Assembly and Disassembly of PKA is Allosterically Controlled by Nucleotides and Metal Ions

Abstract

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.