Cyclic-nucleotide binding (CNB) domains are structurally and evolutionarily conserved signaling modules that regulate proteins with diverse folds and functions. Despite a wealth of structural information, the mechanisms by which CNB domains couple cyclic-nucleotide binding to conformational changes involved in signal transduction remain unknown. Here we combined single-molecule and computational approaches to investigate the conformation and folding energetics of the two CNB domains of the regulatory subunit of protein kinase A (PKA). We found that the CNB domains exhibit different conformational and folding signatures in the apo state, when bound to cAMP, or when bound to the PKA catalytic subunit, underscoring their ability to adapt to different binding partners. Moreover, we show while the two CNB domains have near-identical structures, their thermodynamic coupling signatures are divergent, leading to distinct cAMP responses and differential mutational effects. Specifically, we demonstrate mutation W260A exerts local and allosteric effects that impact multiple steps of the PKA activation cycle. Taken together, these results highlight the complex interplay between folding energetics, conformational dynamics, and thermodynamic signatures that underlies structurally conserved signaling modules in response to ligand binding and mutational effects.