Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Dengue viruses (DENV) have 5'-capped RNA genomes of (+) polarity and encode a single polyprotein precursor that is processed into mature viral proteins. NS2B, NS3 and NS5 proteins catalyse/activate enzyme activities that are required for key processes in the virus life cycle. The heterodimeric NS2B/NS3 is a serine protease required for processing. Using a high-throughput protease assay, we screened a small molecule chemical library and identified -200 compounds having > or = 50% inhibition. Moreover, NS3 exhibits RNA-stimulated NTPase, RNA helicase and the 5'-RNA triphosphatase activities. The NTPase and the 5'-RTPase activities of NS3 are stimulated by interaction with NS5. Moreover, the conserved, positively charged motif in DENV-2 NS3, 184RKRK, is required for RNA binding and modulates the RNA-dependent enzyme activities of NS3. To study viral replication, a variety of methods are used such as the in vitro RNA-dependent RNA polymerase assays that utilize lysates from DENV-2-infected mosquito or mammalian cells or the purified NS5 along with exogenous short subgenomic viral RNAs or the replicative intracellular membrane-bound viral RNAs as templates. In addition, a cell-based DENV-2 replicon RNA encoding a luciferase reporter is also used to examine the role of cis-acting elements within the 3' UTR and the RKRK motif in viral replication.
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PMID:Multiple enzyme activities of flavivirus proteins. 1731 55

Infectious diseases caused by flaviviruses are important emerging public health concerns and new vaccines and therapeutics are urgently needed. The NS3 protein from flavivirus is a multifunctional protein with protease, helicase and nucleoside 5' triphosphatase activities (NTPase). Thus, NS3 plays a crucial role in viral replication and represents an interesting target for the development of specific antiviral inhibitors. We have solved the structure of an enzymatically active fragment of the dengue virus NTPase/ helicase C-terminal catalytic domain in several related crystal forms. The structure is composed of three domains, bears an asymmetric distribution of charges and comprises a tunnel large enough to accommodate single strand RNA. A concave face formed by domains 2 and 3 is proposed to bind a nucleic acid duplex substrate. Comparison of the various copies of dengue and yellow fever virus NS3 NTPase/helicase catalytic domains reveals mobile regions of the enzyme. Such dynamic behaviour is likely to be coupled with directional translocation along the single strand nucleic acid substrate during strand separation. We used structure-based site directed mutagenesis to identify regions of the enzyme that are crucial for its ATPase or nucleic acid duplex unwinding activity.
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PMID:Towards the design of flavivirus helicase/NTPase inhibitors: crystallographic and mutagenesis studies of the dengue virus NS3 helicase catalytic domain. 1731 56

Hepatitis C virus NS3 helicase is an enzyme that unwinds double-stranded polynucleotides in an ATP-dependent reaction. It provides a promising target for small molecule therapeutic agents against hepatitis C. Design of such drugs requires a thorough understanding of the dynamical nature of the mechanochemical functioning of the helicase. Despite recent progress, the detailed mechanism of the coupling between ATPase activity and helicase activity remains unclear. Based on an elastic network model (ENM), we apply two computational analysis tools to probe the dynamical mechanism underlying the allosteric coupling between ATP binding and polynucleotide binding in this enzyme. The correlation analysis identifies a network of hot-spot residues that dynamically couple the ATP-binding site and the polynucleotide-binding site. Several of these key residues have been found by mutational experiments as functionally important, while our analysis also reveals previously unexplored hot-spot residues that are potential targets for future mutational studies. The conformational changes between different crystal structures of NS3 helicase are found to be dominated by the lowest frequency mode solved from the ENM. This mode corresponds to a hinge motion of the highly flexible domain 2. This motion simultaneously modulates the opening/closing of the domains 1-2 cleft where ATP binds, and the domains 2-3 cleft where the polynucleotide binds. Additionally, a small twisting motion of domain 1, observed in both mode 1 and the computed ATP binding induced conformational change, fine-tunes the binding affinity of the domains 1-3 interface for the polynucleotide. The combination of these motions facilitates the translocation of a single-stranded polynucleotide in an inchworm-like manner.
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PMID:Toward the mechanism of dynamical couplings and translocation in hepatitis C virus NS3 helicase using elastic network model. 1737 6

Flaviviral NS3 is a multifunctional protein displaying N-terminal protease activity in addition to C-terminal helicase, nucleoside 5'-triphosphatase (NTPase), and 5'-terminal RNA triphosphatase (RTPase) activities. NS3 is held to support the separation of RNA daughter and template strands during viral replication. In addition, NS3 assists the initiation of replication by unwinding the RNA secondary structure in the 3' non-translated region (NTR). We report here the three-dimensional structure (at 3.1 A resolution) of the NS3 helicase domain (residues 186-619; NS3:186-619) from Kunjin virus, an Australian variant of the West Nile virus. As for homologous helicases, NS3:186-619 is composed of three domains, two of which are structurally related and held to host the NTPase and RTPase active sites. The third domain (C-terminal) is involved in RNA binding/recognition. The NS3:186-619 construct occurs as a dimer in solution and in the crystals. We show that NS3:186-619 displays both ATPase and RTPase activities, that it can unwind a double-stranded RNA substrate, being however inactive on a double-stranded DNA substrate. Analysis of different constructs shows that full length NS3 displays increased helicase activity, suggesting that the protease domain plays an assisting role in the RNA unwinding process. The structural interaction between the helicase and protease domain has been assessed using small angle X-ray scattering on full length NS3, disclosing that the protease and helicase domains build a rather elongated molecular assembly differing from that observed in the NS3 protein from hepatitis C virus.
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PMID:Crystal structure and activity of Kunjin virus NS3 helicase; protease and helicase domain assembly in the full length NS3 protein. 1765 51

Nonstructural (NS) protein 3 is a DEXH/D-box motor protein that is an essential component of the hepatitis C viral (HCV) replicative complex. The full-length NS3 protein contains two functional modules, both of which are essential in the life cycle of HCV: a serine protease domain at the N terminus and an ATPase/helicase domain (NS3hel) at the C terminus. Truncated NS3hel constructs have been studied extensively; the ATPase, nucleic acid binding, and helicase activities have been examined and NS3hel has been used as a target in the development of antivirals. However, a comprehensive comparison of NS3 and NS3hel activities has not been performed, so it remains unclear whether the protease domain plays a vital role in NS3 helicase function. Given that many DEXH/D-box proteins are activated upon interaction with cofactor proteins, it is important to establish if the protease domain acts as the cofactor for stimulating NS3 helicase function. Here we show that the protease domain greatly enhances both the direct and functional binding of RNA to NS3. Whereas electrostatics plays an important role in this process, there is a specific allosteric contribution from the interaction interface between NS3hel and the protease domain. Most importantly, we establish that the protease domain is required for RNA unwinding by NS3. Our results suggest that, in addition to its role in cleavage of host and viral proteins, the NS3 protease domain is essential for the process of viral RNA replication and, given its electrostatic contribution to RNA binding, it may also assist in packaging of the viral RNA.
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PMID:The serine protease domain of hepatitis C viral NS3 activates RNA helicase activity by promoting the binding of RNA substrate. 1792 Nov 46

Hepatitis C virus (HCV) chronic infections represent one of the major and still unresolved health problems because of low efficiency and high cost of current therapy. Therefore, our studies centered on a viral protein, the NS3 helicase, whose activity is indispensable for replication of the viral RNA, and on its peptide inhibitor that corresponds to a highly conserved arginine-rich sequence of domain 2 of the helicase. The NS3 peptide (p14) was expressed in bacteria. Its 50% inhibitory activity in a fluorometric helicase assay corresponded to 725 nM, while the ATPase activity of NS3 was not affected. Nuclear magnetic resonance (NMR) studies of peptide-protein interactions using the relaxation filtering technique revealed that p14 binds directly to the full-length helicase and its separately expressed domain 1 but not to domain 2. Changes in the NMR chemical shift of backbone amide nuclei ((1)H and (15)N) of domain 1 or p14, measured during complex formation, were used to identify the principal amino acids of both domain 1 and the peptide engaged in their interaction. In the proposed interplay model, p14 contacts the clefts between domains 1 and 2, as well as between domains 1 and 3, preventing substrate binding. This interaction is strongly supported by cross-linking experiments, as well as by kinetic studies performed using a fluorometric assay. The antiviral activity of p14 was tested in a subgenomic HCV replicon assay that showed that the peptide at micromolar concentrations can reduce HCV RNA replication.
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PMID:NS3 Peptide, a novel potent hepatitis C virus NS3 helicase inhibitor: its mechanism of action and antiviral activity in the replicon system. 1803 21

Hepatitis C virus (HCV) infects over 170 million persons worldwide. It is the leading cause of liver disease in the U.S. and is responsible for most liver transplants. Current treatments for this infectious disease are inadequate; therefore, new therapies must be developed. Several labs have obtained evidence for a protein complex that involves many of the nonstructural (NS) proteins encoded by the virus. NS3, NS4A, NS4B, NS5A, and NS5B appear to interact structurally and functionally. In this study, we investigated the interaction between the helicase, NS3, and the RNA polymerase, NS5B. Pull-down experiments and surface plasmon resonance data indicate a direct interaction between NS3 and NS5B that is primarily mediated through the protease domain of NS3. This interaction reduces the basal ATPase activity of NS3. However, NS5B stimulates product formation in RNA unwinding experiments under conditions of excess nucleic acid substrate. When the concentrations of NS3 and NS5B are in excess of nucleic acid substrate, NS5B reduces the rate of NS3-catalyzed unwinding. Under pre-steady-state conditions, in which NS3 and substrate concentrations are similar, product formation increased in the presence of NS5B. The increase was consistent with 1:1 complex formed between the two proteins. A fluorescently labeled form of NS3 was used to investigate this interaction through fluorescence polarization binding assays. Results from this assay support interactions that include a 1:1 complex formed between NS3 and NS5B. The modulation of NS3 by NS5B suggests that these proteins may function together during replication of the HCV genome.
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PMID:RNA unwinding activity of the hepatitis C virus NS3 helicase is modulated by the NS5B polymerase. 1817 52

The NS3 protein of Japanese encephalitis virus (JEV) is a large multifunctional protein possessing protease, helicase, and nucleoside 5'-triphosphatase (NTPase) activities, and plays important roles in the processing of a viral polyprotein and replication. To clarify the enzymatic properties of NS3 protein from a structural point of view, an enzymatically active fragment of the JEV NTPase/helicase catalytic domain was expressed in bacteria and the crystal structure was determined at 1.8 A resolution. JEV helicase is composed of three domains, displays an asymmetric distribution of charges on its surface, and contains a tunnel large enough to accommodate single-stranded RNA. Each of the motifs I (Walker A motif), II (Walker B motif) and VI was composed of an NTP-binding pocket. Mutation analyses revealed that all of the residues in the Walker A motif (Gly(199), Lys(200) and Thr(201)), in addition to the polar residues within the NTP-binding pocket (Gln(457), Arg(461) and Arg(464)), and also Arg(458) in the outside of the pocket in the motif IV were crucial for ATPase and helicase activities and virus replication. Lys(200) was particularly indispensable, and could not be exchanged for other amino acid residues without sacrificing these activities. The structure of the NTP-binding pocket of JEV is well conserved in dengue virus and yellow fever virus, while different from that of hepatitis C virus. The detailed structural comparison among the viruses of the family Flaviviridae should help in clarifying the molecular mechanism of viral replication and in providing rationale for the development of appropriate therapeutics.
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PMID:Crystal structure of the catalytic domain of Japanese encephalitis virus NS3 helicase/nucleoside triphosphatase at a resolution of 1.8 A. 1820 43

The development of effective therapies for hepatitis C virus (HCV) must take into account genetic variation among HCV strains. Response rates to interferon-based treatments, including the current preferred treatment of pegylated alpha interferon administered with ribavirin, are genotype specific. Of the numerous HCV inhibitors currently in development as antiviral drugs, nucleoside analogs that target the conserved NS5B active site seem to be quite effective against diverse HCV strains. To test this hypothesis, we examined the effects of a panel of nucleotide analogs, including ribavirin triphosphate (RTP) and several chain-terminating nucleoside triphosphates, on the activities of purified HCV NS5B polymerases derived from genotype 1a, 1b, and 2a strains. Unlike the genotype-specific effects on NS5B activity reported previously for nonnucleoside inhibitors (F. Pauwels, W. Mostmans, L. M. Quirynen, L. van der Helm, C. W. Boutton, A. S. Rueff, E. Cleiren, P. Raboisson, D. Surleraux, O. Nyanguile, and K. A. Simmen, J. Virol. 81:6909-6919, 2007), only minor differences in inhibition were observed among the various genotypes; thus, nucleoside analogs that are current drug candidates may be more promising for treatment of a broader variety of HCV strains. We also examined the effects of RTP on the HCV NS3 helicase/ATPase. As with the polymerase, only minor differences were observed among 1a-, 1b-, and 2a-derived enzymes. RTP did not inhibit the rate of NS3 helicase-catalyzed DNA unwinding but served instead as a substrate to fuel unwinding. NS3 added to RNA synthesis reactions relieved inhibition of the polymerase by RTP, presumably due to RTP hydrolysis. These results suggest that NS3 can limit the incorporation of ribavirin into viral RNA, thus reducing its inhibitory or mutagenic effects.
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PMID:Effects of mutagenic and chain-terminating nucleotide analogs on enzymes isolated from hepatitis C virus strains of various genotypes. 1839 Oct 43

Similar to many flavivirus types including Dengue and yellow fever viruses, the nonstructural NS3 multifunctional protein of West Nile virus (WNV) with an N-terminal serine proteinase domain and an RNA triphosphatase, an NTPase domain, and an RNA helicase in the C-terminal domain is implicated in both polyprotein processing and RNA replication and is therefore a promising drug target. To exhibit its proteolytic activity, NS3 proteinase requires the presence of the cofactor encoded by the upstream NS2B sequence. During our detailed investigation of the biology of the WNV helicase, we characterized the ATPase and RNA/DNA unwinding activities of the full-length NS2B-NS3 proteinase-helicase protein as well as the individual NS3 helicase domain lacking both the NS2B cofactor and the NS3 proteinase sequence and the individual NS3 proteinase-helicase lacking only the NS2B cofactor. We determined that both the NS3 helicase and NS3 proteinase-helicase constructs are capable of unwinding both the DNA and the RNA templates. In contrast, the full-length NS2B-NS3 proteinase-helicase unwinds only the RNA templates, whereas its DNA unwinding activity is severely repressed. Our data suggest that the productive, catalytically competent fold of the NS2B-NS3 proteinase moiety represents an essential component of the RNA-DNA substrate selectivity mechanism in WNV and, possibly, in other flaviviruses. Based on our data, we hypothesize that the mechanism we have identified plays a role yet to be determined in WNV replication occurring both within the virus-induced membrane-bound replication complexes in the host cytoplasm and in the nuclei of infected cells.
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PMID:The two-component NS2B-NS3 proteinase represses DNA unwinding activity of the West Nile virus NS3 helicase. 1844 76


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