Riboswitches are regulatory fragments of mRNA that exert their function upon binding of a ligand or upon change of temperature or pH. Therefore, the activity of the riboswitches is related to their structural changes. We focus on a synthetic riboswitch, named N1, that binds aminoglycosides (Duchardt-Ferner, E. et al., Angew. Chem. Int. Ed., 55:1527, 2016). This riboswitch forms a 27-nucleotide hairpin with a bulge to accomodate the ligand. Experiments indicate that some single-point mutations, especially A17G, change the riboswitch activity (Weigand, J. E. et al., ChemBioChem, 15:1627, 2014). We performed conventional and generalized replica exchange with solute tempering (gREST) all-atom molecular dynamics simulations to understand how the activity of the riboswitch correlates with its flexibility (Chyzy P. et al., Front. Mol. Biosci., 8:633130, 2021). Simulations were performed for the unbound and neomycin-bound N1 riboswitch and its mutants (U14C, U14C+, U15A, and A17G). We found that the reduced activity of the A17G mutant arises from the bulge-apical loop stacking (C6:U7:G17) formed in the free mutant. These interactions decrease the number of A17G conformations predisposed for neomycin to enter its pocket. We also observed that the A17G mutation changes the riboswitch conformation in the neomycin-bound state, due to the weaker C6:G17 stacking interaction that is crucial for the riboswitch regulatory activity. On the contrary, the free U15A mutant displays a higher population of conformations preformed for neomycin binding, which agrees with the enhanced activity of this mutant. In addition, molecular dynamics simulations of the U14C mutant support the protonated state of C14 in the bound state and deprotonated one in the unbound state. Further simulations are being performed to understand the neomycin association paths with the N1 riboswitch using replica-exchange umbrella sampling method.

Structural dynamics and recognition of an aminoglycoside-binding riboswitch

ACS Spring 2022

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