Structural basis for guanidine sensing by the ykkC family of riboswitches

Abstract

Regulation of gene expression by cis-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 ykkC orphan family was recently characterized as guanidine-sensing riboswitches. Herein we present a 2.3 Å crystal structure of the guanidine-bound ykkC riboswitch from Dickeya dadantii 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 sp² 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 ykkC riboswitches and pave the way for a deeper understanding of guanidine detoxification-a previously unappreciated aspect of bacterial physiology.

Keywords: Dickeya dadantii; RNA structure; gene regulation; guanidine; orphan riboswitch.

FIGURE 1.

Secondary structure model and ITC binding curve of Dda_ykkC. (A) 2D-representation of Dda_ykkC with Leontis–Westhof notation describing base interactions observed in the crystal structure. P1.1 (teal), L1 (violet), P1.2 (marine), P2 (gray), and P3 (orange) are shown. Circled residues are 97% conserved in subtype 1 ykkC riboswitches. Residues and phosphate contacting guanidinium (blue triangle) are highlighted in blue. Red residues participate in alternative terminator stem. Gray residues, boxes, and arrows show changes to WT sequence in the crystal construct. (B) ITC analysis of guanidinium binding by WT Dda_ykkC.

FIGURE 2.

Overall structure of Dda_ykkC and detailed cross-helix interactions. (A) Cartoon representation of guanidinium (cyan) bound Dda_ykkC. Surface representation showing boot-like shape (bottom inset). (B) Detailed non-WC contacts in L1 region including AG aDNB and cross-helix contacts (inset). (C) Minor groove interaction between P3 A-loop and L1. Ribose zipper contacts are shown from the front (left) and base-specific contacts from the rear (right).

FIGURE 3.

Guanidinium recognition in the binding pocket. (A) Front (right) and rear (left) views of Dda_ykkC P3 stem. Base pairs are detailed with Leontis–Westhof notation in the middle with arrows indicating the location of the pair in front and rear views. Inset shows magnesium ions coordinated by the P1 and P3 backbones. (B) Side view of the binding pocket showing cation–π stacking interaction between guanidinium (cyan) and G67 (orange). Inset shows omit-map electron density of the binding pocket at 1.5 σ generated by simulated annealing refinement. Black dashes represent hydrogen bonds. Gray dashes represent weak electrostatic interactions. (C) Top view of the binding pocket showing hydrogen-bonding network. Mutants designed for binding affinity experiments are connected by lines to their corresponding residue labels. Interatomic distances are given next to dashes.

FIGURE 4.

Structure-guided mutagenesis evaluated by ITC analysis. (A) 2D-representation of Dda_ykkC showing the positions with which each mutant corresponds. (B) Table displaying estimates of WT and mutant binding affinities for guanidinium.