Structural studies of an RNA-theophylline complex using multidimensional, heteronuclear magnetic resonance spectroscopy

RNAs are known to fold into stable structures capable of binding proteins, ligands, or substrates for a catalytic reaction. Little is known about the specific interactions used by RNA to stabilize these functional conformations in solution. To visualize the interplay of RNA structural interactions in a ligand binding site, the solution structure of a high-affinity RNA-theophylline complex was determined using multidimensional, heteronuclear magnetic resonance spectroscopy and restrained molecular dynamics calculations. The structure provides insight into the ability of this in vitro selected RNA to discriminate theophylline from the structurally similar molecule caffeine. Numerous RNA structural motifs combine to form a well-ordered binding pocket where an intricate network of hydrogen bonds and stacking interactions lock the theophylline into the complex. The RNA contains an S-turn in the backbone that allows two internal loops to interact and form the binding site which consists of a sandwich of three base triples. An important feature of the RNA is that many of the conserved core residues participate in multiple, interdependent tertiary interactions. This complex illustrates how interlocking structural motifs can be assembled into a highly specific ligand binding site that possesses both high levels of affinity and molecular discrimination.; In addition to the three-dimensional structure of the complex, a number of biochemical aspects of the theophylline-binding RNA were investigated. This RNA aptamer requires divalent metal ions for the high-affinity binding of theophylline, and a specific Mn{dollar}sp{lcub}2+{rcub}{dollar}-binding site in the core of the RNA was identified. C27 was shown to be a highly dynamic residue in the otherwise rigid core of the RNA-theophylline complex by measurement of {dollar}sp{lcub}13{rcub}{dollar}C-T{dollar}sb{lcub}1rho{rcub}{dollar} relaxation parameters. C27 was also implicated in the pH dependence of the theophylline-binding activity of this RNA aptamer. At acidic pH, it is likely that residue A7 becomes protonated and is able to interact with C27 in the context of an A{dollar}sp{lcub}+{rcub}{dollar}C base pair to block theophylline binding. The unique structural features and biochemical aspects of the theophylline-binding RNA system illustrate the exquisite complexity of RNA biochemistry.