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Structures of the PKA RI? Holoenzyme with the FLHCC Driver J-PKAc? or Wild-Type PKAc?.

ABSTRACT: Fibrolamellar hepatocellular carcinoma (FLHCC) is driven by J-PKAc?, a kinase fusion chimera of the J domain of DnaJB1 with PKAc?, the catalytic subunit of protein kinase A (PKA). Here we report the crystal structures of the chimeric fusion RI?2:J-PKAc?2 holoenzyme formed by J-PKAc? and the PKA regulatory (R) subunit RI?, and the wild-type (WT) RI?2:PKAc?2 holoenzyme. The chimeric and WT RI? holoenzymes have quaternary structures different from the previously solved WT RI? and RII? holoenzymes. The WT RI? holoenzyme showed the same configuration as the chimeric RI?2:J-PKAc?2 holoenzyme and a distinct second conformation. The J domains are positioned away from the symmetrical interface between the two RI?:J-PKAc? heterodimers in the chimeric fusion holoenzyme and are highly dynamic. The structural and dynamic features of these holoenzymes enhance our understanding of the fusion chimera protein J-PKAc? that drives FLHCC as well as the isoform specificity of PKA.

SUBMITTER: Cao B

PROVIDER: S-EPMC6506387 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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Structures of the PKA RIα Holoenzyme with the FLHCC Driver J-PKAcα or Wild-Type PKAcα.

Cao Baohua B Lu Tsan-Wen TW Martinez Fiesco Juliana A JA Tomasini Michael M Fan Lixin L Simon Sanford M SM Taylor Susan S SS Zhang Ping P

Structure (London, England : 1993) 20190321 5

Fibrolamellar hepatocellular carcinoma (FLHCC) is driven by J-PKAcα, a kinase fusion chimera of the J domain of DnaJB1 with PKAcα, the catalytic subunit of protein kinase A (PKA). Here we report the crystal structures of the chimeric fusion RIα2:J-PKAcα2 holoenzyme formed by J-PKAcα and the PKA regulatory (R) subunit RIα, and the wild-type (WT) RIα2:PKAcα2 holoenzyme. The chimeric and WT RIα holoenzymes have quaternary structures different from the pre ...[more]

PMID: 30905674

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Project description: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 RIIbeta knockout mice have important disease phenotypes, the RIIbeta 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 RIIbeta holoenzyme. To understand how RIIbeta functions as both an inhibitor and a substrate and to elucidate the structural role of the linker, we engineered different RIIbeta constructs. In the absence of nucleotide, RIIbeta(108-268), which contains a single cyclic nucleotide binding domain, bound C subunit poorly, whereas with AMP-PNP, a non-hydrolyzable ATP analog, the affinity was 11 nM. The RIIbeta(108-268) holoenzyme structure (1.62 A) with AMP-PNP/Mn(2+) showed that we trapped the RIIbeta subunit in an enzyme:substrate complex with the C subunit in a closed conformation. The enhanced affinity afforded by AMP-PNP/Mn(2+) 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 RIIbeta(102-265), which contained six additional linker residues. The additional linker residues in RIIbeta(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 alphaF-alphaG 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 RIIbeta holoenzyme.

Dataset Information

Structures of the PKA RI? Holoenzyme with the FLHCC Driver J-PKAc? or Wild-Type PKAc?.

Publications

Structures of the PKA RIα Holoenzyme with the FLHCC Driver J-PKAcα or Wild-Type PKAcα.

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