title: Definition of an electrostatic relay switch critical for the cAMP-dependent activation of protein kinase A as revealed by the D170A mutant of RIalpha.
abstract: The Regulatory (R) subunit of Protein Kinase A (PKA) inhibits its kinase activity by shielding the Catalytic (C) subunit from physiological substrates. This inhibition is reversed in response to extra-cellular signals that increase cAMP levels in the cytoplasm. Upon cAMP binding to R, C is allosterically released from R, activating a spectrum of downstream signaling cascades. Crystallographic data indicated that a series of distinct conformational changes within CBD-A must occur to relay the cAMP signal from the cAMP binding site to the R:C interaction interface. One critical cAMP relay site within the CBD-A of R has been identified as Asp170 because the D170A mutation selectively reduces the negative cooperativity between the cAMP- and C-recognition sites (i.e. the KD for the R:C complex in the presence of cAMP is reduced by more than 12-fold), without significantly compromising the high affinity of R for both binding partners. Here, utilizing an integrated set of comparative NMR analyses we have elucidated how this critical electrostatic switch is able to control the interaction network which transmits the cAMP signal within CBD-A. The D170A-induced variations in backbone chemical shifts as well as in hydrogen-deuterium and hydrogen-hydrogen exchange profiles show that Asp170 not only plays a pivotal role in controlling the local conformation of the phosphate binding cassette (PBC), where cAMP docks, but also significantly affects the long-range cAMP-dependent interaction network that extends from the PBC to the three major sites of C-recognition. We also found that the D170A mutation promotes partial unfolding, thus assisting the uncoupling of the alpha- and beta-subdomains of CBD-A as required for the major alpha-helical conformational re-arrangement necessary for C-binding. Overall, the emerging map of allosteric networks features Asp170 as an essential component of an electrostatic switch mechanism that stabilizes the conformation of the PBC region for optimal interaction with cAMP and that is also crucial for relaying allosteric effects leading to C subunit activation. Taken together, our results consolidate the interdependence between the Asp170 relay site and the R:C interaction interface. Furthermore, they provide insight into the driving forces for the in vivo formation of intermediate PKA ternary complexes. Finally, our current study is relevant for elucidating the antagonistic properties of Rp-cAMPS on PKA by providing a detailed picture of the long-range effects of the altered interaction between this analog and the PBC.
authors.affiliation: Department of Chemistry, McMaster University, Hamilton, Ontario, Canada L8S 4M1.
authors.forename: Mona
authors.initials: M
authors.lastname: Abu-Abed
authors.forename: Rahul
authors.initials: R
authors.lastname: Das
authors.forename: Lijun
authors.initials: L
authors.lastname: Wang
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Melacini
mesh_terms: D001665:Binding Sites; D020134:Catalytic Domain; D000242:Cyclic AMP / Q000378:metabolism*; D054756:Cyclic AMP-Dependent Protein Kinase RIalpha Subunit; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation
chemical_list: D054756:Cyclic AMP-Dependent Protein Kinase RIalpha Subunit; D050505:Mutant Proteins; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases