Activation of PKA via Asymmetric Allosteric Coupling of Structurally Conserved Cyclic Nucleotide Binding Domains

Abstract

Cyclic nucleotide binding (CNB) domains are universally conserved signaling modules that regulate the activities of diverse protein functions. While high-resolution structures of CNB domains have been available for decades, the energetic and dynamic features that enable the cyclic-nucleotide binding signal to regulate other functional domains have remained less understood. We use optical tweezers and molecular dynamics to monitor the signals transduced by cAMP binding between the two CNB domains of the regulatory subunit of protein kinase A (PKA). By mechanically manipulating the CNB domains either as isolated domains or as part of the regulatory subunit, we dissect the changes in the folding energy landscape associated to cAMP binding. We find that the response of the folding energy landscape to cAMP is asymmetric and domain-specific, resulting in unique but mutually coordinated tasks: one CNB domain initiates cAMP binding and cooperativity, while the other triggers inter-domain interactions that lock the active conformation. Inter-domain interactions occur in a stepwise fashion, beginning in intermediate-liganded states between an apo and a cAMP-bound CNB domain. Moreover, we identify a new cAMP-responsive switch, the N3A motif, whose conformational dynamics, stability and selectively depend on cAMP occupancy, and require intact inter-domain contacts. We propose that this dynamic switch serves as a signaling hub, amplifying the cAMP binding signal during the allosteric activation of PKA. Our single-molecule approach combined with simulations may be readily applicable to investigate other kinases regulated by conserved signaling modules such as SH2, SH3 or PH domains.