Global analysis of riboswitches by small-angle X-ray scattering and calorimetry

Abstract

Riboswitches are phylogenetically widespread non-coding mRNA domains that directly bind cellular metabolites and regulate transcription, translation, RNA stability or splicing via alternative RNA structures modulated by ligand binding. The details of ligand recognition by many riboswitches have been elucidated using X-ray crystallography and NMR. However, the global dynamics of riboswitch-ligand interactions and their thermodynamic driving forces are less understood. By compiling the work of many laboratories investigating riboswitches using small-angle X-ray scattering (SAXS) and isothermal titration calorimetry (ITC), we uncover general trends and common themes. There is a pressing need for community-wide consensus experimental conditions to allow results of riboswitch studies to be compared rigorously. Nonetheless, our meta-analysis reveals considerable diversity in the extent to which ligand binding reorganizes global riboswitch structures. It also demonstrates a wide spectrum of enthalpy-entropy compensation regimes across riboswitches that bind a diverse set of ligands, giving rise to a relatively narrow range of physiologically relevant free energies and ligand affinities. From the strongly entropy-driven binding of glycine to the predominantly enthalpy-driven binding of c-di-GMP to their respective riboswitches, these distinct thermodynamic signatures reflect the versatile strategies employed by RNA to adapt to the chemical natures of diverse ligands. Riboswitches have evolved to use a combination of long-range tertiary interactions, conformational selection, and induced fit to work with distinct ligand structure, charge, and solvation properties. This article is part of a Special Issue entitled: Riboswitches.

Keywords: Conformational selection; Enthalpy–entropy compensation; Induced fit; Isothermal titration calorimetry; Small-angle X-ray scattering.

Fig. 1

Structural contexts of cyclic-di-GMP and thiamine pyrophosphate (TPP) binding to their respective cognate riboswitches. (A) Crystal structure of cyclic-di-GMP bound to its riboswitch (3IWN). (B) SAXS-derived molecular envelope of the cyclic-di-GMP riboswitch bound to its cognate ligand in 2.5 mM Mg²⁺, superimposed on the crystal structure in (A). (C) SAXS-derived molecular envelope of the ligand-free cyclic-di-GMP riboswitch in 2.5 mM Mg²⁺. (D) Crystallographic analysis reveals that TPP binds across two parallel RNA helical stacks and stabilizes a compact riboswitch conformation (PDB: 2HOJ). (E) SAXS-derived molecular envelope of the TPP riboswitch bound to TPP in 2.5 mM Mg²⁺, superimposed on the crystal structure in (D). (F) SAXS-derived molecular envelope of the ligand-free TPP riboswitch in 2.5 mM Mg²⁺.

Fig. 2

Global SAXS analysis of riboswitches. (A) The radius of gyration (R_g) of riboswitches in solution correlates linearly with RNA length. Riboswitch data for RNAs in the free (hollow, blue squares) and ligand-bound (filled, black circles) states in the presence of Mg²⁺ is from Table 2 and references therein. Correlation analysis yields a Pearson’s R value of 0.96 for ligand-free riboswitches and 0.93 for ligand-bound riboswitches. Data for tRNA^Phe is shown for comparison and is from [27]. (B) Entropy of ligand binding versus the extent of riboswitch compaction (ΔR_g). Poor correlation (R = 0.44) suggests that the entropy change that accompanies ligand binding is not dominated by the conformational entropy arising from global reorganization.

Fig. 3

Calorimetric analysis of riboswitches. (A–C) Solvent-accessible ligand surface area buried (Ų) by riboswitch binding does not correlate strongly with the binding enthalpy (A), free energy (B), or entropy (C). This contrasts with the strong correlation (R = 0.89) between binding free energy and solvent-accessible area buried by in vitro selected aptamers [55]. (D) A wide spectrum of entropy-enthalpy distributions demonstrate significant compensation (R = 0.98), resulting in a relatively narrow range of ligand binding affinities for riboswitches.

Fig. 4

Dynamic, multi-step mechanisms of ligand binding to riboswitches. Ligand-free riboswitches can exist in two or more structurally and dynamically different forms. A conformational equilibrium between binding-competent and binding-incompetent structures can be modulated by temperature, Mg²⁺ concentration, etc. This allows the riboswitch to sample a variety of distinct structures, some of which are primed to engage the ligand rapidly. Attaining the native, ligand-bound state may also require induced fit to form a ligand-binding site that does not pre-exist in any of the ligand-free structures.