Protein Kinase A (PKA) is a macromolecular assembly composed of a regulatory subunit (PKA-R) that harbors cyclic-nucleotide binding (CNB) domains, and a catalytic subunit (PKA-C) that binds nucleotides and metal ions in its active site. During its phosphorylation cycle, PKA-C experiences different conformations starting with a close conformation when bound to ATP-Mg2+, to a dynamic equilibrium between close and open conformations with ADP-Mg2+, and an open conformation in the absence of nucleotide and Mg2+. How the conformational changes in PKA-C allosterically modulate interactions with the CNB domains of PKA-R remain largely unknown. Here we use optical tweezers to selectively unfold and refold one CNB domain (termed CNB-A) of PKA-R in the presence of PKA-C and different nucleotides. This approach allowed us to dissect the allosteric effects that the PKA-C conformation exert over the folding energy landscape and conformation of CNB-A. When PKA-C is in an open conformation, we find that CNB-A unfolds predominantly at low forces (9 pN) as in the apo state, indicating that CNB-A interactions with PKA-C require nucleotide occupancy. When PKA-C is in a closed conformation bound to ATP-Mg2+, CNB-A predominately unfolds at high forces (19 pN), suggesting that CNB-A and PKA-C are in a single, tightly-bound conformation. Interestingly, in the presence of ADP-Mg2+, CNB-A unfolds at low (9 pN), intermediate (13 pN) and high forces (19 pN), suggesting that CNB-A can be in three conformations: apo, weakly- or tightly-bound to PKA-C. We further dissect the kinetics of interconversions between these three conformations by varying the PKA-C concentration and the refolding and rebinding dwell time. We show that transition kinetics from the apo to the tightly-bound conformation in the presence of ATP-Mg2+ are much faster than in ADP-Mg2+, which has consequences for the timing of assembly of the inactive PKA holoenzyme and dissociation of the PKA complex after cAMP-mediate activation. Altogether, this study directly shows how nucleotides and metal ions at the active site of PKA-C can allosterically modulate the strength and kinetics of interactions with the CNB domains, thereby highlighting the delicate regulation mechanisms for PKA to function as a proper switch in cell signaling processes.
lt;section id="fsb2202034s100621-sec-0002"gt;This work was supported by a grant from the National Institutes of Health 1R15GM135866 to R.A.M. lt;/sectiongt;
