Structural basis for guanidine sensing by the family of riboswitches.

Regulation of gene expression by encoded riboswitches is a prevalent theme in bacteria. Of the hundreds of riboswitch families identified, the majority of them remain as orphans, without a clear ligand assignment. The orphan family was recently characterized as guanidine-sensing riboswitches. Herein we present a 2.3 Å crystal structure of the guanidine-bound riboswitch from The riboswitch folds into a boot-shaped structure, with a coaxially stacked P1/P2 stem forming the boot, and a 3'-P3 stem-loop forming the heel. Sophisticated base-pairing and cross-helix tertiary contacts give rise to the ligand-binding pocket between the boot and the heel. The guanidine is recognized in its positively charged guanidinium form, in its sp hybridization state, through a network of coplanar hydrogen bonds and by a cation-π stacking contact on top of a conserved guanosine residue. Disruption of these contacts resulted in severe guanidinium-binding defects. These results provide the structural basis for specific guanidine sensing by riboswitches and pave the way for a deeper understanding of guanidine detoxification-a previously unappreciated aspect of bacterial physiology.