cAMP is an allosteric modulator of DNA-binding specificity in the cAMP receptor protein from Mycobacterium...

Allosteric proteins with multiple subunits and ligand-binding sites are central in regulating biological signals. The cAMP receptor protein from Mycobacterium tuberculosis (CRP_MTB) is a global regulator of transcription composed of two identical subunits, each one harboring structurally conserved cAMP- and DNA-binding sites. The mechanisms by which these four binding sites are allosterically coupled in CRP_MTB remain unclear. Here, we investigate the binding mechanism between CRP_MTB and cAMP, and the linkage between cAMP and DNA interactions. Using calorimetric and fluorescence-based assays, we find that cAMP binding is entropically driven and displays negative cooperativity. Fluorescence anisotropy experiments show that apo-CRP_MTB forms high-order CRP_MTB-DNA oligomers through interactions with nonspecific DNA sequences or preformed CRP_MTB-DNA complexes. Moreover, we find that cAMP prevents and reverses the formation of CRP_MTB-DNA oligomers, reduces the affinity of CRP_MTB for nonspecific DNA sequences, and stabilizes a 1-to-1 CRP_MTB-DNA complex, but does not increase the affinity for DNA like in the canonical CRP from Escherichia coli (CRP_Ecoli). DNA-binding assays as a function of cAMP concentration indicate that one cAMP molecule per homodimer dissociates high-order CRP_MTB-DNA oligomers into 1-to-1 complexes. These cAMP-mediated allosteric effects are lost in the double-mutant L47P/E178K found in CRP from Mycobacterium bovis Bacille Calmette-Guérin (CRP_BCG). The functional behavior, thermodynamic stability, and dimerization constant of CRP_BCG are not due to additive effects of L47P and E178K, indicating long-range interactions between these two sites. Altogether, we provide a previously undescribed archetype of cAMP-mediated allosteric regulation that differs from CRP_Ecoli, illustrating that structural homology does not imply allosteric homology.