines. Strikingly, processivity assays documented that in the presence of CTP as a sole nucleotide, the enzyme terminates after adding two cytidines only. Comparison of our CutA 3D model to selected noncanonical NTases of known structures revealed substantial differences in the nucleotide recognition motif (NRM) within PAP/OAS1 SBD. We demonstrate that CutA specificity toward CTP can be partially changed to PAP or PUP by rational mutagenesis within NRM and, analogously, Cid1 PUP can be converted into a C/U-adding enzyme. Collectively, we suggest that a short cluster of amino acids within NRM is a determinant of NTases' substrate preference, which may allow us to predict their specificity.
Asunto(s)
Aspergillus nidulans/enzimología , Biología Computacional/métodos , Citidina Trifosfato/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Nucleotidiltransferasas/química , Nucleotidiltransferasas/metabolismo , Secuencia de Aminoácidos , Citidina/química , Citidina Trifosfato/química , Modelos Moleculares , Homología de Secuencia , Especificidad por SustratoRESUMEN
P2X receptors are trimeric ATP-gated cation channels involved in diverse physiological processes, ranging from muscle contraction to nociception. Despite the recent structure determination of the ATP-bound P2X receptors, the molecular mechanism of the nucleotide base specificity has remained elusive. Here, we present the crystal structure of zebrafish P2X4 in complex with a weak affinity agonist, CTP, together with structure-based electrophysiological and spectroscopic analyses. The CTP-bound structure revealed a hydrogen bond, between the cytosine base and the side chain of the basic residue in the agonist binding site, which mediates the weak but significant affinity for CTP. The cytosine base is further recognized by two main chain atoms, as in the ATP-bound structure, but their bond lengths seem to be extended in the CTP-bound structure, also possibly contributing to the weaker affinity for CTP over ATP. This work provides the structural insights for the nucleotide base specificity of P2X receptors.