Abstract
The N7-methylated guanosine (m7G) cap structure, which is found at the 5' ends of mature eukaryotic mRNAs, is critical to a myriad of biological processes. The twenty structures of complexes of cap nucleosides and nucleotides and methylated bases with the vaccinia virus VP39, a cap-specific RNA 2'-O-methyltransferase, which we have determined previously, have revealed the atomic basis of cap binding. The precise insertion and tight fitting of the m7Gua moiety of the cap between two parallel aromatic residues that are spaced only 6.8 A apart governs the high specificity of binding. Here we report the investigation of the reaction mechanism of VP39 with three capped ligands (m7G, m7GpppG, and m7GpppGA3) by fluorescence stopped-flow technique. Cap binding is a simple one-step mechanism with very fast association rate constant (approximately 10(7) M-1 s-1). Moreover, the pH dependence on the association rate constant of m7G binding indicates that only the positively charged keto tautomer of the cap is recognized and bound. The association and dissociation rate constants and affinity constants of the three ligands do not vary greatly, demonstrating that binding is achieved almost entirely by the interactions of m7Gua with two aromatic residues in a cation-pi sandwich.
Highlights
The ability of proteins to discriminate alkylated from nonalkylated nucleic acids is of paramount importance in numerous biological processes, including DNA repair, pre-mRNA splicing, nucleocytoplasmic transport, mRNA translation, cap-dependent ribose methylation, and influenza virus transcriptional priming (briefly summarized in (1))
Our determination of over 20 x-ray structures of VP39 complexed with a variety of methylated nucleobases, nucleotides, and oligonucleotides have provided a comprehensive understanding at the atomic level of the process by which a protein interacts with a methylated, positively charged nucleobase of the cap and with an mRNA transcript in a sequence-nonspecific manner (1, 5– 8)
The F180W VP39 showed no apparent defects in methyltransferase catalytic activity or VP39-capped RNA interactions and no perturbation in the cap-binding pocket structure (6, 7)
Summary
One-step Binding Mechanism—Cap analog binding studies in solution have been greatly facilitated by replacing one of the two aromatic residues (Phe-180) that sandwich the m7Gua moiety of capped nucleotides and oligonucleotides with a Trp residue to provide a fluorescent reporter group (Fig. 1) (6, 8). The fluorescence emission maximum is at 330 nm with 55% quenching, with no shift in the emission maximum, occurring upon the addition of excess m7G. Ligand binding and functional activities of the mutant protein (named F180W VP39) have been characterized by a variety of techniques, including x-ray crystallographic, fluorescence measurements, isothermal calorimetry, and surface plasmon resonance. The F180W VP39 showed no apparent defects in methyltransferase catalytic activity or VP39-capped RNA interactions and no perturbation in the cap-binding pocket structure (6, 7). The structures of the wild-type and F180W VP39s complexed with m7G
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