c-di-AMP binds the ydaO riboswitch in two pseudo-symmetry-related pockets

The ydaO riboswitch, involved in sporulation, osmotic stress responses and cell wall metabolism, targets the second messenger c-di-AMP with subnanomolar affinity. We have solved the structure of c-di-AMP bound to the Thermoanaerobacter tengcongenesis ydaO riboswitch, thereby identifying a five-helical scaffold containing a zippered-up bubble, a pseudoknot and long-range tertiary base pairs. Highlights include the identification of two c-di-AMP binding pockets on the same face of the riboswitch, related by pseudo two-fold symmetry, with potential for cross-talk between sites mediated by adjacently-aligned base stacking alignments connecting pockets. The adenine rings of bound c-di-AMP molecules are wedged between bases and stabilized by stacking, base-sugar and sugar-sugar intermolecular hydrogen bonding interactions. The structural studies are complemented by ITC-based binding studies of mutants mediating key tertiary intermolecular contacts. The T. tengcongenesis ydaO riboswitch, like its B. subtilis counterpart, likely functions through a transcription termination mechanism, with the c-di-AMP bound state representing an ‘off’ switch.