Ionic Cooperativity between Lysine and Potassium in the Lysine Riboswitch: Single-Molecule Kinetic and Thermodynamic Studies.

Functionality in many biological systems, including proteins and nucleic acid structures, including protein and nucleic acid riboswitch structures, can depend on cooperative kinetic behavior between multiple small molecule ligands. In this work, single-molecule FRET data on the Bacillus subtilis lysine riboswitch reveals that affinity for the cognate lysine ligand increases significantly with K + , providing evidence for synergism between lysine/K + binding to the aptamer and successful folding of the riboswitch. To describe/interpret this more complex kinetic scenario, we explore the conventional 4-state ("square") model for aptamer binding as a function of K + . Extension into this additional dimension generates a novel "cube" model for riboswitch folding dynamics with respect to lysine/K + binding, revealing that riboswitch folding ( k fold ) and unfolding ( k unfold ) rate constants increase and decrease dramatically with K + , respectively. Furthermore, temperature-dependent single-molecule kinetic studies indicate that the presence of K + entropically enhances the transition state barrier to folding but partially compensates for this by increasing the overall exothermicity for lysine binding. We rationalize this behavior as evidence that K + facilitates hydrogen bonding between the negatively charged carboxyl group of lysine and the RNA, increasing structural rigidity and lowering entropy in the binding pocket. Finally, we explore the effects of cation size with Na + and Cs + studies to demonstrate that K + is optimally suited for bridging interactions between lysine and the riboswitch aptamer domain. Regulation of lysine production and transport, dictated by the riboswitch's ability to recognize and bind lysine, is therefore intimately tied to the presence of K + in the binding pocket and is strongly modulated by local cation conditions. The results suggest an increase in lysine riboswitch functionality by sensitivity to additional species in the cellular riboswitch environment.