scholarly journals West Nile Virus Methyltransferase Catalyzes Two Methylations of the Viral RNA Cap through a Substrate-Repositioning Mechanism

2008 ◽  
Vol 82 (9) ◽  
pp. 4295-4307 ◽  
Author(s):  
Hongping Dong ◽  
Suping Ren ◽  
Bo Zhang ◽  
Yangsheng Zhou ◽  
Francesc Puig-Basagoiti ◽  
...  
Keyword(s):  
West Nile Virus ◽  
Binding Site ◽  
Binding Sites ◽  
Biochemical Analysis ◽  
Rna Binding ◽  
Binding Pocket ◽  
Viral Rna ◽  
West Nile ◽  
Rna Molecules ◽  
Gtp Binding

ABSTRACT Flaviviruses encode a single methyltransferase domain that sequentially catalyzes two methylations of the viral RNA cap, GpppA-RNA→m7GpppA-RNA→m7GpppAm-RNA, by using S-adenosyl-l-methionine (SAM) as a methyl donor. Crystal structures of flavivirus methyltransferases exhibit distinct binding sites for SAM, GTP, and RNA molecules. Biochemical analysis of West Nile virus methyltransferase shows that the single SAM-binding site donates methyl groups to both N7 and 2′-O positions of the viral RNA cap, the GTP-binding pocket functions only during the 2′-O methylation, and two distinct sets of amino acids in the RNA-binding site are required for the N7 and 2′-O methylations. These results demonstrate that flavivirus methyltransferase catalyzes two cap methylations through a substrate-repositioning mechanism. In this mechanism, guanine N7 of substrate GpppA-RNA is first positioned to SAM to generate m7GpppA-RNA, after which the m7G moiety is repositioned to the GTP-binding pocket to register the 2′-OH of the adenosine with SAM, generating m7GpppAm-RNA. Because N7 cap methylation is essential for viral replication, inhibitors designed to block the pocket identified for the N7 cap methylation could be developed for flavivirus therapy.

scholarly journals Cell Proteins TIA-1 and TIAR Interact with the 3′ Stem-Loop of the West Nile Virus Complementary Minus-Strand RNA and Facilitate Virus Replication

2002 ◽  
Vol 76 (23) ◽  
pp. 11989-12000 ◽  
Author(s):  
W. Li ◽  
Y. Li ◽  
N. Kedersha ◽  
P. Anderson ◽  
M. Emara ◽  
...  
Keyword(s):  
West Nile Virus ◽  
Rna Binding ◽  
Peptide Sequencing ◽  
Viral Rna ◽  
Cell Protein ◽  
Affinity Column ◽  
Rna Recognition Motif ◽  
The West ◽  
West Nile ◽  
Stem Loop

ABSTRACT It was reported previously that four baby hamster kidney (BHK) proteins with molecular masses of 108, 60, 50, and 42 kDa bind specifically to the 3′-terminal stem-loop of the West Nile virus minus-stand RNA [WNV 3′(−) SL RNA] (P. Y. Shi, W. Li, and M. A. Brinton, J. Virol. 70:6278-6287, 1996). In this study, p42 was purified using an RNA affinity column and identified as TIAR by peptide sequencing. A 42-kDa UV-cross-linked viral RNA-cell protein complex formed in BHK cytoplasmic extracts incubated with the WNV 3′(−) SL RNA was immunoprecipitated by anti-TIAR antibody. Both TIAR and the closely related protein TIA-1 are members of the RNA recognition motif (RRM) family of RNA binding proteins. TIA-1 also binds to the WNV 3′(−) SL RNA. The specificity of these viral RNA-cell protein interactions was demonstrated using recombinant proteins in competition gel mobility shift assays. The binding site for the WNV 3′(−) SL RNA was mapped to RRM2 on both TIAR and TIA-1. However, the dissociation constant (Kd ) for the interaction between TIAR RRM2 and the WNV 3′(−) SL RNA was 1.5 × 10−8, while that for TIA-1 RRM2 was 1.12 × 10−7. WNV growth was less efficient in murine TIAR knockout cell lines than in control cells. This effect was not observed for two other types of RNA viruses or two types of DNA viruses. Reconstitution of the TIAR knockout cells with TIAR increased the efficiency of WNV growth, but neither the level of TIAR nor WNV replication was as high as in control cells. These data suggest a functional role for TIAR and possibly also for TIA-1 during WNV replication.

scholarly journals West Nile virus strain Kunjin NS5 polymerase is a phosphoprotein localized at the cytoplasmic site of viral RNA synthesis

2007 ◽  
Vol 88 (4) ◽  
pp. 1163-1168 ◽  
Author(s):  
Jason M. Mackenzie ◽  
Mark T. Kenney ◽  
Edwin G. Westaway
Keyword(s):  
West Nile Virus ◽  
Rna Polymerase ◽  
Virus Strain ◽  
Viral Rna ◽  
West Nile ◽  
Replication Complex ◽  
Virus Rna ◽  
West Nile Virus Strain ◽  
First Time ◽  
Cytoplasmic Location

Using West Nile virus strain Kunjin virus (WNVKUN) as a model system for flavivirus replication, we showed that the virus replication complex (RC) is associated with the dsRNA template located in induced membranes only in the cytoplasm. In this report we established for the first time that the RNA-dependent RNA polymerase NS5 is located in flavivirus-induced membranes, including the site of viral RNA replication. We found no evidence for nuclear localization of the essential RC components NS5 and its dsRNA template for WNVKUN or the closely related WNV strain Sarafend, by immuno-electron microscopy or by immunofluorescence. Metabolic radiolabelling with [32P]orthophosphate revealed that WNVKUN NS5 was phosphorylated and this was confirmed by Western blotting with antibodies specific for phosphorylated serine and threonine only. These observations of a cytoplasmic location for the WNV polymerase and its phosphorylation state correspond to the characteristics of the hepatitis C virus RNA polymerase NS5B.

scholarly journals One Intact Transmembrane Substrate Binding Site Is Sufficient for the Function of the Homodimeric Type I ATP-Binding Cassette Importer for Positively Charged Amino Acids Art(MP) 2 of Geobacillus stearothermophilus

2018 ◽  
Vol 200 (12) ◽  
Author(s):  
Johanna Heuveling ◽  
Heidi Landmesser ◽  
Erwin Schneider
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Substrate Binding ◽  
Binding Pocket ◽  
Atp Binding ◽  
Content Type ◽  
Charged Amino Acids ◽  
Substrate Binding Sites ◽  
Positively Charged ◽  
Atp Binding Cassette

ABSTRACT ATP-binding cassette (ABC) transport systems comprise two transmembrane domains/subunits that form a translocation path and two nucleotide-binding domains/subunits that bind and hydrolyze ATP. Prokaryotic canonical ABC import systems require an extracellular substrate-binding protein for function. Knowledge of substrate-binding sites within the transmembrane subunits is scarce. Recent crystal structures of the ABC importer Art(QN) 2 for positively charged amino acids of Thermoanerobacter tengcongensis revealed the presence of one substrate molecule in a defined binding pocket in each of the transmembrane subunits, ArtQ (J. Yu, J. Ge, J. Heuveling, E. Schneider, and M. Yang, Proc Natl Acad Sci U S A 112:5243–5248, 2015, https://doi.org/10.1073/pnas.1415037112 ). This finding raised the question of whether both sites must be loaded with substrate prior to initiation of the transport cycle. To address this matter, we first explored the role of key residues that form the binding pocket in the closely related Art(MP) 2 transporter of Geobacillus stearothermophilus , by monitoring consequences of mutations in ArtM on ATPase and transport activity at the level of purified proteins embedded in liposomes. Our results emphasize that two negatively charged residues (E153 and D160) are crucial for wild-type function. Furthermore, the variant Art[M(L67D)P] 2 exhibited strongly impaired activities, which is why it was considered for construction of a hybrid complex containing one intact and one impaired substrate-binding site. Activity assays clearly revealed that one intact binding site was sufficient for function. To our knowledge, our study provides the first biochemical evidence on transmembrane substrate-binding sites of an ABC importer. IMPORTANCE Canonical prokaryotic ATP-binding cassette importers mediate the uptake of a large variety of chemicals, including nutrients, osmoprotectants, growth factors, and trace elements. Some also play a role in bacterial pathogenesis, which is why full understanding of their mode of action is of the utmost importance. One of the unsolved problems refers to the chemical nature and number of substrate binding sites formed by the transmembrane subunits. Here, we report that a hybrid amino acid transporter of G. stearothermophilus , encompassing one intact and one impaired transmembrane binding site, is fully competent in transport, suggesting that the binding of one substrate molecule is sufficient to trigger the translocation process.

scholarly journals Evolutionary meandering of intermolecular interactions along the drift barrier

2014 ◽  
Vol 112 (1) ◽  
pp. E30-E38 ◽  
Author(s):  
Michael Lynch ◽  
Kyle Hagner
Keyword(s):  
Intermolecular Interactions ◽  
Binding Sites ◽  
Rna Binding ◽  
Fine Tuning ◽  
Evolutionary Divergence ◽  
Sequence Motif ◽  
Cellular Functions ◽  
Mutation Pressure ◽  
Rna Molecules ◽  
The Face

Many cellular functions depend on highly specific intermolecular interactions, for example transcription factors and their DNA binding sites, microRNAs and their RNA binding sites, the interfaces between heterodimeric protein molecules, the stems in RNA molecules, and kinases and their response regulators in signal-transduction systems. Despite the need for complementarity between interacting partners, such pairwise systems seem to be capable of high levels of evolutionary divergence, even when subject to strong selection. Such behavior is a consequence of the diminishing advantages of increasing binding affinity between partners, the multiplicity of evolutionary pathways between selectively equivalent alternatives, and the stochastic nature of evolutionary processes. Because mutation pressure toward reduced affinity conflicts with selective pressure for greater interaction, situations can arise in which the expected distribution of the degree of matching between interacting partners is bimodal, even in the face of constant selection. Although biomolecules with larger numbers of interacting partners are subject to increased levels of evolutionary conservation, their more numerous partners need not converge on a single sequence motif or be increasingly constrained in more complex systems. These results suggest that most phylogenetic differences in the sequences of binding interfaces are not the result of adaptive fine tuning but a simple consequence of random genetic drift.

scholarly journals Monoclonal antibodies to the West Nile virus NS5 protein map to linear and conformational epitopes in the methyltransferase and polymerase domains

2009 ◽  
Vol 90 (12) ◽  
pp. 2912-2922 ◽  
Author(s):  
Roy A. Hall ◽  
Si En Tan ◽  
Barbara Selisko ◽  
Rachael Slade ◽  
Jody Hobson-Peters ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
West Nile Virus ◽  
Binding Sites ◽  
Synthetic Peptides ◽  
Structural Model ◽  
Full Length ◽  
Physical Interaction ◽  
Rdrp Activity ◽  
The West ◽  
West Nile

The West Nile virus (WNV) NS5 protein contains a methyltransferase (MTase) domain involved in RNA capping and an RNA-dependent RNA polymerase (RdRp) domain essential for virus replication. Crystal structures of individual WNV MTase and RdRp domains have been solved; however, the structure of full-length NS5 has not been determined. To gain more insight into the structure of NS5 and interactions between the MTase and RdRp domains, we generated a panel of seven monoclonal antibodies (mAbs) to the NS5 protein of WNV (Kunjin strain) and mapped their binding sites using a series of truncated NS5 proteins and synthetic peptides. Binding sites of four mAbs (5D4, 4B6, 5C11 and 6A10) were mapped to residues 354–389 in the fingers subdomain of the RdRp. This is consistent with the ability of these mAbs to inhibit RdRp activity in vitro and suggests that this region represents a potential target for RdRp inhibitors. Using a series of synthetic peptides, we also identified a linear epitope (bound by mAb 5H1) that mapped to a 13 aa stretch surrounding residues 47 and 49 in the MTase domain, a region predicted to interact with the palm subdomain of the RdRp. The failure of one mAb (7G6) to bind both N- and C-terminally truncated NS5 recombinants indicates that the antibody recognizes a conformational epitope that requires the presence of residues in both the MTase and RdRp domains. These data support a structural model of the full-length NS5 molecule that predicts a physical interaction between the MTase and the RdRp domains.

scholarly journals Energetics of RNA binding by the West Nile virus RNA triphosphatase

FEBS Letters ◽  
2006 ◽  
Vol 580 (3) ◽  
pp. 867-877 ◽  
Author(s):  
Ines Benzaghou ◽  
Isabelle Bougie ◽  
Frédéric Picard-Jean ◽  
Martin Bisaillon
Keyword(s):  
West Nile Virus ◽  
Rna Binding ◽  
The West ◽  
West Nile ◽  
Virus Rna ◽  
Rna Triphosphatase

scholarly journals Anti-diabetic drug binding site in KATP channels revealed by Cryo-EM

2017 ◽  
Author(s):  
Gregory M. Martin ◽  
Balamurugan Kandasamy ◽  
Frank DiMaio ◽  
Craig Yoshioka ◽  
Show-Ling Shyng
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Katp Channels ◽  
Binding Pocket ◽  
Drug Binding ◽  
Channel Activity ◽  
Nucleotide Binding Domain ◽  
High Affinity ◽  
Drug Binding Site ◽  
First Time

AbstractSulfonylureas are anti-diabetic medications that act by inhibiting pancreatic KATP channels composed of SUR1 and Kir6.2. The mechanism by which these drugs interact with and inhibit the channel has been extensively investigated, yet it remains unclear where the drug binding pocket resides. Here, we present a cryo-EM structure of the channel bound to a high-affinity sulfonylurea drug glibenclamide and ATP at 3.8Å resolution, which reveals in unprecedented details of the ATP and glibenclamide binding sites. Importantly, the structure shows for the first time that glibenclamide is lodged in the transmembrane bundle of the SUR1-ABC core connected to the first nucleotide binding domain near the inner leaflet of the lipid bilayer. Mutation of residues predicted to interact with glibenclamide in our model led to reduced sensitivity to glibenclamide. Our structure provides novel mechanistic insights of how sulfonylureas and ATP interact with the KATP channel complex to inhibit channel activity.

scholarly journals Improving CLIP-Seq Data Analysis by Incorporating Transcript Information

2020 ◽  
Author(s):  
Michael Uhl ◽  
Dinh Van Tran ◽  
Rolf Backofen
Keyword(s):  
Data Analysis ◽  
Binding Site ◽  
Binding Sites ◽  
Rna Binding ◽  
Rna Binding Protein ◽  
Peak Calling ◽  
Genomic Context ◽  
Site Prediction ◽  
Prediction Tools ◽  
Downstream Analysis

Abstract Background: Current peak callers for identifying RNA-binding protein (RBP) binding sites from CLIP-seq data take into account genomic read profiles, but they ignore the underlying transcript information, that is information regarding splicing events. So far, there are no studies available that closer observe this issue. Results: Here we show that current peak callers are susceptible to false peak calling near exon borders. We further quantify its extent in publicly available datasets, which turns out to be substantial. Finally, by providing a tool called CLIPcontext for automatic transcript and genomic context sequence extraction, we demonstrate that context choice also affects the performances of RBP binding site prediction tools. Conclusions: Our results demonstrate the importance of incorporating transcript information in CLIP-seq data analysis. Taking advantage of the underlying transcript information should therefore become an integral part of future peak calling and downstream analysis tools.

scholarly journals Overcoming the design, build, test (DBT) bottleneck for synthesis of nonrepetitive protein-RNA binding cassettes for RNA applications

2019 ◽  
Author(s):  
Noa Katz ◽  
Eitamar Tripto ◽  
Sarah Goldberg ◽  
Orna Atar ◽  
Zohar Yakhini ◽  
...  
Keyword(s):  
Binding Site ◽  
High Throughput ◽  
Binding Sites ◽  
Mammalian Cells ◽  
Rna Binding ◽  
Coat Proteins ◽  
Binding Affinities ◽  
Predictive Capability ◽  
Design Build ◽  
Major Bottleneck

AbstractThe design-build-test (DBT) cycle in synthetic biology is considered to be a major bottleneck for progress in the field. The emergence of high-throughput experimental techniques, such as oligo libraries (OLs), combined with machine learning (ML) algorithms, provide the ingredients for a potential “big-data” solution that can generate a sufficient predictive capability to overcome the DBT bottleneck. In this work, we apply the OL-ML approach to the design of RNA cassettes used in gene editing and RNA tracking systems. RNA cassettes are typically made of repetitive hairpins, therefore hindering their retention, synthesis, and functionality. Here, we carried out a high-throughput OL-based experiment to generate thousands of new binding sites for the phage coat proteins of bacteriophages MS2 (MCP), PP7 (PCP), and Qβ (QCP). We then applied a neural network to vastly expand this space of binding sites to millions of additional predicted sites, which allowed us to identify the structural and sequence features that are critical for the binding of each RBP. To verify our approach, we designed new non-repetitive binding site cassettes and tested their functionality in U2OS mammalian cells. We found that all our cassettes exhibited multiple trackable puncta. Additionally, we designed and verified two additional cassettes, the first containing sites that can bind both PCP and QCP, and the second with sites that can bind either MCP or QCP, allowing for an additional orthogonal channel. Consequently, we provide the scientific community with a novel resource for rapidly creating functional non-repetitive binding site cassettes using one or more of three phage coat proteins with a variety of binding affinities for any application spanning bacteria to mammalian cells.

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