STRUCTURAL INSIGHTS INTO THE MECHANISM OF THE ALLOSTERIC TRANSITIONS OF THE MYCOBACTERIUM TUBERCULOSIS cAMP RECEPTOR PROTEIN

The cyclic AMP receptor protein (CRP) from Mycobacterium tuberculosis is a cAMPresponsive global transcriptional regulator, responsible for the regulation of a multitude of diverse proteins. We have determined the crystal structures of the CRP-cAMP and CRPN6-cAMP derivative bound forms of the enzyme to 2.2 and 2.3 Å resolution, respectively, in order to investigate cAMP mediated conformational and structural changes. The allosteric switch from the open, inactive conformation to the closed, active conformation begins with a number of changes in the ligand binding cavity upon cAMP binding. These subtle structural changes and numerous non-bonding interactions between cAMP, the N-domain residues and the Cdomain helices demonstrate that the N-domain hairpin loop acts as a structural mediator of the allosteric switch. Based on the CRP-N6-cAMP crystal structure, binding of N6-cAMP with a bulkier methyl-phenyl-ethyl extension from the N6 atom, also stabilizes the cAMP-binding domain, N-domain hairpin and C-terminal domain in a similar manner as that of the CRPcAMP structure, maintaining structural integrity within the subunits. However, the bulkier N6 extension of N6-cAMP (in R conformation) is accommodated only in subunit A with minor changes whereas in subunit B, N6 extension is in S conformation hindering with the hinge region of the central helix. As a result, the entire N-domain and the C-domain of subunit B integrated by the cAMP portion of this ligand, together tilt away (~7 ̊ tilt) from central helix C, positioning the helix-turn-helix (HTH) motif in an unfavorable position for the DNA substrate, asymmetrically. Together these crystal structures demonstrate the mechanism of action of the cAMP molecule and its role in integrating the active CRP structure.