Using Markov state models to develop a mechanistic understanding of protein kinase A regulatory subunit RIα...

Protein kinase A (PKA) holoenzyme consists of two catalytic (C) subunits and a regulatory (R) subunit dimer (R(2)C(2)). The kinase is activated by the binding of cAMPs to the two cyclic nucleotide binding domains (CBDs), A and B, on each R-subunit. Despite extensive study, details of the allosteric mechanisms underlying the cooperativity of holoenzyme activation remain unclear. Several Markov state models of PKA-RIα were developed to test competing theories of activation for the R(2)C(2) complex. We found that CBD-B plays an essential role in R-C interaction and promotes the release of the first C-subunit prior to the binding to CBD-A. This favors a conformational selection mechanism for release of the first C-subunit of PKA. However, the release of the second C-subunit requires all four cAMP sites to be occupied. These analyses elucidate R-C heterodimer interactions in the cooperative activation of PKA and cAMP binding and represent a new mechanistic model of R(2)C(2) PKA-RIα activation.