Differential analogue binding by two classes of c-di-GMP riboswitches

The ability of bacteria to adapt to a changing environment is essential for their survival. One mechanism bacteria have evolved to sense environmental cues and translate these signals into phenotypic changes uses the second messenger signaling molecule, cyclic diguanosine monophosphate (c-di-GMP). In addition to several classes of protein receptors, two classes of c-di-GMP-binding riboswitches (class I and class II) have been identified as downstream targets of the second messenger in this signaling pathway. The crystal structures of both riboswitch classes bound to c-di-GMP were previously reported. Here, we further investigate the mechanisms that RNA has evolved for recognition and binding of this second messenger. Using a series of c-di-GMP analogues, we probed the interactions made in the RNA-ligand complex for both classes of riboswitches to identify the most critical elements of c-di-GMP for binding. We found that the structural features of c-di-GMP required for binding differ between these two effectors and that the class II riboswitch is much less discriminatory in ligand binding than the class I riboswitch. These data suggest an explanation for the predicted preferential use of the class I motif over the class II motif in the c-di-GMP signaling pathway.