Identifying Allosteric Hotspots in cAMP Receptor Protein through Structural Homology.

Understanding the mechanisms of allosteric regulation in response to second messengers is crucial for advancing basic and applied research. This study focuses on the differential allosteric regulation by the ubiquitous signaling molecule, cAMP, in the cAMP receptor protein from (CRP) and from (CRP). By introducing structurally homologous mutations from allosteric hotspots previously identified in CRP into CRP and examining their effects on protein solution structure, stability and function, we aimed to determine the factors contributing to their differential allosteric regulation. Our results demonstrate that the mutations did not significantly alter the overall fold, assembly and thermodynamic stability of CRP, but had varying effects on cAMP binding affinity and cooperativity. Interestingly, the mutations had minimal impact on the specific binding of CRP to DNA promoter sites. However, we found that cAMP primarily reduces nonspecific CRP-DNA complexes and that the mutants largely lose this ability. Furthermore, our experiments revealed that CRP-DNA complexes serve as a nucleation point for additional binding of CRP proteins to form high-order oligomers with the DNA. Overall, our findings highlight the importance of both cAMP and DNA interactions in modulating the allosteric regulation of CRP and provide insights into the differential responses of CRP and CRP to cAMP.