PKA RIα Holoenzyme Structure Reveals ATP‐Dependent Dynamics and Allosteric Regulation in Isoform‐Specific Manner

Abstract

Protein Kinase A (PKA) is the master switch for cAMP‐mediated signaling. PKA holoenzyme (R2C2) comprised a cAMP‐binding regulatory (R)‐subunit dimer and 2 catalytic (C)‐subunits, while cAMP binding to the R‐subunits unleashes the activity of the C‐subunit. Of the 4 functional non‐redundant R‐subunits (RIα, RIβ, RIIα, RIIβ), RIα and RIβ contain PKA pseudo‐substrate sites, in contrast to RIIα and RIIβ have substrate sites in their inhibitor sequences.Understanding how protein kinases serve as molecular switches is one of the holy grails in kinase biology. We provide new molecular features of PKA RIα allostery by identifying two conformational states of the holoenzyme (R2C2), one with and the other without ATP.RIα, in contrast to RIIβ, contains PKA pseudo‐substrate sequence, and our structures, with and without ATP, show how ATP functions as an allosteric inhibitor of RIα activation. The conformational changes of RIα holoenzyme are driven by ATP. ATP also facilitates RIα holoenzyme locks into inactive state and become more resistant to cAMP.In contrast to the RIIβ holoenzyme, ATP has no effect on RIIβ holoenzyme conformations; however, it serves as a substrate and can facilitate the phosphorylation of the inhibitor sequence in RIIβ. When the RIIβ holoenzyme is phosphorylated, it is easier to be activated by cAMP and unleash kinase activity.The isoform‐specific quaternary structures reveal a novel interface that helps to define domain crosstalk and a previously unappreciated allosteric pathway for cAMP activation. The results allow us to further appreciate the diversity of PKA isoforms and to understand the mechanism for allosteric activation.