Insertion of an N7-methylguanine mRNA Cap between Two Coplanar Aromatic Residues of a Cap-binding Protein Is Fast and Selective for a Positively Charged Cap*

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 Å 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 (∼107 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-π sandwich.