Determination of metal ion binding sites within the hairpin ribozyme domains by NMR

Cations play an important role in RNA folding and stabilization. The hairpin ribozyme is a small catalytic RNA consisting of two domains,A and B, which interact in the transition state in an ion-dependent fashion. Here we describe the interaction of mono-, di-, and trivalent cations with the domains of the ribozyme, as studied by homo- and heteronuclear NMR spectroscopy. Paramagnetic Line broadening, chemical shift mapping, and intermolecular NOEs indicate that the B domain contains four to five metal binding sites, which bind Mn2+, Mg2+, and Co(NH3)(6)(3+). There is no significant structural change in the B domain upon the addition of Co(NH3)(6)(3+) or Mg2+. No specific monovalent ion binding sites exist on the B domain, as determined by (NH4+)-N-15 binding studies. In contrast to the B domain, there are no observable metal ion interactions within the internal loop of the A domain. Model structure calculations of Mn2+ interactions at two sites within the B domain indicate that the binding sites comprise major groove pockets lined with functional groups oriented so that multiple hydrogen bonds can be formed between the RNA and Mn(H2O)(6)(2+) or CO(NH3)(6)(3+). Site 1 is very similar in geometry to a site within the PLD;PG domain of the Tetrahymena group I intron, while site 2 is unique among known ion binding sites. The site 2 ion interacts with a catalytically essential nucleotide and bridges two phosphates. Due to its location and geometry, this ion may play an important role in the docking of the A and B domains.