An uncommon [K⁺(Mg²⁺)₂] metal ion triad imparts stability and selectivity to the Guanidine-I riboswitch

Abstract

The widespread ykkC-I riboswitch class exemplifies divergent riboswitch evolution. To analyze how natural selection has diversified its versatile RNA fold, we determined the X-ray crystal structure of the Burkholderia sp. TJI49 ykkC-I subtype-1 (Guanidine-I) riboswitch aptamer domain. Differing from the previously reported structures of orthologs from Dickeya dadantii and Sulfobacillus acidophilus, our Burkholderia structure reveals a chelated K⁺ ion adjacent to two Mg²⁺ ions in the guanidine-binding pocket. Thermal melting analysis shows that K⁺ chelation, which induces localized conformational changes in the binding pocket, improves guanidinium-RNA interactions. Analysis of ribosome structures suggests that the [K⁺(Mg²⁺)₂] ion triad is uncommon. It is, however, reminiscent of metal ion clusters found in the active sites of ribozymes and DNA polymerases. Previous structural characterization of ykkC-I subtype-2 RNAs, which bind the effector ligands ppGpp and PRPP, indicate that in those paralogs, an adenine responsible for K⁺ chelation in the Burkholderia Guanidine-I riboswitch is replaced by a pyrimidine. This mutation results in a water molecule and Mg²⁺ ion binding in place of the K⁺ ion. Thus, our structural analysis demonstrates how ion and solvent chelation tune divergent ligand specificity and affinity among ykkC-I riboswitches.