Catalytic Mechanism of Vaccinia Virus Protein in VP39 in 2'-O-Methylation of the mRNA 5' Cap Structure

Texas A&M Research Foundation, College Station TX

Investigators

Abstract

0091260 Gershon Recently, the P.I. collaborated in determining a high-resolution crystal structure for a prototypical nucleic acid sugar methyltransferase complexed with its cofactor and RNA substrate. This enzyme, vaccinia virus protein VP39, methylates messenger RNA at the 2'OH of a ribose within the 5' end cap structure. The structural data afford an unusual opportunity to identify the chemical processes underlying nucleic acid ribose methylation. Atomic-scale examination of the enzyme's catalytic center with bound substrate and cofactor suggests a mechanism whereby the charged oxygen generated by deprotonation of the target ribose hydroxyl attacks the trivalent sulfur of the methyl-donating cofactor (S-adenosylmethionine), effecting displacement of cofactor-product (S-adenosylhomocysteine). Although the sidechain of VP39 residue Lysine 175 is well-positioned to effect deprotonation of the hydroxyl, lysine 175's pKa would need to be depressed ~three orders of magnitude. The sidechain of a neighboring residue, arginine 209 may cause this depression to occur, and aspartate 138 may act as the proton sink (assuming a pKa elevation in the aspartate 138 sidechain). To determine whether the target hydroxyl is indeed deprotonated in the context of VP39, NMR will be used to investigate hydroxyl-deprotonation of the VP39-bound isotope-enriched RNA substrate. Next, VP39 sidechain pKa values will be examined after (1) modification of a unique-cysteine-175 substitution mutant with an isotope-enriched aminoethylation reagent and/or (2) generating proteins in which lysine 175, arginine 209 or aspartate 138 each comprise half of a uniquely 13C/15N enriched dipeptide. In combination with mutagenesis, these selective labeling strategies will facilitate the determination of sidechain pKa in the presence and absence of cofactor-product.