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

Abstract

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.

FIGURE 1.

Substrate contacts of the N-terminal lid peptide of vaccinia cap methyltransferase. The figure shows a stereo view of a model of the Michaelis complex of vaccinia D1-C active site with AdoMet and GTP bound (Zheng and Shuman 2008). The model was generated by importing the GTP ligand from the aligned structure of GTP-bound cellular cap methyltransferase (PDB ID 1ri2) and adding a methyl group to the AdoHcy ligand in the otherwise unperturbed D1-C structure (PDB ID 2vdw). 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 Tyr555, Phe556, and Tyr683 with the methyl donor are depicted as green dashed lines. The modeled contacts between D1-C side chains and the GTP substrate are drawn as blue dashed lines.

FIGURE 2.

Recombinant wild-type and mutant D1-C/D12 heterodimers. Aliquots of the nickel-agarose preparations (containing 1.8 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.

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.