mRNA cap recognition: Dominant role of enhanced stacking interactions between methylated bases and protein aromatic side chains

We have determined, by high resolution x-raj analysis, 10 structures compri sing the mRNA cap-specific methyltransferase VP39 or specific mutants there of in the presence of methylated nucleobase analogs (N1-methyladenine, N3-m ethyladenine, N1-methylcytosine, N3-methylcytosine) and their unmethylated counterparts, or nucleoside N7-methylguanosine. Together with solution affi nity studies and previous crystallographic data for N7-methylguanosine and its phosphorylated derivatives, these data demonstrate that only methylated , positively charged bases are bound, indicating that their enhanced stacki ng with two aromatic side chains of VP39 (Tyr 22 and Phe 180) plays a domin ant role in cap recognition. Four key features characterize this stacking i nteraction: (i) near perfect parallel alignment between the sandwiched meth ylated bases and aromatic side chains, (ii) substantial areas of overlap in the two-stacked rings, (iii) a 3.4-Angstrom interplanar spacing within the overlapping region, and (iv) positive charge in the heterocyclic nucleobas e.