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