Molecular interactions and dynamics in cyclic AMP signalling

Abstract

The key role of cAMP in mammalian cells is mediated through the activation of cAMP dependent Protein Kinase A (PKA). cAMP binding induces large conformational changes within the R-subunit leading to dissociation of the active C-subunit. Although crystal structures of end-point, inactive and active states are available, the molecular basis for cooperativity in cAMP-dependent activation of PKA is not clear. In this study (Chapter 1) application of amide hydrogen/deuterium exchange (HDX) mass spectrometry (MS) on tracking the stepwise cAMP-induced conformational changes has been reported. Amide exchange results reveal that binding of one molecule of cAMP enhances dynamics of two key regions α:C/C¿:A and αA:B helix coupling the two CNBs and this forms the basis for positive cooperativity in the cAMP-dependent activation of PKA. While extensive structural and biochemical studies have provided molecular insights into the mechanism of PKA activation, little is known about signal termination and the role of PDEs in regulatory feedback. In this study (Chapter 2) a novel mode of AKAP (A-kinase-anchoring protein)-independent feedback regulation between RegA and the PKA regulatory (RIα) subunit has been identified. Results indicate that RegA, in addition to its well-known role as a PDE for bulk cAMP in solution, is also capable of hydrolyzing cAMP-bound to RIα. Furthermore results indicate that binding of RIα activates PDE catalysis several fold demonstrating a dual function of RIα, both as an inhibitor of the C-subunit and as an activator for PDEs. Deletion mutagenesis and amide HDXMS results revealed that the cAMP-binding site (phosphate binding cassette) along with proximal regions important for relaying allosteric changes mediated by cAMP, are important for interactions with the PDE catalytic domain of RegA. These sites of interactions together with measurements of cAMP dissociation rates demonstrate that binding of RegA facilitates dissociation of cAMP followed by hydrolysis of the released cAMP to 5¿AMP. In Chapter 3 the key regions in PDE important for interactions with RIα followed by activation are identified. The amide HDXMS data reveals the regions critical for RegA-RIα interactions include the metal binding M site and substrate binding Q pocket in RegA. Results from the pull down experiment show that RegA binding primes cAMP-bound RIα for reassociation with the C-subunit. When RegA interacts and hydrolyses the bound cAMP from RIα, the cAMP-free RIα generated as an end product remains bound to RegA. The PKA C-subunit then displaces RegA and reassociates with cAMP-free RIα to regenerate the inactive PKA holoenzyme thereby completing the termination step of cAMP signaling. These results reveal a novel mode of regulatory feedback between PDEs and RIα which has important consequences for PKA regulation and cAMP signal termination. cAMP specific phosphodiesterase (PDE), RegA in Dictyostelium discoideum tightly regulates the intracellular levels of cAMP through various stages of cell growth. RegA is known to be activated through the two-component system in which phospho-transfer occurs from RdeA to the receiver domain of RegA. But the mechanistic basis by which the enzyme gets activated is not yet well understood. In this study (Chapter 4), phosphorylation dependent conformational changes in RegA has been mapped using amide HDXMS. Dynamics within RegA and the conformational changes due to accompanying phosphorylation at D212 suggests that phosphorylation stabilizes regions within RegA and keeps the molecule in active state, whereas the unphosphorylated RegA might exist in equilibrium between inactive and active conformations.