Novel Isoform-Specific Interfaces Revealed by PKA RIIβ Holoenzyme Structures

The cAMP-dependent protein kinase catalytic (C) subunit is inhibited by two classes of functionally nonredundant regulatory (R) subunits, RI and RII. Unlike RI subunits, RII subunits are both substrates and inhibitors. Because RII beta knockout mice have important disease phenotypes, the RII beta holoenzyme is a target for developing isoform-specific agonists and/or antagonists. We also know little about the linker region that connects the inhibitor site to the N-terminal dimerization domain, although this linker determines the unique globular architecture of the RII beta holoenzyme. To understand how RII beta functions as both an inhibitor and a substrate and to elucidate the structural role of the linker, we engineered different RII beta constructs. In the absence of nucleotide, RII beta(108-268), which contains a single cyclic nucleotide binding domain, bound C subunit poorly, whereas with AMP-PNTP, a non-hydrolyzable ATP analog, the affinity was 11 nM. The RII beta (108-268) holoenzyme structure (1.62 angstrom) with AMP-PNP/Mn2+ showed that we trapped the RII beta subunit in an enzyme:substrate complex with the C subunit in a closed conformation. The enhanced affinity afforded by AMP-PNP/Mn2+, may be a useful strategy for increasing affinity and trapping other protein substrates with their cognate protein kinase. Because mutagenesis predicted that the region N-terminal to the inhibitor site might dock differently to RI and RII, we also engineered RII beta(102-265), which contained six additional linker residues. The additional linker residues in RII beta(102-265) increased the affinity to 1.6 nM, suggesting that docking to this surface may also enhance catalytic efficiency. In the corresponding holoenzyme structure, this linker docks as an extended strand onto the surface of the large lobe. This hydrophobic pocket, formed by the alpha F-alpha G loop and conserved in many protein kinases, also provides a docking site for the amphipathic helix of PKI. This novel orientation of the linker peptide provides the first clues as to how this region contributes to the unique organization of the M holoenzyme. (C) 2009 Elsevier Ltd. All rights reserved.