Cyclic-3',5 '-adenosine monophosphate (cAMP) is an ancient signaling molecule that is found in a variety of species from prokaryotes to eukaryotes and translates extra-cellular stimuli into tightly controlled intra-cellular responses. The two major mammalian cAMP sensors are protein kinase A (PKA), for the phosphorylation of the downstream effectors, and the exchange protein directly activated by cAMP (Epac ), for the guanine nucleotide exchange in the small GTPase Rap proteins. In this study, we investigated the intra-molecular cAMP dependent allosteric network of Epac cyclic nucleotide binding domain (CBD) via solution NMR spectroscopy. Epac proteins have been shown to employ an auto-inhibition strategy in the control of the equilibrium between the active and the inactive states. In the absence of cAMP, the periphery of the Rap recognition site is masked via an ionic interface provided by the N-terminus of the CBD. Binding of cAMP at the distal Phosphate Binding Cassette (PBC), results in weakening of this interface. Here we show that the cAMP binding signal is propagated to the sites important in Epac activation, i.e. the ionic interface, via two key allosteric spots within the CBD. We have also determined the dynamics as a key carrier of cAMP effects to the region forming the ionic interface (ionic latch). Hence entropic enhancements emerged as a key effector in the cAMP mediated ionic latch weakening. We have also provided initial evidence of a negative allosteric contribution from the C-terminal Hinge-Lid region (CHLR) on the cAMP induced Epac activation. In addition to these findings, we also observed critical differences in the mode of cAMP recognition and inter-subdomain communication between the Epac and PKA. A detailed understanding of these two ubiquitous systems, will aid in the development of agonists and antagonists that are relevant in the drug lead development for related diseases, such as Alzheimer's and diabetes. / Thesis / Master of Science (MSc)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/21617 |
| Date | 07 1900 |
| Creators | Mazhab-Jafari, Mohammad |
| Contributors | Melacini, Giuseppe, Biochemistry and Biomedical Sciences |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
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