Detection of cyclic dinucleotide binding proteins

Abstract

Cyclic dinucleotides are a family of secondary messenger molecules that regulate bacterial physiology, cell division, motility, and biofilm formation. In response to stimuli, activated dinucleotide cyclases synthesize cyclic dinucleotides. Once made, cyclic dinucleotides bind macromolecule receptors, including proteins and RNA, to allosterically regulate downstream functions. Many important classes of cyclic di-GMP protein receptors have been identified including the PilZ domain, various degenerate enzymatic domains (GGDEF, EAL, and HD-GYP), the MshE domain, the AAA+ domain containing DNA binding proteins, as well as many unique examples. The identification of these cyclic di-GMP binding proteins and their cyclic di-GMP binding sites allows the generation of binding-defective alleles for interrogating the importance of cyclic di-GMP signaling in these regulated pathways. Using these tools, the field has revealed that cyclic di-GMP directly regulates many cellular functions through allosteric binding. Despite the success in the field of identifying protein receptors in the past few decades, cyclic dinucleotide receptors often can only be experimentally identified due to their diversity. To address these challenges, a number of experimental techniques have been utilized to empirically demonstrate interactions between cyclic dinucleotide and protein receptors. Here we will review the techniques used for the discovery and validation of these interactions by (1) affinity pull-down, (2) screening of proteins encoded by the genome, and (3) biochemical and structural methods. The use of these techniques will enable future development of predictive computational approaches that allow rapid identification and validation of cyclic dinucleotide receptor proteins. The identity of cyclic dinucleotide receptors will allow for a detailed understanding of the molecular mechanisms of cyclic dinucleotide signaling on cellular physiology.