Two PKA RIα holoenzyme states define ATP as an isoform-specific orthosteric inhibitor that competes with the allosteric activator, cAMP

Abstract

Protein kinase A (PKA) holoenzyme, comprised of a cAMP-binding regulatory (R)-subunit dimer and 2 catalytic (C)-subunits, is the master switch for cAMP-mediated signaling. Of the 4 R-subunits (RIα, RIβ, RIIα, RIIβ), RIα is most essential for regulating PKA activity in cells. Our 2 RIα2C2 holoenzyme states, which show different conformations with and without ATP, reveal how ATP/Mg2+ functions as a negative orthosteric modulator. Biochemical studies demonstrate how the removal of ATP primes the holoenzyme for cAMP-mediated activation. The opposing competition between ATP/cAMP is unique to RIα. In RIIβ, ATP serves as a substrate and facilitates cAMP-activation. The isoform-specific RI-holoenzyme dimer interface mediated by N3A–N3A′ motifs defines multidomain cross-talk and an allosteric network that creates competing roles for ATP and cAMP. Comparisons to the RIIβ holoenzyme demonstrate isoform-specific holoenzyme interfaces and highlights distinct allosteric mechanisms for activation in addition to the structural diversity of the isoforms.

Structural analyses of the PKA RIIβ holoenzyme containing the oncogenic DnaJB1-PKAc fusion protein reveal protomer asymmetry and fusion-induced allosteric perturbations in fibrolamellar hepatocellular carcinoma

“…Once activated by cAMP, the B/C/N-helix divides into 3 segments (B-helix, C-helix, and N-helix) ( Fig 2F and 2G). Several studies have already pointed out the importance of the flexibility of the B/C/N-helix in activation of the RIα holoenzyme [10,17,18]. Here, we show that the B/C/ N-helix is also very dynamic, but different, in the RIIβ holoenzyme.…”

“…The activity of PKA can be stimulated by the elevated concentration of cAMP, through binding of cAMP to R subunits which unleashes the activity. Each of 4 functionally nonredundant R subunit isoforms (RIα, RIβ, RIIα, and RIIβ) has distinct quaternary holoenzyme structures, cAMP sensitivity, and cellular localization [7][8][9][10][11]. All 4 R subunit isoforms share a similar domain organization which consists of an N-terminal Dimerization/Docking (D/D) domain followed by an intrinsically disordered linker that connects to 2 tandem cyclic nucleotide binding (CNB) domains.…”

Activation of PKA via asymmetric allosteric coupling of structurally conserved cyclic nucleotide binding domains

“…The allosteric networks we describe here may be amplified in the PKA hetero-tetramer composed of two regulatory and two catalytic subunits, where the potential cross-talk between PKA subunits is expanded 12,34,40 . Structural studies of the PKA hetero-tetramer formed with RI isoforms showed the N3A motif of one regulatory subunit stacked against the N3A motif of the neighboring one, forming a helical bundle with several hydrophobic interactions 41,42 . Therefore, it is possible that the dynamic switching response of the N3A motif described here may play additional allosteric regulatory roles by communicating the two regulatory subunits in the PKA hetero-tetramer via quaternary interactions.…”

Allosteric pluripotency as revealed by protein kinase A

“…As a result, the mixed A off -B on excited state sampled by R AB :Rp 2 drives Rp to act as an antagonist when C binds R AB with sufficiently high affinity to selectively stabilize A off -B on relative to A on B on and A on -B on , resulting in a stable C:R AB :Rp 2 complex and PKA inhibition. However, if the affinity of C for R AB is reduced, for example, due to lower MgATP levels (28)(29)(30), the inhibitory A off -B on state remains excited in the presence of C, while the ground, most populated state is still A on B on , which, being inhibition incompetent, leads to PKA activation. Hence, the model of Fig.…”