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

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.