A kind of pinky promise: Chain interlocking in the guanidine-II riboswitch observed in atomistic simulations and network analysis

Riboswitches are dynamic RNA structures that regulate gene expression in response to ligand binding. The guanidine-II riboswitch consists of two hairpin loops, connected by a flexible linker, which can dimerize in the presence of a cognate ligand. Both processes, ligand binding and dimerization, are governed by the first three bases of each of the tetraloops, while the role of the extrahelical fourth base of the tetraloop, conserved to a purine, is less well understood. An influence of base identity in this position on ligand binding affinity and cooperativity has been shown in experiment, but no molecular-level explanation of this influence has yet been attempted. To investigate the interactions underlying this observation, we performed molecular dynamics simulations of the guanidine-II riboswitch with varied base identity of these extrahelical bases. The simulations show base-identity dependence in the formation of a recently described conformational change that involves the extrahelical bases and interlocks both hairpins. Tuning an established network-analysis-based approach to be sensitive to delicate changes in hydrogen bonding patterns enables us to classify the conformational states involved and show how base identity affects interlocking stability. These interactions may underpin the influence of the extrahelical bases on the structural equilibrium of the riboswitch and suggest a rationale for base-identity preference at this position.