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bioRxiv · 10.1101/505347

Structures of the PKA RIα holoenzyme with the FLHCC driver J-PKAcα or wild type PKAcα

Cao, B.·Lu, T.-W.·Martinez Fiesco, J.·Tomasini, M.·Fan, L.·Simon, S.·Taylor, S.·Zhang, P.
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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 RI2:J-PKAc2 holoenzyme formed by J-PKAc and the PKA regulatory (R) subunit RI, and the wild type (wt) RI2:PKAc2 holoenzyme. The chimeric and wt RI holoenzymes have quaternary structures different from the previously solved wt RI{beta} and RII{beta} holoenzymes. The chimeric holoenzyme shows an isoform-specific interface dominated by antiparallel interactions between the N3A-N3A motifs of RI that serves as an anchor for RI structural rearrangements during cAMP activation. The wt RI holoenzyme showed the same configuration as well as a distinct second conformation. In the structure of the chimeric fusion RI2:J-PKAc2 holoenzyme, the presence of the J-domain does not prevent formation of the holoenzymes, and is positioned away from the symmetrical interface between the two RI:J-PKAc heterodimers in the holoenzyme. The J-domains have significantly higher temperature factors than the rest of the holoenzyme, implying a large degree of conformational flexibility. Furthermore molecular dynamics simulations were applied to analyze the conformational states of chimeric fusion and wt RI holoenzymes, and showed an ensemble of conformations in the majority of which the J-domain was dynamic and rotated away from the R:J-PKAc interface. Thus, rather than affecting the interactions with the regulatory subunits, the fusion of the J-domain to the PKAc alters the conformational landscape of the chimeric fusion holoenzymes and potentially, as result, the interactions with other molecules. 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.

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Cao, B., Lu, T.-W., Martinez Fiesco, J., Tomasini, M., Fan, L., Simon, S., Taylor, S., Zhang, P.. 2018-12-22. Structures of the PKA RIα holoenzyme with the FLHCC driver J-PKAcα or wild type PKAcα. https://doi.org/10.1101/505347

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