The ability of bacteria to sense environmental cues and adapt is essential for their survival. The use of second-messenger signaling molecules to translate these cues into a physiological response is a common mechanism employed by bacteria. The second messenger 3′–5′-cyclic diadenosine monophosphate (c-di-AMP) has been linked to a diverse set of biological processes involved in maintaining cell viability and homeostasis, as well as pathogenicity. A complex network of both protein and RNA receptors inside the cell activates specific pathways and mediates phenotypic outputs in response to c-di-AMP. Structural analysis of these RNA and protein receptors has revealed the different recognition elements employed by these effectors to bind the same small molecule. Herein, using a series of c-di-AMP analogues, we probed the interactions made with a riboswitch and a phosphodiesterase protein to identify the features important for c-di-AMP binding and recognition. We found that the ydaO riboswitch binds c-di-AMP in two discrete sites with near identical affinity and a Hill coefficient of 1.6. The ydaO riboswitch distinguishes between c-di-AMP and structurally related second messengers by discriminating against an amine at the C2 position more than a carbonyl at the C6 position. We also identified phosphate-modified analogues that bind both the ydaO RNA and GdpP protein with high affinity, whereas symmetrically modified ribose analogues exhibited a substantial decrease in ydaO affinity but retained high affinity for GdpP. These ligand modifications resulted in increased resistance to enzyme-catalyzed hydrolysis by the GdpP enzyme. Together, these data suggest that these c-di-AMP analogues could be useful as chemical tools to specifically target subsections of second-messenger signaling pathways.

The bacterial second messenger signaling molecule bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) controls important biological processes such as biofilm formation, virulence response, and motility. This second messenger is sensed by macromolecular targets inside the cell, both protein and RNA, which induce specific phenotypic responses critical for bacterial survival. One class of enzymes responsible for regulating the intracellular concentration of c-di-GMP, and therefore the physiological behavior of the cell, consists of the EAL domain phosphodiesterases, which degrade the second messenger to its linear form, pGpG. Here, we investigate how base and backbone modifications of c-di-GMP affect the rate of cyclic dinucleotide degradation by an EAL domain protein (CC3396 from Caulobacter crescentus). The doubly substituted thiophosphate analogue is highly resistant to hydrolysis by this metabolizing enzyme but can still bind c-di-GMP riboswitch targets. We used these findings to develop a novel ribosyl phosphate-modified derivative of c-di-GMP containing 2′-deoxy and methylphosphonate substitutions that is charge neutral and demonstrate that this analogue is also resistant to EAL domain-catalyzed degradation. This suggests a general strategy for designing c-di-GMP derivatives with increased enzymatic stability that also possess desirable properties for development as chemical probes of c-di-GMP signaling.