Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2008 Apr;14(4):696-705.
doi: 10.1261/rna.928208. Epub 2008 Feb 6.

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

Sushuang Zheng  1 , Stewart Shuman
Affiliations

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

Sushuang Zheng et al. RNA. 2008 Apr.

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.

PubMed Disclaimer

Figures

FIGURE 1.
FIGURE 1.
Tertiary structures of cellular and poxvirus cap methyltransferases and a model of the Michaelis complex of the vaccinia virus enzyme. (A) The structures of GTP-bound Ecm1 (PDB ID 1ri2) and AdoHcy-bound D1-C (PDB ID 2vdw) were superimposed and then offset horizontally. The folds are shown with β strands colored magenta and α helices in cyan. The GTP and AdoHcy ligands are depicted as stick models. A sulfate ion in the active site of vaccinia D1-C occupies the same position as the GTP γ-phosphate in the Ecm1 active site. (B) Stereo view of a model of the vaccinia D1-C active site with both substrates bound, generated by importing the GTP ligand from the aligned Ecm1 structure and adding a methyl group to the AdoHcy ligand in the otherwise unperturbed D1-C structure. The sulfur atom of the methyl donor is colored green. Actual polar atomic contacts of D1-C with the methyl donor and contacts between amino acid side chains are depicted as black dashed lines; Van der Waals contacts of Tyr683 and Asp598 with the adenosine moiety of the methyl donor are depicted as beige dashed lines. The modeled contacts between D1-C side chains and the GTP substrate are drawn as blue dashed lines.
FIGURE 2.
FIGURE 2.
Recombinant wild-type and mutant D1-C/D12 heterodimers. Aliquots of the nickel-agarose preparations (containing 3 μg of the D1-C polypeptide) were analyzed by SDS-PAGE. The polypeptides were visualized by staining with Coomassie Blue dye. The positions and sizes (kDa) of marker polypeptides are indicated on the left. Alanine mutants are shown in A; conservative mutants are shown in B.
FIGURE 3.
FIGURE 3.
Mutational effects on vaccinia virus cap methyltransferase activity. The extents of 3H-m7GpppA formation by wild-type and mutant D1-C/D12 heterodimers are plotted with alanine mutants surveyed in A and conservative mutants in B. Each datum is the average of three experiments; standard error bars are shown.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Bujnicki, J.M., Feder, M., Radlinska, M., Rychlewski, L. mRNA:guanine-N7 methyltransferases: Identification of novel members of the family, evolutionary analysis, homology modeling, and analysis of sequence–structure–function relationships. BMC Bioinformatics. 2001;2:2. - PMC - PubMed
    1. Chrebet, G.L., Wisniewski, D., Perkins, A.L., Deng, Q., Kurtz, M.B., Marcy, A., Parent, S.A. Cell-based assays to detect inhibitors of fungal mRNA capping enzymes and characterization of sinefungin as a cap methyltransferase inhibitor. J. Biomol. Screen. 2005;10:355–364. - PubMed
    1. Cong, P., Shuman, S. Methyltransferase and subunit association domains of vaccinia virus mRNA capping enzyme. J. Biol. Chem. 1992;267:16424–16429. - PubMed
    1. De la Peña, M., Kyrielis, O., Cusack, S. Structural insights into the mechanism and evolution of the vaccinia virus mRNA cap N7 methyltransferase. EMBO J. 2007;26:4913–4925. - PMC - PubMed
    1. Fabrega, C., Hausmann, S., Shen, V., Shuman, S., Lima, C.D. Structure and mechanism of cap (guanine-N7) methyltransferase. Mol. Cell. 2004;13:77–89. - PubMed

Publication types

MeSH terms

LinkOut - more resources