Tertiary Interactions in the Unbound Guanine-Sensing Riboswitch Focus Functional Conformational Variability on the Binding Site.

Riboswitches are genetic regulatory elements mainly found in bacteria, which regulate gene expression based on the availability of a ligand. Purine-sensing riboswitches, including the guanine-sensing riboswitch (Gsw), possess tertiary interactions connecting the L2 and L3 loops. These interactions are important for ligand binding to the aptamer. However, atomic-level structural knowledge about the unbound state and how the tertiary interactions influence the conformational heterogeneity of the aptamer is still scarce. We performed replica exchange molecular dynamics simulations of the aptamer domain of wild-type Gsw and a G37A/C61U mutant, which exhibits destabilized tertiary interactions, at different Mg concentrations with an aggregate simulation time of ∼16 μs, and subsequently obtained free-energy landscapes. Our data provide evidence that suggests that the unbound state of wild-type Gsw is conformationally rather homogeneous from a global viewpoint, yet the ligand binding site shows functionally necessary mobility required for ligand binding. For the mutant, the data suggest a heterogeneous ensemble, in particular without Mg. Hence, the tertiary interactions focus functional conformational variability on the binding site region of wild-type Gsw. Our data allow speculating that already the weakening of the tertiary interactions by two hydrogen bonds shifts the kinetics of folding from downhill folding without traps or intermediate states for wild-type Gsw to a folding including intermediates and misfolded structures for the mutant. A slowed-down folding of the aptamer might favor a decision during transcriptional regulation for the off-path, even if the ligand binds.