Interplay of 'induced fit' and preorganization in the ligand induced folding of the aptamer domain of the guanine binding riboswitch

Abstract

Riboswitches are highly structured elements in the 5'-untranslated regions (5'-UTRs) of messenger RNA that control gene expression by specifically binding to small metabolite molecules. They consist of an aptamer domain responsible for ligand binding and an expression platform. Ligand binding in the aptamer domain leads to conformational changes in the expression platform that result in transcription termination or abolish ribosome binding. The guanine riboswitch binds with high-specificity to guanine and hypoxanthine and is among the smallest riboswitches described so far. The X-ray-structure of its aptamer domain in complex with guanine/hypoxanthine reveals an intricate RNA-fold consisting of a three-helix junction stabilized by long-range base pairing interactions. We analyzed the conformational transitions of the aptamer domain induced by binding of hypoxanthine using high-resolution NMR-spectroscopy in solution. We found that the long-range base pairing interactions are already present in the free RNA and preorganize its global fold. The ligand binding core region is lacking hydrogen bonding interactions and therefore likely to be unstructured in the absence of ligand. Mg2+-ions are not essential for ligand binding and do not change the structure of the RNA-ligand complex but stabilize the structure at elevated temperatures. We identified a mutant RNA where the long-range base pairing interactions are disrupted in the free form of the RNA but form upon ligand binding in an Mg2+-dependent fashion. The tertiary interaction motif is stable outside the riboswitch context.

Figure 1

Structure of the complex of the aptamer domain of the guanine riboswitch with hypoxanthine. (A) Secondary structure of the aptamer domain of the guanine binding riboswitch found in the 5′-UTR of the xpt-pbuX mRNA from B.subtilis. Helices I, II and III and the loops II and III are labeled accordingly. Long range base pairing interactions are indicated by solid lines for Watson–Crick base pairs and dashed lines for non-canonical base pairs. Nucleotides added to facilitate in vitro transcription are given in lower case letters and a stabilizing mutation in helix II that has no effect on ligand binding (14) is shaded. (B) Base quadruple formed by nucleotides U34 and G37 of loop II and C61 and A65 of loop III. G37 and C61 are involved in a long-range Watson–Crick base pair, whereas U34 and A65 form a reversed Hoogsteen base pair. Hydrogen bonds are indicated by dashed lines. (C) Base quadruple formed by nucleotides A33 and G38 of loop II and C60 and A66 of loop III. G38 and C60 are involved in a long-range Watson–Crick base pair, whereas A33 and A66 form an A:A base pair. Hydrogen bonds are indicated by dashed lines.

Figure 2

Hypoxanthine binding to the aptamer domain and NMR resonance assignments for the RNA–hypoxanthine complex. (A) HNN-COSY spectrum for a complex of ¹⁵N-labeled RNA and ¹⁵N,¹³C-labeled hypoxanthine at 10°C in 25 mM KPO₄-buffer (pH 6.2), 50 mM KCl in the absence of Mg²⁺-ions. Dashed lines highlight the correlations between hydrogen bond donor imino groups and hydrogen bond acceptor nitrogens due to the intermolecular hydrogen bonds between the N1H1 imino group of hypoxanthine and the C74 N3 nitrogen of the RNA as well as between the N3 of hypoxanthine and the U51 imino group of the RNA. The signal for the N9H9 imino group of bound hypoxanthine that is observable due to hydrogen bonding to a carbonyl group of the RNA is also labeled. (B) Schematic drawing of the intermolecular base pairing between hypoxanthine and the aptamer domain as derived from our NMR-data in the absence of Mg²⁺ and the X-ray-structure of the hypoxanthine–RNA complex in the presence of Mg²⁺. (C) Secondary structure and numbering scheme of the hypoxanthine–RNA complex. The ligand is colored blue, nucleotides in the helices are colored black, nucleotides in the ligand binding pockets are shown in green and nucleotides in the loops are highlighted in red. (D) Imino group region of an ¹H,¹⁵N-HSQC-spectrum of the hypoxanthine–RNA complex at 10°C in the absence of Mg²⁺. Signals for the imino groups of the ligand are labeled in blue. Signals of the uridine and guanine imino groups are labeled in black for nucleotides in the helices, in green for nucleotides in the ligand binding pockets and in red for nucleotides in the loops.

Figure 3

Divalent cation binding to the hypoxanthine–RNA complex. (A) Overlay of the imino group regions of ¹H,¹⁵N-HSQC-spectra for the hypoxanthine–RNA complex at 10°C in the absence of Mg²⁺-ions (black) and the presence of 5 mM MgCl₂ (red). Only small chemical shift changes and no new signals are observed upon addition of Mg²⁺. Resonances shifting by more then three line widths are labeled. (B) Overlay of the imino group regions of ¹H,¹⁵N-HSQC-spectra for the hypoxanthine–RNA complex at 10°C in the presence of 5 mM Mg²⁺ (black) and in the presence of 5 mM Mg²⁺ and 5 μM Mn²⁺ (red). Resonances that are strongly broadened or disappear due to paramagnetic line broadening in the presence of 8 μM Mn²⁺ are labeled. (C) Mapping of the position of the imino groups experiencing line broadening upon Mn²⁺-addition (8 μM) on the 3D structure of the hypoxanthine–RNA complex. The affected imino groups are highlighted as orange spheres. The RNA is shown as white lines. Four well-defined divalent cation binding sites are observed. The position of [Co(NH₃)₆]³⁺-ions of cobalt hexamine groups found in the X-ray structure of the complex is indicated by gray spheres.

Figure 4

Conformational changes of the RNA upon hypoxanthine binding and the conformation of the free RNA. (A) Overlay of the imino group regions of ¹H,¹⁵N-HSQC-spectra for the hypoxanthine–RNA complex (black) and the free RNA (red) at 10°C. Signals of imino groups are labeled in green for nucleotides in the ligand binding core and in red for nucleotides of loop II and III. The signals for the core nucleotides, such as U22, G45, G46, U47, U49, U51, G72 and U75 are only present in the bound state. In contrast, the imino groups of G32, U34, G37 and G38 in loop II as well as those for the closing base pairs of helix II G31 are observable in both the free and the bound form and have virtually identical chemical shifts. (B) HNN-COSY spectrum for the free RNA at 10°C in 25 mM KPO₄-buffer, 50 mM KCl in the absence of Mg²⁺-ions. A solid line indicates a correlation corresponding to a hydrogen bond between an uridine imino group and an adenine N7-nitrogen due to the presence of the reversed Hoogsteen base pair formed by U34 in loop II and A65 in loop III as shown schematically in the inset. A dashed line indicates the hydrogen bond between the imino group of G38 in loop II and the N3 nitrogen of C60 in loop III as expected in a canonical Watson–Crick G:C base pair. The presence of these hydrogen bonds indicates that the two base quadruples that stabilize the loop–loop interaction are present already in the free RNA. (C) HNN-COSY spectrum for the free RNA at 10°C in 25 mM KPO₄-buffer, 50 mM KCl in the presence of 5 mM magnesium. The cross peak corresponding to the hydrogen bond in the reversed Hoogsteen base pair formed by U34 in loop II and A65 in loop III is stronger as in the absence of Mg²⁺ and the correlation between the imino group of G37 and the N3 of C61 due to the presence of the G37:C61 Watson–Crick base pair becomes detectable indicating that these base pairs are further stabilized in the presence of divalent cations.

Figure 5

Ligand binding and conformational changes of the G37A/C61U-mutant. (A) Location of the G37A/C61U-mutations in the secondary structure of the aptamer domain. It replaces a long-range Watson–Crick G:C base pair with a weaker A:U Watson–Crick base pair. (B) Consequences of the double mutation on hydrogen bonding of the base quadruple formed between loop II and III. The mutation results in the formal loss of two hydrogen bonds in the base quadruple. (C) Imino group region of an ¹H,¹⁵N-HSQC-spectrum of the free G37A/C61U-RNA in the presence of Mg²⁺. The imino group signals for nucleotides G32 and G38 are missing in the mutant due to the absence of a loop–loop interaction. They are observable only in the free wild-type RNA due to the presence of the loop–loop interaction there. In addition, no signal is observed for U61 in the free mutant RNA. The introduction of the G37A-mutation also eliminates the signal for G37 imino group. Inset: Native gel electrophoresis comparing the wild-type RNA (left) and the G37A/C61U-mutant (right). The lower electrophoretic mobility of the mutant indicates that the mutant is less compact compared to the wild-type in agreement with the absence of a loop–loop interaction. (D) Overlay of the imino group regions of ¹H,¹⁵N-HSQC-spectra for the hypoxanthine–RNA complexes at 10°C for the G37A/C61U-mutant RNA (red) and the wild-type RNA (black) in the presence of 5 mM Mg²⁺. In both cases, signals for the bound hypoxanthine are observable (labeled Hyp N1H1 and Hyp N9H9) as well as signals for nucleotides in the ligand binding core. This demonstrates that both RNAs are capable of ligand binding under these conditions. In addition, G32 and G38 are now observable in both RNA's indicating that upon ligand binding the loop–loop interaction forms also in the G37A/C61U-mutant. In addition, an imino group signal is observable for U61 in the mutant due to the formation of the A37:U61 Watson–Crick base pair instead of the G37:C61 Watson–Crick base pair in the wild-type RNA.

Figure 6

A loop–loop interaction outside the riboswitch context. (A) RNA-constructs containing only helices II and III with the wild-type and a modified (boxed) linker sequence. Nucleotides that correspond to sequences different from the natural riboswitch are given in lower-case letters. The numbering is analogous to the aptamer domain. (B) Schematic drawing of a reversed Hoogsteen A:U base pair with the uridine N3H3 adenine N7 hydrogen bond (dashed red line) and the expected NOE contact between uridine H3 and adenine H8 (red curved arrow) indicated. (C) Detail of the HNN-COSY spectrum for the RNA at 10°C at the position of the U34 imino group showing the correlation expected due to the hydrogen bond to the adenine N7-nitrogen. (D) Detail of a 2D-Watergate-¹H,¹H-NOESY-spectrum at 10°C at the position of the U34 imino group showing the NOE-cross peak to the adenine H8 in agreement with the formation of a reversed Hoogsteen A:U base pair. (E) 1D-¹H-imino proton spectra of the RNAs shown in (A) at 10°C reveal the presence of a signal for the G32 imino proton in both RNAs.