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

Cations play an important role in RNA folding and stabilization. The hairpi n ribozyme is a small catalytic RNA consisting of two domains,A and B, whic h interact in the transition state in an ion-dependent fashion. Here we des cribe the interaction of mono-, di-, and trivalent cations with the domains of the ribozyme, as studied by homo- and heteronuclear NMR spectroscopy. P aramagnetic Line broadening, chemical shift mapping, and intermolecular NOE s indicate that the B domain contains four to five metal binding sites, whi ch 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 spec ific 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 obser vable metal ion interactions within the internal loop of the A domain. Mode l structure calculations of Mn2+ interactions at two sites within the B dom ain indicate that the binding sites comprise major groove pockets lined wit h 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 simila r 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 i on interacts with a catalytically essential nucleotide and bridges two phos phates. Due to its location and geometry, this ion may play an important ro le in the docking of the A and B domains.