Full-Length NAD⁺-I Riboswitches Bind a Single Cofactor but Cannot Discriminate against Adenosine Triphosphate

Bacterial riboswitches are structured RNAs that bind small metabolites to control downstream gene expression. Two riboswitch classes have been reported to sense nicotinamide adenine dinucleotide (NAD⁺), which plays a key redox role in cellular metabolism. The NAD⁺-I (class I) riboswitch stands out because it comprises two homologous, tandemly arranged domains. However, previous studies examined the isolated domains rather than the full-length riboswitch. Crystallography and ligand binding analyses led to the hypothesis that each domain senses NAD⁺ but with disparate equilibrium binding constants (K_D) of 127 μM (domain I) and 3.4 mM (domain II). Here, we analyzed individual domains and the full-length riboswitch by isothermal titration calorimetry to quantify the cofactor affinity and specificity. Domain I senses NAD⁺ with a K_D of 24.6 ± 8.4 μM but with a reduced ligand-to-receptor stoichiometry, consistent with nonproductive domain self-association observed by gel-filtration chromatography; domain II revealed no detectable binding. By contrast, the full-length riboswitch binds a single NAD⁺ with a K_D of 31.5 ± 1.5 μM; dinucleotides NADH and AP₂-ribavirin also bind with one-to-one stoichiometry. Unexpectedly, the full-length riboswitch also binds a single ATP equivalent (K_D = 11.0 ± 3.5 μM). The affinity trend of the full-length riboswitch is ADP = ATP > NAD⁺ = AP₂-ribavirin > NADH. Although our results support riboswitch sensing of a single NAD⁺ at concentrations significantly below the intracellular levels of this cofactor, our findings do not support the level of specificity expected for a riboswitch that exclusively senses NAD⁺. Gene regulatory implications and future challenges are discussed.