cAMP activation of PKA defines an ancient signaling mechanism.
cAMP and the cAMP binding domain (CBD) constitute a ubiquitous regulatory switch that translates an extracellular signal into a biological response. The CBD contains alpha- and beta-subdomains with cAMP binding to a phosphate binding cassette (PBC) in the beta-sandwich. The major receptors for cAMP in mammalian cells are the regulatory subunits (R-subunits) of PKA where cAMP and the catalytic subunit compete for the same CBD. The R-subunits inhibit kinase activity, whereas cAMP releases that inhibition. Here, we use NMR to map at residue resolution the cAMP-dependent interaction network of the CBD-A domain of isoform Ialpha of the R-subunit of PKA. Based on H/D, H/H, and N(z) exchange data, we propose a molecular model for the allosteric regulation of PKA by cAMP. According to our model, cAMP binding causes long-range perturbations that propagate well beyond the immediate surroundings of the PBC and involve two key relay sites located at the C terminus of beta(2) (I163) and N terminus of beta(3) (D170). The I163 site functions as one of the key triggers of global unfolding, whereas the D170 locus acts as an electrostatic switch that mediates the communication between the PBC and the B-helix. Removal of cAMP not only disrupts the cap for the B' helix within the PBC, but also breaks the circuitry of cooperative interactions stemming from the PBC, thereby uncoupling the alpha- and beta-subdomains. The proposed model defines a signaling mechanism, conserved in every genome, where allosteric binding of a small ligand disrupts a large protein-protein interface.
Das
Rahul
R
Department of Chemistry, Biochemistry, and Biomedical Sciences, McMaster University, 1280 Main Street West, Hamilton, ON, Canada.
Esposito
Veronica
V
Abu-Abed
Mona
M
Anand
Ganesh S
GS
Taylor
Susan S
SS
Melacini
Giuseppe
G
D001665:Binding Sites; D000242:Cyclic AMP / Q000502:physiology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry* / Q000502:physiology*; D004789:Enzyme Activation; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D015398:Signal Transduction / Q000502:physiology*
D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases