Abstract
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.