Elbow Flexibility of the kt38 RNA Kink-Turn Motif Investigated by Free-Energy Molecular Dynamics Simulations

Kink-turns (K-turns) are common structural motifs that can introduce sharp kinks into double-stranded RNA, and have been proposed to mediate large-scale motions in the ribosome. K-turns consist of a bulge loop region flanked by trans sugar-Hoogsteen G:A pairs, and the sharp kink conformation is stabilized by A-minor interactions (adenine contacting a G:C basepair in the minor groove). Umbrella-sampling molecular dynamics simulations were used to disrupt an A-minor interaction in the ribosomal kt38 turn and to calculate the associated free-energy change. Coupling of umbrella sampling with replica exchanges between neighboring umbrella-sampling intervals could further improve the convergence of the free-energy calculations. The simulations revealed a coupled A-minor disruption and global opening of the K-turn motif, and allowed us to characterize several intermediate A-minor conformations. The calculated free-energy profile indicated a meta-stable, semi-open structure of slightly higher free energy (∼1 kcal mol−1), separated by a small free-energy barrier (∼1.5 kcal mol−1) from the closed (highly kinked) form. Both K-turn states are stabilized by distinct variants of the A-minor interaction. Further opening of the K-turn structure required significantly larger free-energy changes. The semi-open form had a reduced kink angle compatible with experimental data on K-turn solution structures, and opening was coupled to a continuous global unwinding of the K-turn motif. The range of free-energy changes associated with kt38 opening and unwinding are compatible with the idea that K-turns may facilitate biologically relevant motions during large-scale ribosome dynamics.