Defining the contours of cyclic nucleaotide mediated regulatory switches from prokaryotes to eukaryotes

Abstract

cAMP signal transduction is a critical second messenger pathway in both prokaryotes and eukaryotes. We have used a combination of crystallography, amide hydrogen/deuterium exchange (HDX) and ion mobility mass spectrometry to study the cAMP mediated activation in eukaryotic PKA (Protein kinase A) as well as prokaryotic CNB (cyclic nucleotide binding domain) containing MSMEG_5458 and cynaobacterial GAF-B domain. Our study on isolated CNB-A of PKA provides evidence for conformational selection as the basis of cAMP action, contradicting earlier reports of induced fit mechanism alone. Furthermore, our study on PKA with tandem CNBs, reports that the close allosteric coupling between CNB-A and CNB-B with the C-subunit provides an important molecular insight into the function of CNB-B domain. Our HDX results of the MSMEG_5458 revealed a mechanism of cAMP mediated activation, in which binding of cAMP induces significant conformational changes that spatially rearranges the catalytic domain through a linker region, for activity. Our HDX results on GAF-B domain showed that isolated GAF-B bound to cAMP displayed an increased dynamics in comparison to cGMP, suggesting higher ordering of the protein in presence of cGMP. cAMP and cGMP induce distinct changes on the structural fold of the GAF-B domain. Extention of our analysis with GAF-AB has shown that GAF-AB lost its binding ability for cGMP. This suggests that the regulatory elements for ligand specificity are associated with GAF-A and the dimerization domain.