Molecular dynamics in multidimensional space explains how mutations affect the association path of neomycin to a riboswitch

Riboswitches are messenger RNA (mRNA) fragments binding specific small moleculesto regulate gene expression. A synthetic N1 riboswitch, inserted into yeast mRNAcontrols the translation of a reporter gene in response to neomycin. However, itsregulatory activity is sensitive to single-point RNA mutations, even those distantfrom the neomycin binding site. While the association paths of neomycin to N1and its variants remain unknown, recent fluorescence kinetic experiments indicatea two-step process driven by conformational selection. This raises the question ofwhich step is affected by mutations. To address this, we performed all-atom two-dimensional replica-exchange molecular dynamics simulations for N1 and U14C,U14C+, U15A, and A17G mutants, ensuring extensive conformational sampling ofboth RNA and neomycin. The obtained neomycin association and binding paths, alongwith multidimensional free-energy profiles, revealed a two-step binding mechanism,consisting of conformational selection and induced fit. Neomycin binds to a preformedN1 conformation upon identifying a stable upper stem and U-turn motif in theriboswitch hairpin. However, the positioning of neomycin in the binding site occurs atdifferent RNA-neomycin distances for each mutant, which may explain their differentregulatory activities. The subsequent induced fit arises from the interactions of theneomycin's N3 amino group with RNA, causing the G9 backbone to rearrange. Inthe A17G mutant, the critical C6-A17/G17 stacking forms at a closer RNA-neomycindistance compared to N1. These findings together with estimated binding free energiescoincide with experiments and elucidate why the A17G mutation decreases and U15Aenhances N1 activity in response to neomycin