Excited States of apo -Guanidine-III Riboswitch Contribute to Guanidinium Binding through Both Conformational and Induced-Fit Mechanisms.

Riboswitches are mRNA segments that regulate gene expression through conformational changes driven by their cognate ligand binding. The ykkC motif forms a riboswitch class that selectively senses a guanidinium ion (Gdm + ) and regulates the downstream expression of proteins which aid in the efflux of excess Gdm + from the cells. The aptamer domain (AD) of the guanidine-III riboswitch forms an H-type pseudoknot with a triple helical domain that binds a Gdm + . We studied the binding of Gdm + to the AD of the guanidine ( ykkC )-III riboswitch using computer simulations to probe the specificity of the riboswitch to Gdm + binding. We show that Gdm + binding is a fast process occurring on the nanosecond time scale, with minimal conformational changes to the AD. Using machine learning and Markov-state models, we identified the excited conformational states of the AD, which have a high Gdm + binding propensity, making the Gdm + binding landscape complex exhibiting both conformational selection and induced-fit mechanisms. The proposed apo -AD excited states and their role in the ligand-sensing mechanism are amenable to experimental verification. Further, targeting these excited-state conformations in discovering new antibiotics can be explored.