An intermolecular G-quadruplex as the basis for GTP recognition in the class V-GTP aptamer

Abstract

Many naturally occurring or artificially created RNAs are capable of binding to guanine or guanine derivatives with high affinity and selectivity. They bind their ligands using very different recognition modes involving a diverse set of hydrogen bonding and stacking interactions. Apparently, the potential structural diversity for guanine, guanosine, and guanine nucleotide binding motifs is far from being fully explored. Szostak and coworkers have derived a large set of different GTP-binding aptamer families differing widely in sequence, secondary structure, and ligand specificity. The so-called class V-GTP aptamer from this set binds GTP with very high affinity and has a complex secondary structure. Here we use solution NMR spectroscopy to demonstrate that the class V aptamer binds GTP through the formation of an intermolecular two-layered G-quadruplex structure that directly incorporates the ligand and folds only upon ligand addition. Ligand binding and G-quadruplex formation depend strongly on the identity of monovalent cations present with a clear preference for potassium ions. GTP binding through direct insertion into an intermolecular G-quadruplex is a previously unobserved structural variation for ligand-binding RNA motifs and rationalizes the previously observed specificity pattern of the class V aptamer for GTP analogs.

Keywords: G-quadruplex; GTP; NMR; RNA structure; aptamer; ligand binding.

FIGURE 1.

The GTP–class V aptamer binds GTP through non-Watson-Crick interactions. (A) Secondary structure of the class V–GTP-binding aptamer as predicted by Szostak and coworkers (Carothers et al. 2004). Based on the previously reported sequences of this aptamer, the degree of nucleotide conservation was calculated and is indicated by different colors with completely conserved nucleotides shown in red and nucleotides conserved in >95% of the reported sequences shown in orange. (B) 1D-¹H imino proton spectra in potassium phosphate buffer for the GTP–class V aptamer in its free form (top), in the presence of 2 mM Mg²⁺ (middle), and in the presence of 2 mM Mg²⁺ and 1.2 eq of GTP (bottom). (C) Overlay of ¹H,¹⁵N-HSQC spectra of the ¹⁵N-guanine-labeled class V aptamer RNA in the presence of 2 mM Mg²⁺ (black) and in the presence of 2 mM Mg²⁺ and 1.2 eq of ¹⁵N,¹³C-labeled GTP (red). The spectra are slightly displaced with respect to each other. A gray box highlights the area where a number of guanine imino group signals appear only upon addition of the ligand GTP. (D) The 1D slice of a ¹H,¹⁵N-HSQC spectrum recorded with a sample consisting of unlabeled RNA and ¹⁵N-labeled GTP reveals the imino group signal of the bound GTP with a chemical shift of 11.2 ppm outside the range typically found for guanine imino protons in Watson-Crick base pairs.

FIGURE 2.

GTP binding to the class V aptamer RNA depends on the identity of the monovalent cations. (A) Overlay of 1D-¹H imino proton spectra of the GTP–class V RNA in a buffer containing 25 mM NaPO₄, 50 mM NaCl, 2 mM Mg²⁺ in the absence of GTP (black) and in the presence of 1.2 (red), and 5.3 eq of GTP. In contrast to the observations in the equivalent potassium phosphate buffer, ligand addition induces only minor changes in the spectra. (B) 1D slices of ¹H,¹⁵N-HSQC spectra recorded for samples containing unlabeled RNA and 1.2 (red) or 5.3 eq (green) of ¹⁵N-labeled GTP. Only a weak imino group signal corresponding to bound GTP is observed in comparison to the corresponding signal observed in potassium phosphate buffer (gray). (C) ITC thermograms and derived binding curves for GTP binding to the class V aptamer RNA in buffers of equivalent ionic strengths containing potassium, sodium, or thallium ions.

FIGURE 3.

NMR evidence for the formation of an intermolecular G-quadruplex structure upon GTP binding to the class V aptamer RNA. (A) Structure of a single G-tetrad layer from a G-quadruplex including the bound ligand (red). Hydrogen bonds are indicated by dashed lines. Connecting lines denote expected NOE connectivities between guanine nucleotides in the quadruplex (black) including those expected for the bound GTP (red). Note that similar NOE connectivities are also observable between nucleotides in neighboring layers of a quadruplex. (B) 2D-¹H,¹H NOESY spectrum recorded for an unlabeled RNA bound to unlabeled GTP in potassium phosphate buffer showing NOEs involving imino protons. NOEs typical for G-quadruplex structures are highlighted by boxes and labeled accordingly. (C) 2D-¹H,¹H-plane from a ¹³C-edited NOESY-HSQC spectrum recorded with a ¹⁵N,¹³C-guanine-labeled RNA bound to unlabeled GTP. This experiment allows the selective detection of NOEs from guanine H8C8 aromatic moieties to imino protons that are typical for G-quadruplexes. (D) Intermolecular NOEs involving the imino group (left) and the H8C8 group of the bound GTP as detected in either ¹⁵N- (left) or ¹³C-edited (right) NOESY-HSQC experiments recorded for samples containing ¹⁵N,¹³C-labeled ligand GTP and unlabeled RNA. (E) Schematic structure of a G-quadruplex tetrad bound to an ammonium ion. (F) The GTP–class V aptamer stably binds an ammonium ion in the presence (top, red) but not in the absence of bound GTP (top, black), as seen from 1D slices of ¹H,¹⁵N-HSQC spectra recorded for samples containing ¹⁵N-labeled ammonium chloride and unlabeled aptamer RNA or the unlabeled RNA-GTP complex. A single signal with a nitrogen chemical shift of ∼30.8 ppm corresponding to the bound ammonium ion is detectable in a 2D-¹H,¹⁵N-HSQC experiment with the RNA-GTP complex (bottom). Free ammonium ions are not detectable in this experiment due to the fast exchange of their protons with the bulk solvent. (G) The ammonium ion is located in the center of the G-quadruplex structure as indicated by NOEs between the ammonium ion protons and guanine imino protons seen in 2D-NOESY experiments (left) or in ¹⁵N-edited NOESY-HSQC experiments with a sample containing ¹⁵N-ammonium chloride and unlabeled RNA/GTP (middle). The imino proton of the bound GTP also shows an NOE to the protons of the ammonium ion as seen in a ¹⁵N-edited NOESY-HSQC recorded with a sample containing ¹⁵N-labeled GTP and unlabeled ammonium chloride and unlabeled RNA (right).

FIGURE 4.

The G-quadruplex structure is formed by seven guanine nucleotides from the two asymmetric internal bulges and the bound GTP. (A) Comparison of 1D- ¹H imino proton (top) and ¹H,¹⁵N-HSQC spectra (bottom) for the GTP-aptamer complex before (black) and 30 min after exchange of the sample into D₂O (red). A box highlights the region of guanine residues involved in G-quadruplex formation. The gray inset shows a 1D slice of a ¹H,¹⁵N-HSQC spectrum recorded for a sample containing ¹⁵N-GTP and unlabeled RNA 30 min after exchange into D₂O to demonstrate that the imino proton of the bound ligand is also protected against exchange with the solvent. (B) Overlay of the G-quadruplex imino group region of ¹H,¹⁵N-HSQC spectra of the GTP-aptamer complex prior to (black) and 30 min (red) or 4 h (yellow) after exchange of the sample into D₂O, respectively. The signal of the imino group of the bound GTP is indicated by an arrow. The area shown corresponds to the boxed region in A. Resonances marked with an asterisk (*) correspond to guanine imino groups in G:U wobble base pairs. (C) Secondary structure of the bipartite GTP–class V aptamer construct used for strand-selective labeling experiments. A dashed line separates the 5′-strand (left) from the 3′-strand (right) of the RNA. The nucleotides of the “lower bulge” are colored in green and those of the “upper bulge” in blue. (D) Overlay of the G-quadruplex imino group region of ¹H,¹⁵N-HSQC spectra of the GTP-aptamer complex with those recorded for the bipartite RNA construct ¹⁵N-labeled in either the 5′-strand (green) or in the 3′-strand (blue). The imino group signal for the bound GTP (arrow) is absent in the spectra of the bipartite construct since in this experiment only unlabeled ligand was used. (E) Secondary structure of the bipartite GTP–class V aptamer construct with the G61A, G62A double mutation. (F) Comparison of 1D-¹H imino proton spectra of the GTP-bound form of the WT–class V aptamer (bottom), the bipartite aptamer construct with the WT sequence, and the G61A, G62A double mutant aptamer (top). Note that in comparison to the WT aptamer, the bipartite aptamer constructs both lack the apical UUCG tetraloop and therefore the diagnostic G imino proton resonance at 9.6 ppm as well as a G:U base pair in the upper stem.

FIGURE 5.

Mapping the guanine residues participating in ligand binding and intermolecular G-quadruplex formation by G to A point mutations. Shown is the secondary structure of the GTP–class V aptamer. Mutation sites that abolish binding completely are colored red (no bdg. = no binding observable even in NMR experiments with an RNA concentration of 150 µM and a 6.7-fold excess of GTP). Nucleotides where mutation reduces the K_D significantly are colored in orange, and nucleotides that can be mutated without an effect on the GTP affinity are colored green. Note that the K_D’s for the G40A and G41A mutants are in the µM range. The corresponding NMR and ITC data are shown in Supplemental Figure 9.

FIGURE 6.

Influence of the length of the central helix on the GTP affinity of the aptamer. The secondary structure of the aptamer is shown on the left. A gray box highlights the central helix. The secondary structures of the shortened and extended helices are shown on the right with the respective K_D given below. Added base pairs are shown in red; gray base pairs were deleted in the shortened constructs. “No bdg.” indicates that no binding is observable even in NMR experiments with an RNA concentration of 150 µM and an excess of GTP (see Supplemental Fig. 10).