Adaptive Ligand Binding by the Purine Riboswitch in the Recognition of Guanine and Adenine Analogs  Sunny D. Gilbert, Francis E. Reyes, Andrea L. Edwards, Robert T. Batey  Structure  Volume 17, Issue 6, Pages 857-868 (June 2009) DOI: 10.1016/j.str.2009.04.009 Copyright © 2009 Elsevier Ltd Terms and Conditions

Figure 1 Ligand Binding Site of the Purine Riboswitch and Chemicals (A) Details of hypoxanthine (top, PDB 1U8D) and adenine (bottom, PDB 1Y26) bound to the guanine and adenine riboswitches, respectively. The specificity pyrimidine (C74 or U74, yellow) forms hydrogen bonds (red dashed lines) to the Watson-Crick face of the ligand (pink), whereas U51, U47, and U22 interact with the other faces of the purine nucleobase. Residues are colored according to their strand position in the junction region. Note that the hydrogen bonding pattern for ligand recognition is identical. (B) Chemical structures of purine derivatives characterized in this study. Structure 2009 17, 857-868DOI: (10.1016/j.str.2009.04.009) Copyright © 2009 Elsevier Ltd Terms and Conditions

Figure 2 2-Position Purine Derivatives Bound to the Purine Riboswitch (A) Chemical structure of xanthine, emphasizing the keto-enol tautomerization of the N3/O2 atoms and the deprotonation of the enolic proton. (B) Simulated annealing omit map of the X:GR complex in which xanthine, U51, and C74 are removed and contoured at 1.0σ (orange cage); the X:GR model is depicted with blue carbon atoms. Superposition of the xanthine-bound (blue) and hypoxanthine-bound (pink) guanine riboswitches shows that base positioning within the binding pocket is unaltered. (C) Simulated-annealing omit map of the 2FA:GRA complex with 2FA, U51, and C74 omitted and contoured at 1.0σ (model of 2FA:GRA complex is pink). Superposition of the 2FA-bound (cyan) and 2,6-diaminpurine-bound (pink) GRA RNAs reveals the binding pocket is also unaltered. Structure 2009 17, 857-868DOI: (10.1016/j.str.2009.04.009) Copyright © 2009 Elsevier Ltd Terms and Conditions

Figure 3 6-Position Derivatives Binding to a Guanine Binding RNA (A) Binding pocket of the 6ClG:GR structure. The electron density map of the ligand and surrounding nucleotides (orange mesh, contoured at 1σ) is a simulated annealing omit map in which the shown nucleotides have been omitted from the model used to calculate the map. Hydrogen bonding interactions between the ligand and the specificity pyrimidine (C/U74) are depicted as gray dashed lines. Similar maps are shown throughout Figures 3, 5, and 6. (B) Ligand and binding pocket of the 6OMeG:GR structure. (C) Superposition of structures of wild-type hypoxanthine:GR (red), 6ClG:GR (blue), and 6OMeG:GR (green). Although U22, U47, and U51 in each structure are in nearly identical positions, the ligand and C74 in the 6OMeG:GR have shifted. The largest positional shift is C74, which is moved toward the minor groove, allowing for an altered hydrogen bonding pattern with the ligand. (D) Ligand and binding pocket of the 2AP:GR structure. Structure 2009 17, 857-868DOI: (10.1016/j.str.2009.04.009) Copyright © 2009 Elsevier Ltd Terms and Conditions

Figure 4 Energetic Cost of Minor-Groove Wobbling (A) Calculation of the energetic penalty associated with shifting C74 into the minor groove by directly comparing the same ligand, 2AP, binding to the GRA in the unshifted form and GR in the shifted position. (B) A second independent means of this calculation yields the apparent energetic penalty by a direct measure of the loss of a third hydrogen bond. Structure 2009 17, 857-868DOI: (10.1016/j.str.2009.04.009) Copyright © 2009 Elsevier Ltd Terms and Conditions

Figure 5 6-Position Derivatives Complexed to an Adenine Binding RNA (A) Structure of 6ClG bound to GRA; double arrow represents a potential halogen bond between the chloro group and U74. (B) Binding pocket of the 6OMeG:GRA complex; arrow denotes a long hydrogen bond (3.5 Å) between N1(6OMeG) and N3(U74). (C) Superposition of structures of the wild-type hypoxanthine:GR complex (red), 6ClG:GRA (blue), and 6OMeG:GRA (green). Most atoms in each structure superimpose well, except for a minor repulsion between the ligand and U74 in the 6OMeG:GRA complex to accommodate the methyl group at the Watson-Crick interface. Structure 2009 17, 857-868DOI: (10.1016/j.str.2009.04.009) Copyright © 2009 Elsevier Ltd Terms and Conditions

Figure 6 Structure of 6ClG Bound to GR RNA Containing an A21G/U75C Mutation (A) Binding pocket and mutated pair (cyan). C74 is clearly shifted toward the minor groove (compare with Figure 4A), to allow for an alternative hydrogen-bonding pattern to occur. (B) Superposition of the wild-type HX:GR (red) and 6ClG:GR(A21G/U75G) (blue) complexes emphasizing the ligand:C74 interaction and the base pair (21–75) below it. Mutation of the 21–75 pair does not cause this pair to adjust its position, but allows C74 to shift toward the minor groove when binding 6ClG. Structure 2009 17, 857-868DOI: (10.1016/j.str.2009.04.009) Copyright © 2009 Elsevier Ltd Terms and Conditions