Definition of an electrostatic relay switch critical for the cAMP‐dependent activation of protein kinase A as revealed by the D170A mutant of RIα

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.

Cyclic AMP- and (Rp)-cAMPS-induced Conformational Changes in a Complex of the Catalytic and Regulatory (RIα) Subunits of Cyclic AMP-dependent Protein Kinase

“…It is important that the results confirm the importance of the Arg 209 /Asp 170 relay in propagating the effects of cAMP binding to the R-C interface. This is one region that showed decreased exchange upon (R p )-cAMPS binding and strongly suggests that the basis for the inability of (R p )-cAMPS to facilitate dissociation of the C-subunit is the uncoupling of the charge relay linking Arg 209 and Asp 170 and is consistent with previous mutagenesis data (26,29). In the C-subunit, changes in amide exchange occurred almost globally with single deuteron increases in fragments associated with the ␣A-␤1 loop, ␣F, ␣G, ␣G-␣H loop, ␣H-␣I loop, and the C-terminal tail.…”

“…Substitution of Lys for Arg at residue 209 blocks binding to the CNB-A site (26) and transforms (R p )-cAMPS into an agonist (27) Functionally, the binding of cAMP to the PBC can be considered as a phosphorylation event that acts as an electrostatic switch. Arguably, the most important moiety of cAMP involved in the activation process is its phosphate oxygen in the equatorial position (R p ) anchored by a critical, conserved Arg 209 (29). As discussed above, cAMP binding activates this electrostatic switch.…”

Allosteric Communication between cAMP Binding Sites in the RI Subunit of Protein Kinase A Revealed by NMR

“…5) , and Ile 206 in the PBC, Asp 172 (which interacts with the conserved Arg in the PBC), and the ␣C:A and ␣C':A helices are all sensitive to cAMP binding in the A site (14, 39 -41). Previous NMR studies on R truncated at position 246 also revealed communication between A site binding and the ␣C:A helix (11,12,30,42). However, the cumulative and sequential structural effect on the interdomain helices affected by binding of cAMP to both sites, as well as the communication of cAMP binding in the A site to the B domain, is observed here for the first time.…”

Using Markov State Models to Develop a Mechanistic Understanding of Protein Kinase A Regulatory Subunit RIα Activation in Response to cAMP Binding

“…First, the R 2 C 2 holoenzyme contains four binding sites for cAMP that are capable of communicating between each other, creating a cooperative effect of cAMP binding (9). Second, each CBD undergoes major structural changes during activation (10), and two of them (both CBD-As) have extended interfaces with the C-subunits that can interfere with the conformational changes (11,12). Thus, the unleashing of the C-subunits in the R 2 C 2 complex must also be a factor in the activation process.…”

“…Thus, the unleashing of the C-subunits in the R 2 C 2 complex must also be a factor in the activation process. Because of the complexity of the system, it has been difficult to expand the NMR and crystal structure studies beyond a single CBD or an R-C heterodimer to the whole R 2 C 2 complex (12,13).…”