PROBING ALLOSTERY IN THE EXCHANGE PROTEIN DIRECTLY ACTIVATED BY cAMP (EPAC) USING NMR SPECTROSCOPY

Abstract

<p>Exchange proteins directly activated by cAMP (EPAC) are guanine nucleotide exchange factors for the small GTPases, Rap1 and Rap2. The central regulatory module of EPAC is a cAMP binding domain (CBD), which in the absence of cAMP provides auto-inhibition of the catalytic guanine nucleotide exchange activity. Binding of the allosteric effector, cAMP, removes the auto-inhibition exerted by the CBD of EPAC. Herein, we investigate through NMR spectroscopy the structural and dynamical basis of auto-inhibition and cAMP-dependent allosteric activation in the CBD of EPAC. Specifically, the work described in this dissertation proposes novel methods that utilize NMR chemical shifts to define the network of residues that mediates long-range intra-molecular signalling, <em>i.e.</em> the chemical shift covariance analysis (CHESCA) and the chemical shift projection analysis (CHESPA). Using CHESCA as explained in Chapter 2, we identified an allosteric network that bridges the sites of cAMP-binding and cAMP-dependent structural changes to those of cAMP-dependent dynamical changes, which are critical for the release of auto-inhibition. The CHESCA results therefore rationalize how cAMP leads to activation through modulation of both structure and dynamics. In order to dissect the determinants of auto-inhibition in the absence of cAMP, several mutations along the signaling pathways identified by CHESCA were implemented and their effect on the auto-inhibitory conformational equilibrium of the apo-CBD was assessed through CHESPA, as outlined in Chapters 3 and 4. Overall, we have shown how CHESCA and CHESPA provide unprecedented insight into the allosteric networks underlying auto-inhibition and cAMP dependent activation in the CBD of EPAC. In addition, the methods employed here to map EPAC allostery are likely to be generally applicable to other systems.</p>