Palmitic acid, a saturated free fatty acid, has been shown to be a potent inducer of cellular apoptosis in various types of cells, although the mechanisms are unclear. Recently, our group found that palmitic acid (PA), but not oleic acid (OA), an unsaturated free fatty acid, reduced the activity of double-stranded RNA-dependent protein kinase (PKR) by decreasing the level of autophosphorylation at Thr451 residue. In the present study, we investigated how palmitic acid affects the activity of PKR protein at the molecular and biophysical level. Using a fluorescent palmitic acid, Bodipy-FL-PA, binding assays were performed with four types of recombinant PKR proteins, namely, the double-stranded RNA (dsRNA) binding domain on PKR (PKR-dsRBD), the phosphorylated form of the wild-type PKR protein (PKR-wt), the unphosphorylated form of PKR-wt, and a catalytic inactive mutant of the PKR protein (PKR-K296R). We found that both the PKR-wt (phosphorylated) and PKR-K296R (mutant) proteins have high affinity for Bodipy-FL-PA, with KD of 1503.67±79.96 nM and 1211.33±49.70 nM, respectively. PKR-dsRBD did not show a significant increase in the fluorescence intensity, suggesting that the palmitic acid did not bind to the dsRNA binding domain. Computational docking experiments suggested that PA may dock onto the ATP binding site on PKR, near the K296 residue (within a sphere of 5Å around the side chain of K296) with a higher docking score (34.9) than OA (23.9). We experimentally confirmed through a competition assay with ATP, that PA binds to the ATP binding site on the PKR proteins. Furthermore, a fluorescence polarization based competition assay using Bodipy-ATP suggests PA had a lower inhibitory concentration (IC50) than OA, suggesting that PA binds more strongly than OA to the ATP binding site. This study provides a novel mechanism by which PA downregulates the phosphorylation level, and in turn activity, of PKR. These findings raise the question of whether palmitic acid may have broader effects, with a potential of modulating kinases with similar ATP binding sites. This has potential implications on signaling pathways, in particular kinases, that are targeted by drugs.

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