Identifying Allosteric Hotspots in <i>Mycobacterium tuberculosis</i> 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 Escherichia coli (CRP Ecoli ) and from Mycobacterium tuberculosis (CRP MTB ). By introducing structurally homologous mutations from allosteric hotspots previously identified in CRP Ecoli into CRP MTB 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 MTB , but had varying effects on cAMP binding affinity and cooperativity. Interestingly, the mutations had minimal impact on the specific binding of CRP MTB to DNA promoter sites. However, we found that cAMP primarily reduces nonspecific CRP MTB -DNA complexes and that the mutants largely lose this ability. Furthermore, our experiments revealed that CRP MTB -DNA complexes serve as a nucleation point for additional binding of CRP MTB 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 MTB and provide insights into the differential responses of CRP Ecoli and CRP MTB to cAMP.