# N3A motifs in RIβ mediate allosteric crosstalk between cAMP and ATP in PKA activation

## TL;DR

This paper explores how specific motifs in the RIβ subunit of PKA help regulate enzyme activation through interactions with cAMP and ATP.

## Contribution

The study reveals new structural insights into the RIβ subunit and its role in allosteric activation of PKA.

## Key findings

- The N3A motifs and linker in RIβ mediate crosstalk between ATP and cAMP during PKA activation.
- Oxidation or AKAP binding alters the structure of the RIβ holoenzyme, affecting nucleotide binding.
- A structural change involving W260 in RIβ may represent an early step in cAMP activation.

## Abstract

The RIβ subunit of cAMP‐dependent protein kinase (PKA) is highly expressed in the brain, yet it remains the least studied of the PKA regulatory subunits (R). As pathologic variants of its gene are increasingly implicated in neurodevelopmental disorders, neurodegeneration, and cancer, gaining more information about the structure/function of RIβ, and how it differs from RIα, has become increasingly important. We previously reported the structure of the RIβ2C2 holoenzyme, which revealed a novel conformation where ATP binding was stabilized by a head‐to‐head anti‐parallel packing of the C‐tail wrapped around the N‐lobe of the catalytic subunit (C). Although visible, the Dimerization/Docking Domain was poorly folded and reduced. Since RIβ is oxidized in brain tissues, we asked if oxidation or binding of an A Kinase Anchoring Protein (AKAP) would affect the holoenzyme structure. Oxidation or addition of an AKAP peptide to crystals led to the release of nucleotide. To capture this at higher resolution we crystallized RIβ2C2 in the presence of an AKAP peptide. This new structure represents an RIβ:C heterodimer. Density for the D/D domain was missing; ATP was absent, the kinase adopted an open conformation, and the C‐terminus of the RIβ subunit was no longer resolved. Because the crosstalk between ATP and cAMP in the R:C complex appears to be mediated by the two N3A motifs (N3AA and N3AB) as well as by the linker, which in free RIβ is intrinsically disordered, we describe the conserved features of these two motifs as well as the linker and show how each contributes in a unique but coordinated way to allosteric activation of RIβ holoenzymes by cAMP. A key difference in our RIβ:C structure is the rotation of the side chain of W260 at the N‐terminus of the αA Helix in N3AB. W260, at the R:C interface in the holoenzyme, is also the capping residue for cAMP bound to CNB‐A, so we may have actually captured the first step in cAMP activation.