Dissecting the influence of Mg2+ on 3D architecture and ligand-binding of the guanine-sensing riboswitch aptamer domain

Abstract

Long-range tertiary interactions determine the three-dimensional structure of a number of metabolite-binding riboswitch RNA elements and were found to be important for their regulatory function. For the guanine-sensing riboswitch of the Bacillus subtilis xpt-pbuX operon, our previous NMR-spectroscopic studies indicated pre-formation of long-range tertiary contacts in the ligand-free state of its aptamer domain. Loss of the structural pre-organization in a mutant of this RNA (G37A/C61U) resulted in the requirement of Mg(2+) for ligand binding. Here, we investigate structural and stability aspects of the wild-type aptamer domain (Gsw) and the G37A/C61U-mutant (Gsw(loop)) of the guanine-sensing riboswitch and their Mg(2+)-induced folding characteristics to dissect the role of long-range tertiary interactions, the link between pre-formation of structural elements and ligand-binding properties and the functional stability. Destabilization of the long-range interactions as a result of the introduced mutations for Gsw(loop) or the increase in temperature for both Gsw and Gsw(loop) involves pronounced alterations of the conformational ensemble characteristics of the ligand-free state of the riboswitch. The increased flexibility of the conformational ensemble can, however, be compensated by Mg(2+). We propose that reduction of conformational dynamics in remote regions of the riboswitch aptamer domain is the minimal pre-requisite to pre-organize the core region for specific ligand binding.

Figure 1.

(a) Secondary structure of the guanine-sensing riboswitch aptamer domain (Gsw) of the B. subtilis xpt-pbuX operon and the mutant aptamer domain (G37A/C61U, Gsw^loop) following the nomenclature of Mandal et al. (22). Nucleotides in the loop regions L2 and L3 that differ in the two constructs are highlighted in red [for further construct details, see refs (27,29)]; (b) X-ray structure of the Gsw-hypoxanthine complex (pdb: 1U8D) (24), loop regions (L2 and L3) are color coded in black, mutated residues at nucleotide positions 37 and 61 in the Gsw^loop-construct are color coded in red, the ligand hypoxanthine is highlighted in blue; the base-quadruple including the mutation sites for Gsw^loop is enlarged next to the X-ray structure; (c) ¹H,¹⁵N-HSQC spectrum of Gsw^loop with annotated NMR imino proton resonance assignment ([RNA]:[Mg²⁺] ratio = 1:7, T = 283 K).

Figure 2.

Mg²⁺-induced effects on Gsw^loop structure formation; (a) Overlay of ¹H,¹⁵N-HSQC spectra of Gsw^loop (T = 283 K) at [RNA]:[Mg²⁺] ratios of 1:8 (black) and 1:33 (red). INSET: secondary structure of Gsw^loop. RNA imino proton resonances that are exclusively detectable at a high [RNA]:[Mg²⁺] ratio (1:33) are annotated and color coded in red. Imino proton resonances with a chemical shift perturbation Δδ of >40 Hz within [RNA]:[Mg²⁺] ratios ranging from 1:8 to 1:33 are annotated in blue; (b) chemical shift perturbation (CSP) Δδ [Hz] of resolved imino proton resonances upon Mg²⁺ titration, gray bar: titration of Gsw^loop to a final [RNA]:[Mg²⁺] ratio of 1:8, black bar: titration of Gsw^loop to a final [RNA]:[Mg²⁺] ratio of 1:33; (c) CSP [Hz] of two exemplary imino proton resonances (G55 and G57) as a function of the [Mg²⁺]:[RNA] ratio; the K_D-values are determined from the correlation up to a [Mg²⁺]:[RNA] ratio of ∼18:1. The dashed gray line indicates the minimal [RNA]:[Mg²⁺] ratio for which all signals of the tertiary loop–loop interaction can be detected in the NMR spectra of Gsw^loop.

Figure 3.

Mg²⁺-induced effects on Gsw^loop–hypoxanthine complexes; (a) overlay of ¹H,¹⁵N-HSQC spectra of the Gsw^loop–hypoxanthine complex with (ii) 7 eq (black) and (iii) 33 eq (red) of Mg²⁺. Residues that show a chemical shift perturbation Δδ of >25 Hz or can exclusively be detected in the presence of high Mg²⁺ concentrations are annotated; (b) residues depicted in (a) are highlighted as red spheres on the X-ray structure of the Gsw–hypoxanthine complex [pdb: 1U8D (24)].

Figure 4.

Temperature-dependent characteristics of Gsw–hypoxanthine complexes; overlay of temperature-dependent ¹H,¹⁵N-HSQC spectra (black: 10°C, blue: 20°C, red: 30°C) of (a) the Gsw–hypoxanthine complex, (b) the Gsw–hypoxanthine complex in the presence of Mg²⁺ (¹⁵N-labeled RNA, unlabeled hypoxanthine). Imino proton signals not detectable at 30°C are annotated; (c) normalized CD melting profiles of the Gsw–hypoxanthine complex in the presence and absence of Mg²⁺.