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.