Tuberculosis is a lethal disease that affects millions of people each year. Interestingly, an estimated 60-80% of infections stem from reinfection by the causative agent, Mycobacterium tuberculosis. While the mechanism of resurgence of dormant M. tuberculosis is largely understudied, one gene thought to be involved is rpfA. rpfA encodes a resuscitation-promoting factor whose expression is associated with the activation of latent bacteria.
rpfA is regulated by the M. tuberculosis cAMP receptor protein (CRPMt) which has recently been implicated in the virulence of the bacteria as deletion of the CRP-encoding gene resulted in failure to infect macrophages and mice. These findings, taken together with the high persistence of global M. tuberculosis infection and reinfection, make the understanding of CRP allostery and DNA-binding in M. tuberculosis extremely important. Additionally, upon invasion of host cells, M. tuberculosis releases a large burst of cAMP which promotes bacterial survival. This necessitates further study of CRPMt with respect to cAMP-mediated transcriptional regulation and its target genes.
The Mycobacterium tuberculosis cAMP receptor protein (CRPMt) is a 438-residue protein containing 6 alpha-helices and 12 beta-sheets . It is composed of two subunits, each made of : an N-terminal cAMP binding domain, a C-terminal DNA binding domain , and a hinge region . The hinge region links the cAMP-binding and DNA-binding domains and acts as an axis by which cAMP-induced conformational change occurs. The subunits also contain a 27-residue C helix which mediates intersubunit interaction.
Extensive interactions between residues of the cAMP-binding domain and the cAMP molecule stabilize the ligand’s position within a in each subunit.
Interactions with adenine
The hydroxyl group of Thr-134 in the C-helix forms a hydrogen bond with the N6-amino group of cAMP . Other residues also with the adenine ring. Among them are Leu-69, Thr-70, and Met-72.
Interactions with ribose
The backbone nitrogen of Leu-81 hydrogen bonds with the O3’ atom of cAMP and the backbone nitrogen of Gly-79 hydrogen bonds with the O2’ atom . Phe-38, Ile-57, and Phe-78 residues also participate in with cAMP’s sugar ring.
Interactions with the phosphate group
A terminal nitrogen atom of Arg-89 hydrogen bonds with the hydrogen atom bound to the axial phosphate oxygen. This oxygen atom also forms a hydrogen bond with the sidechain oxygen of Ser-82 and the backbone nitrogen . The equatorial phosphate oxygen atom of cAMP forms a hydrogen bond with the backbone nitrogen of Thr-90 .
In the absence of cAMP, cAMP-binding sites are occupied by Arg-130 , whose guanidino group occludes the adenine binding site, and Glu-80 which occupies the sites where ribose would bind. In the absence of cAMP, Asp-76 in the cAMP-binding domain and Arg-160 in the D-helix of the DNA-binding domain form a salt bridge . In the unbound state, the cAMP-binding and DNA-binding domains are in close proximity which creates a consisting of Leu and Ile residues.
When cAMP binds (not shown in this pdb file), the ligand displaces Arg-130 which promotes ionic interactions between Glu-80, Asp-76, and the mainchain carbonyl oxygen of Met-77 . Consequently, beta-strand 6 and the rest of the cAMP-binding domain move 27° closer to the C-helix by way of hinge region rotation . Beta-strands 4 and 5 of the cAMP-binding domain thus occlude the E helix , inducing the repositioning of the DNA-binding domain, as well.
Within the DNA-binding domain, there is a helix turn helix motif which contains the E helix and the F helix , mediated by the D helix . The F-helix specifically recognizes DNA.
The most extensively studied CRP is that of E. coli (CRPEc) which shares vast functional and structural similarities with CRPMt. However, the mechanism of allosteric regulation of DNA-binding by cAMP is not a conserved characteristic. In E. coli, CRP only binds to DNA when also bound to cAMP. Interestingly, the affinity of CRPMt for DNA promoters remains largely unchanged by cAMP binding, an uncommon phenomenon with respect to transcription factors. In fact, CRPMt binds nonspecific DNA sequences in the absence of cAMP as well as forming larger multi-protein and DNA molecule complexes. This is consistent with the finding that cAMP induces relatively small conformational changes in CRPMt. Importantly, while cAMP binding does not affect the affinity of CRPMt for DNA promoters, it decreases the occurrence of these nonspecific interactions and high-order complexes.
The contrasting allosteric properties of the CRP of E. coli and M. tuberculosis taken with their strong structural homology poses a curious conundrum. There are few slight differences between their structures that may play a role in their different cAMP and DNA binding properties.
Bai, G., Schaak, D. D., & McDonough, K. A. (2009). cAMP levels within Mycobacterium tuberculosis and Mycobacterium bovis BCG increase upon infection of macrophages. FEMS Immunology & Medical Microbiology, 55(1), 68-73. Gárate, F., Dokas, S., Lanfranco, M. F., Canavan, C., Wang, I., Correia, J. J., & Maillard, R. A. (2021). cAMP is an allosteric modulator of DNA-binding specificity in the cAMP receptor protein from Mycobacterium tuberculosis. Journal of Biological Chemistry, 296. Mathiesen, J. M., Vedel, L., & Bräuner-Osborne, H. (2013). cAMP biosensors applied in molecular pharmacological studies of G protein-coupled receptors. In Methods in enzymology (Vol. 522, pp. 191-207). Academic Press. Reddy, M. C., Palaninathan, S. K., Bruning, J. B., Thurman, C., Smith, D., & Sacchettini, J. C. (2009). Structural insights into the mechanism of the allosteric transitions of Mycobacterium tuberculosis cAMP receptor protein. Journal of Biological Chemistry, 284(52), 36581-36591. Rickman, L., Scott, C., Hunt, D. M., Hutchinson, T., Menéndez, M. C., Whalan, R., ... & Buxton, R. S. (2005). A member of the cAMP receptor protein family of transcription regulators in Mycobacterium tuberculosis is required for virulence in mice and controls transcription of the rpfA gene coding for a resuscitation promoting factor. Molecular microbiology, 56(5), 1274-1286. Stapleton, M., Haq, I., Hunt, D. M., Arnvig, K. B., Artymiuk, P. J., Buxton, R. S., & Green, J. (2010). Mycobacterium tuberculosis cAMP receptor protein (Rv3676) differs from the Escherichia coli paradigm in its cAMP binding and DNA binding properties and transcription activation properties. Journal of Biological Chemistry, 285(10), 7016-7027. Back to Top
Gárate, F., Dokas, S., Lanfranco, M. F., Canavan, C., Wang, I., Correia, J. J., & Maillard, R. A. (2021). cAMP is an allosteric modulator of DNA-binding specificity in the cAMP receptor protein from Mycobacterium tuberculosis. Journal of Biological Chemistry, 296.
Mathiesen, J. M., Vedel, L., & Bräuner-Osborne, H. (2013). cAMP biosensors applied in molecular pharmacological studies of G protein-coupled receptors. In Methods in enzymology (Vol. 522, pp. 191-207). Academic Press.
Reddy, M. C., Palaninathan, S. K., Bruning, J. B., Thurman, C., Smith, D., & Sacchettini, J. C. (2009). Structural insights into the mechanism of the allosteric transitions of Mycobacterium tuberculosis cAMP receptor protein. Journal of Biological Chemistry, 284(52), 36581-36591.
Rickman, L., Scott, C., Hunt, D. M., Hutchinson, T., Menéndez, M. C., Whalan, R., ... & Buxton, R. S. (2005). A member of the cAMP receptor protein family of transcription regulators in Mycobacterium tuberculosis is required for virulence in mice and controls transcription of the rpfA gene coding for a resuscitation promoting factor. Molecular microbiology, 56(5), 1274-1286.
Stapleton, M., Haq, I., Hunt, D. M., Arnvig, K. B., Artymiuk, P. J., Buxton, R. S., & Green, J. (2010). Mycobacterium tuberculosis cAMP receptor protein (Rv3676) differs from the Escherichia coli paradigm in its cAMP binding and DNA binding properties and transcription activation properties. Journal of Biological Chemistry, 285(10), 7016-7027.
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