title: Understanding cAMP-dependent allostery by NMR spectroscopy: comparative analysis of the EPAC1 cAMP-binding domain in its apo and cAMP-bound states.
abstract: cAMP (adenosine 3',5'-cyclic monophosphate) is a ubiquitous second messenger that activates a multitude of essential cellular responses. Two key receptors for cAMP in eukaryotes are protein kinase A (PKA) and the exchange protein directly activated by cAMP (EPAC), which is a recently discovered guanine nucleotide exchange factor (GEF) for the small GTPases Rap1 and Rap2. Previous attempts to investigate the mechanism of allosteric activation of eukaryotic cAMP-binding domains (CBDs) at atomic or residue resolution have been hampered by the instability of the apo form, which requires the use of mixed apo/holo systems, that have provided only a partial picture of the CBD apo state and of the allosteric networks controlled by cAMP. Here, we show that, unlike other eukaryotic CBDs, both apo and cAMP-bound states of the EPAC1 CBD are stable under our experimental conditions, providing a unique opportunity to define at an unprecedented level of detail the allosteric interactions linking two critical functional sites of this CBD. These are the phosphate binding cassette (PBC), where cAMP binds, and the N-terminal helical bundle (NTHB), which is the site of the inhibitory interactions between the regulatory and catalytic regions of EPAC. Specifically, the combined analysis of the cAMP-dependent changes in chemical shifts, 2 degrees structure probabilities, hydrogen/hydrogen exchange (H/H) and hydrogen/deuterium exchange (H/D) protection factors reveals that the long-range communication between the PBC and the NTHB is implemented by two distinct intramolecular cAMP-signaling pathways, respectively, mediated by the beta2-beta3 loop and the alpha6 helix. Docking of cAMP into the PBC perturbs the NTHB inner core packing and the helical probabilities of selected NTHB residues. The proposed model is consistent with the allosteric role previously hypothesized for L273 and F300 based on site-directed mutagenesis; however, our data show that such a contact is part of a significantly more extended allosteric network that, unlike PKA, involves a tight coupling between the alpha- and beta-subdomains of the EPAC CBD. The proposed mechanism of allosteric activation will serve as a basis to understand agonism and antagonism in the EPAC system and provides also a general paradigm for how small ligands control protein-protein interfaces.
authors.affiliation: Department of Chemistry, McMaster University, 1280 Main Street West, Hamilton, Ontario L8S 4M1, Canada.
authors.forename: Mohammad T
authors.initials: MT
authors.lastname: Mazhab-Jafari
authors.forename: Rahul
authors.initials: R
authors.lastname: Das
authors.forename: Steven A
authors.initials: SA
authors.lastname: Fotheringham
authors.forename: Soumita
authors.initials: S
authors.lastname: SilDas
authors.forename: Somenath
authors.initials: S
authors.lastname: Chowdhury
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Melacini
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000737:chemistry / Q000378:metabolism; D000494:Allosteric Regulation; D001059:Apoproteins / Q000737:chemistry / Q000378:metabolism*; D020134:Catalytic Domain; D000242:Cyclic AMP / Q000737:chemistry / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D003903:Deuterium / Q000737:chemistry; D005057:Eukaryotic Cells / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry / Q000378:metabolism*; D006859:Hydrogen / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008958:Models, Molecular; D010710:Phosphates / Q000737:chemistry / Q000378:metabolism; D011953:Receptors, Cyclic AMP / Q000737:chemistry / Q000378:metabolism*; D020668:rap GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D020827:rap1 GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D018528:ATP-Binding Cassette Transporters; D001059:Apoproteins; D020662:Guanine Nucleotide Exchange Factors; D010710:Phosphates; D011953:Receptors, Cyclic AMP; D006859:Hydrogen; D003903:Deuterium; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; C116756:RAP2A protein, human; D020668:rap GTP-Binding Proteins; D020827:rap1 GTP-Binding Proteins