Cyclic AMP (cAMP) is a second messenger that translates extracellular signals into tightly regulated biological responses. The cAMP binding domain (CBD) is a conserved regulatory switch that binds to cAMP and allosterically controls multiple cellular functions. All CBDs share a common architecture comprised of α- and β-subdomains. cAMP binds to the phosphate binding cassette (PBC) nested within the β-subdomain. In mammals the main cAMP receptors are protein kinase A (PKA), guanine exchange factors (EPAC) and ion channel proteins, including both the hyperpolarization-activated cyclic nucleotide-dependent channels (HCN channels) and the cyclic nucleotide-gated channels (CNG channels). Impaired activities of these proteins are associated with diabetes, cardiovascular diseases, cancer and Alzheimer's disease. Therefore, these proteins represent promising therapeutical targets. However, the mechanism of their cAMP-dependent allosteric control is not completely understood. In the present thesis we have studied the allosteric mechanism of activation in PKA and EPAC using an NMR-based approach and we have proposed a model explaining how cAMP allosterically controls the activity of PKA and EPAC.
Binding of cAMP to the Regulatory (R) subunit of PKA facilitates the release of the Catalytic (C) subunit. According to our model, binding of cAMP triggers long range perturbations that propagate from the PBC to the R:C interface through both direct and indirect pathways. The indirect pathway involves two key relay sites located at the C-terminus of β2 (1163) and at the N-terminus of β3 (D170). D170 functions as an electrostatic switch that mediates the communication between the PBC and the helical subdomain, whereas 1163 controls the global unfolding. Hence, removal of cAMP uncouples the α- and β-subdomains by breaking the circuitry of cooperative interactions radiating from the PBC. The proposed model was further validated by the cAMP agonist Sp-cAMPS and the cAMP antagonist Rp-cAMPS. It was observed that Rp-cAMPS, in which the equatorial exocylic oxygen is replaced by sulphur, does not activate a necessary indirect allosteric pathway, while its diastereoisomer (Sp-cAMPS) with opposite phosphorus chirality behaves similarly to cAMP activating all allosteric pathways. Our data also showed that the cAMP-antagonist stabilizes a ternary inhibitory complex between the effector ligand and both the regulatory and the catalytic subunits of PKA. At this point it is still not understood how the proposed model of cAMP allostery is conserved in other cAMP binding proteins such as EPAC.
EPAC is a multidomain guanine nucleotide exchange factor specific for small GTP-binding proteins and is directly activated by cAMP. We have probed how cAMP docks into the EPAC1 CBD and how its signal allosterically propagates from the cAMP binding site to the helical subdomain, which mediates the inhibitory interactions between the regulatory and catalytic regions of EPAC. Our comparative NMR investigation of cAMP signalling in PKA and EPAC revealed key functionally significant differences between these two systems that will facilitate the design of EPAC-selective therapeutics. / Thesis / Doctor of Philosophy (PhD)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/23679 |
| Date | 04 1900 |
| Creators | Das, Rahul |
| Contributors | Melacini, Giuseppe, Biochemistry and Biomedical Sciences |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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