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Structure-function analysis of vaccinia virus mRNA cap (guanine-N7) methyltransferase.

ABSTRACT: The guanine-N7 methyltransferase domain of vaccinia virus mRNA capping enzyme is a heterodimer composed of a catalytic subunit and a stimulatory subunit. Structure-function analysis of the catalytic subunit by alanine scanning and conservative substitutions (49 mutations at 25 amino acids) identified 12 functional groups essential for methyltransferase activity in vivo, most of which were essential for cap methylation in vitro. Defects in cap binding were demonstrated for a subset of lethal mutants that displayed residual activity in vitro. We discuss our findings in light of a model of the Michaelis complex derived from crystal structures of AdoHcy-bound vaccinia cap methyltransferase and GTP-bound cellular cap methyltransferase. The structure-function data yield a coherent picture of the vaccinia cap methyltransferase active site and the determinants of substrate specificity and affinity.

SUBMITTER: Zheng S

PROVIDER: S-EPMC2271365 | biostudies-literature | 2008 Apr

REPOSITORIES: biostudies-literature

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Structure-function analysis of vaccinia virus mRNA cap (guanine-N7) methyltransferase.

Zheng Sushuang S Shuman Stewart S

RNA (New York, N.Y.) 20080206 4

The guanine-N7 methyltransferase domain of vaccinia virus mRNA capping enzyme is a heterodimer composed of a catalytic subunit and a stimulatory subunit. Structure-function analysis of the catalytic subunit by alanine scanning and conservative substitutions (49 mutations at 25 amino acids) identified 12 functional groups essential for methyltransferase activity in vivo, most of which were essential for cap methylation in vitro. Defects in cap binding were demonstrated for a subset of lethal muta ...[more]

PMID: 18256245

Similar Datasets

Structure-function analysis of severe acute respiratory syndrome coronavirus RNA cap guanine-N7-methyltransferase.

Project description:Coronaviruses possess a cap structure at the 5' ends of viral genomic RNA and subgenomic RNAs, which is generated through consecutive methylations by virally encoded guanine-N7-methyltransferase (N7-MTase) and 2'-O-methyltransferase (2'-O-MTase). The coronaviral N7-MTase is unique for its physical linkage with an exoribonuclease (ExoN) harbored in nonstructural protein 14 (nsp14) of coronaviruses. In this study, the structure-function relationships of the N7-MTase were analyzed by deletion and site-directed mutagenesis of severe acute respiratory syndrome coronavirus (SARS-CoV) nsp14. The results showed that the ExoN domain is closely involved in the activity of the N7-MTase, suggesting that coronavirus N7-MTase is different from all other viral N7-MTases, which are separable from other structural domains located in the same polypeptide. Two of the 12 critical residues identified to be essential for the N7-MTase were located at the N terminus of the core ExoN domain, reinforcing a role of the ExoN domain in the N7-MTase activity of nsp14. The other 10 critical residues were distributed throughout the N7-MTase domain but localized mainly in the S-adenosyl-l-methionine (SAM)-binding pocket and key structural elements of the MTase fold of nsp14. The sequence motif DxGxPxA (amino acids [aa] 331 to 338) was identified as the key part of the SAM-binding site. These results provide insights into the structure and functional mechanisms of coronaviral nsp14 N7-MTase.

| S-EPMC3648086 | biostudies-literature

Mutational analysis of vaccinia virus mRNA cap (guanine-N7) methyltransferase reveals essential contributions of the N-terminal peptide that closes over the active site.

Project description:RNA guanine-N7 methyltransferase catalyzes the third step of eukaryal mRNA capping, the transfer of a methyl group from AdoMet to GpppRNA to form m(7)GpppRNA. Mutational and crystallographic analyses of cellular and poxvirus cap methyltransferases have yielded a coherent picture of a conserved active site and determinants of substrate specificity. Models of the Michaelis complex suggest a direct in-line mechanism of methyl transfer. Because no protein contacts to the guanine-N7 nucleophile, the AdoMet methyl carbon (Cepsilon) or the AdoHcy sulfur (Sdelta) leaving group were observed in ligand-bound structures of cellular cap methyltransferase, it was initially thought that the enzyme facilitates catalysis by optimizing proximity and geometry of the donor and acceptor. However, the structure of AdoHcy-bound vaccinia virus cap methyltransferase revealed the presence of an N-terminal "lid peptide" that closes over the active site and makes multiple contacts with the substrates, including the AdoMet sulfonium. This segment is disordered in the vaccinia apoenzyme and is not visible in the available structures of cellular cap methyltransferase. Here, we conducted a mutational analysis of the vaccinia virus lid peptide ((545)DKFRLNPEVSYFTNKRTRG(563)) entailing in vivo and in vitro readouts of the effects of alanine and conservative substitutions. We thereby identified essential functional groups that interact with the AdoMet sulfonium (Tyr555, Phe556), the AdoMet adenine (Asn550), and the cap triphosphate bridge (Arg560, Arg562). The results suggest that van der Waals contacts of Tyr555 and Phe556 to the AdoMet Sdelta and C epsilon atoms, and the electron-rich environment around the sulfonium, serve to stabilize the transition state of the transmethylation reaction.

| S-EPMC2578867 | biostudies-literature

Computational design and experimental characterization of a photo-controlled mRNA-cap guanine-N7 methyltransferase.

Project description:The spatial and temporal control of gene expression at the post-transcriptional level is essential in eukaryotic cells and developing multicellular organisms. In recent years optochemical and optogenetic tools have enabled the manipulation and investigation of many steps in the involved processes. However, examples for light-mediated control of eukaryotic mRNA processing and the responsible enzymes are still rare. In particular, methylation of the 5' cap of mRNA is required for ribosome assembly, and the responsible guanine-N7 methyltransferase (MTase) from E. cuniculi (Ecm1) proved suitable for activating translation. Here, we report on a photoswitchable MTase obtained by bridging the substrate-binding cleft of Ecm1 with a tetra-ortho-methoxy-azobenzene. This azobenzene derivative is characterized by efficient trans-to-cis isomerization using red light at 615 nm. Starting from a cysteine-free Ecm1 variant (ΔCys), we used a computational approach to identify suitable conjugation sites for the azobenzene moiety. We created and characterized the four best-ranked variants, each featuring two appropriately positioned cysteines close to the substrate-binding cleft. Conjugating and crosslinking the azobenzene between C149/C155 in a designed Ecm1 variant (VAR3-Az) enabled light-dependent modulation of the MTase activity and showed a 50% higher activity for the cis form than the trans-form of the azobenzene conjugated to VAR3-Az.

| S-EPMC8495969 | biostudies-literature

Identification and evaluation of potential SARS-CoV-2 antiviral agents targeting mRNA cap guanine N7-Methyltransferase.

Project description:SARS-CoV-2, the cause of the currently ongoing COVID-19 pandemic, encodes its own mRNA capping machinery. Insights into this capping system may provide new ideas for therapeutic interventions and drug discovery. In this work, we employ a previously developed Py-FLINT screening approach to study the inhibitory effects of compounds against the cap guanine N7-methyltransferase enzyme, which is involved in SARS-CoV-2 mRNA capping. We screened five commercially available libraries (7039 compounds in total) to identify 83 inhibitors with IC50 < 50 μM, which were further validated using RP HPLC and dot blot assays. Novel fluorescence anisotropy binding assays were developed to examine the targeted binding site. The inhibitor structures were analyzed for structure-activity relationships in order to define common structural patterns. Finally, the most potent inhibitors were tested for antiviral activity on SARS-CoV-2 in a cell based assay.

| S-EPMC8299157 | biostudies-literature

Structure-function analysis of the nsp14 N7-guanine methyltransferase reveals an essential role in Betacoronavirus replication.

Project description:As coronaviruses (CoVs) replicate in the host cell cytoplasm, they rely on their own capping machinery to ensure the efficient translation of their messenger RNAs (mRNAs), protect them from degradation by cellular 5' exoribonucleases (ExoNs), and escape innate immune sensing. The CoV nonstructural protein 14 (nsp14) is a bifunctional replicase subunit harboring an N-terminal 3'-to-5' ExoN domain and a C-terminal (N7-guanine)-methyltransferase (N7-MTase) domain that is presumably involved in viral mRNA capping. Here, we aimed to integrate structural, biochemical, and virological data to assess the importance of conserved N7-MTase residues for nsp14's enzymatic activities and virus viability. We revisited the crystal structure of severe acute respiratory syndrome (SARS)-CoV nsp14 to perform an in silico comparative analysis between betacoronaviruses. We identified several residues likely involved in the formation of the N7-MTase catalytic pocket, which presents a fold distinct from the Rossmann fold observed in most known MTases. Next, for SARS-CoV and Middle East respiratory syndrome CoV, site-directed mutagenesis of selected residues was used to assess their importance for in vitro enzymatic activity. Most of the engineered mutations abolished N7-MTase activity, while not affecting nsp14-ExoN activity. Upon reverse engineering of these mutations into different betacoronavirus genomes, we identified two substitutions (R310A and F426A in SARS-CoV nsp14) abrogating virus viability and one mutation (H424A) yielding a crippled phenotype across all viruses tested. Our results identify the N7-MTase as a critical enzyme for betacoronavirus replication and define key residues of its catalytic pocket that can be targeted to design inhibitors with a potential pan-coronaviral activity spectrum.

| S-EPMC8670481 | biostudies-literature

Structural insights into the mechanism and evolution of the vaccinia virus mRNA cap N7 methyl-transferase.

Project description:The vaccinia virus mRNA capping enzyme is a multifunctional heterodimeric protein associated with the viral polymerase that both catalyses the three steps of mRNA capping and regulates gene transcription. The structure of a subcomplex comprising the C-terminal N7-methyl-transferase (MT) domain of the large D1 subunit, the stimulatory D12 subunit and bound S-adenosyl-homocysteine (AdoHcy) has been determined at 2.7 A resolution and reveals several novel features of the poxvirus capping enzyme. The structure shows for the first time the critical role played by the proteolytically sensitive N-terminus of the MT domain in binding the methyl donor and in catalysis. In addition, the poxvirus enzyme has a completely unique mode of binding of the adenosine moiety of AdoHcy, a feature that could be exploited for design of specific anti-poxviral compounds. The structure of the poxvirus-specific D12 subunit suggests that it was originally an RNA cap 2'O-MT that has evolved to a catalytically inactive form that has been retained for D1 stabilisation and MT activity enhancement through an allosteric mechanism.

| S-EPMC2099473 | biostudies-literature

A second type of N7-guanine RNA cap methyltransferase in an unusual locus of a large RNA virus genome.

Project description:The order Nidovirales is a diverse group of (+)RNA viruses, with a common genome organization and conserved set of replicative and editing enzymes. In particular, RNA methyltransferases play a central role in mRNA stability and immune escape. However, their presence and distribution in different Nidovirales families is not homogeneous. In Coronaviridae, the best characterized family, two distinct methytransferases perform methylation of the N7-guanine and 2'-OH of the RNA-cap to generate a cap-1 structure (m7GpppNm). The genes of both of these enzymes are located in the ORF1b genomic region. While 2'-O-MTases can be identified for most other families based on conservation of both sequence motifs and genetic loci, identification of the N7-guanine methyltransferase has proved more challenging. Recently, we identified a putative N7-MTase domain in the ORF1a region (N7-MT-1a) of certain members of the large genome Tobaniviridae family. Here, we demonstrate that this domain indeed harbors N7-specific methyltransferase activity. We present its structure as the first N7-specific Rossmann-fold (RF) MTase identified for (+)RNA viruses, making it remarkably different from that of the known Coronaviridae ORF1b N7-MTase gene. We discuss the evolutionary implications of such an appearance in this unexpected location in the genome, which introduces a split-off in the classification of Tobaniviridae.

| S-EPMC9638943 | biostudies-literature

Yeast-based assays for the high-throughput screening of inhibitors of coronavirus RNA cap guanine-N7-methyltransferase.

Project description:The 5'-cap structure is a distinct feature of eukaryotic mRNAs and is important for RNA stability and protein translation by providing a molecular signature for the distinction of self or non-self mRNA. Eukaryotic viruses generally modify the 5'-end of their RNAs to mimic the cellular mRNA structure, thereby facilitating viral replication in host cells. However, the molecular organization and biochemical mechanisms of the viral capping apparatus typically differ from its cellular counterpart, which makes viral capping enzymes attractive targets for drug discovery. Our previous work showed that SARS coronavirus (SARS-CoV) non-structural protein 14 represents a structurally novel and unique guanine-N7-methyltransferase (N7-MTase) that is able to functionally complement yeast cellular N7-MTase. In the present study, we developed a yeast-based system for identifying and screening inhibitors against coronavirus N7-MTase using both 96-well and 384-well microtiter plates. The MTase inhibitors previously identified by in vitro biochemical assays were tested, and some, such as sinefungin, effectively suppressed N7-MTase in the yeast system. However, other compounds, such as ATA and AdoHcy, did not exert an inhibitory effect within a cellular context. These results validated the yeast assay system for inhibitor screening yet also demonstrated the difference between cell-based and in vitro biochemical assays. The yeast system was applied to the screening of 3000 natural product extracts, and three were observed to more potently inhibit the activity of coronavirus than human N7-MTase.

| S-EPMC7119097 | biostudies-literature

Synthesis of adenine dinucleosides SAM analogs as specific inhibitors of SARS-CoV nsp14 RNA cap guanine-N7-methyltransferase.

Project description:The spreading of new viruses is known to provoke global human health threat. The current COVID-19 pandemic caused by the recently emerged coronavirus SARS-CoV-2 is one significant and unfortunate example of what the world will have to face in the future with emerging viruses in absence of appropriate treatment. The discovery of potent and specific antiviral inhibitors and/or vaccines to fight these massive outbreaks is an urgent research priority. Enzymes involved in the capping pathway of viruses and more specifically RNA N7- or 2'O-methyltransferases (MTases) are now admitted as potential targets for antiviral chemotherapy. We designed bisubstrate inhibitors by mimicking the transition state of the 2'-O-methylation of the cap RNA in order to block viral 2'-O MTases. This work resulted in the synthesis of 16 adenine dinucleosides with both adenosines connected by various nitrogen-containing linkers. Unexpectedly, all the bisubstrate compounds were barely active against 2'-O MTases of several flaviviruses or SARS-CoV but surprisingly, seven of them showed efficient and specific inhibition against SARS-CoV N7-MTase (nsp14) in the micromolar to submicromolar range. The most active nsp14 inhibitor identified is as potent as but particularly more specific than the broad-spectrum MTase inhibitor, sinefungin. Molecular docking suggests that the inhibitor binds to a pocket formed by the S-adenosyl methionine (SAM) and cap RNA binding sites, conserved among SARS-CoV nsp14. These dinucleoside SAM analogs will serve as starting points for the development of next inhibitors for SARS-CoV-2 nsp14 N7-MTase.

| S-EPMC7291971 | biostudies-literature

A N7-guanine RNA cap methyltransferase signature-sequence as a genetic marker of large genome, non-mammalian Tobaniviridae.

Project description:The order Nidovirales is a diverse group of (+)RNA viruses, classified together based on their common genome organisation and conserved replicative enzymes, despite drastic differences in size and complexity. One such difference pertains to the mechanisms and enzymes responsible for generation of the proposed viral 5' RNA cap. Within the Coronaviridae family, two separate methytransferases (MTase), nsp14 and nsp16, perform the RNA-cap N7-guanine and 2'-OH methylation respectively for generation of the proposed m7GpppNm type I cap structure. For the majority of other families within the Nidovirales order, the presence, structure and key enzymes involved in 5' capping are far less clear. These viruses either lack completely an RNA MTase signature sequence, or lack an N7-guanine methyltransferase signature sequence, obscuring our understanding about how RNA-caps are N7-methylated for these families. Here, we report the discovery of a putative Rossmann fold RNA methyltransferase in 10 Tobaniviridae members in Orf1a, an unusual genome locus for this gene. Multiple sequence alignments and structural analyses lead us to propose this novel gene as a typical RNA-cap N7-guanine MTase with substrate specificity and active-site organization similar to the canonical eukaryotic RNA-cap N7-guanine MTase.

| S-EPMC7107822 | biostudies-literature

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