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

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.

Structural and Functional Characterization of Mycobacterium tuberculosis Homoserine Transacetylase

“…Given the dimeric nature and likely allostery between the two HTA active sites in the homodimer, negative cooperativity may be operative between the dimer active sites (Figure S10). This is congruent with similar phenomena in other multimeric enzymes involved in primary metabolism. − …”

The global regulation of c‐di‐GMP and cAMP in bacteria

“…The apo‐CRP MTB dimer binds to both specific and nonspecific DNA sequences to generate high‐order CRP MTB ‐DNA complexes in the absence of cAMP 127 . After macrophage infection, intracellular cAMP levels increase significantly, resulting in all apo‐CRP MTB dimers being bound by a single cAMP molecule 127 .…”

Structural and functional diversity of bacterial cyclic nucleotide perception by CRP proteins

“…Therefore, the mycobacterial Crp might be evolutionary adapted to sense high cAMP levels by lowering the binding affinity for this ligand (Green et al 2014 ). A recent study suggests that apo-Rv3637 forms high-order oligomers with DNA, through nonspecific interactions with DNA or through preformed protein–DNA complexes (Gárate et al 2021 ). Binding of cAMP binding to Rv3676 reduced oligomerization and nonspecific binding but did not increase the DNA affinity, suggesting an allosteric regulation mechanism distinct from that of CAP–cAMP (Gárate et al 2021 ).…”