(260d) Experimental and Computational Analyses of Palmitic Acid-Binding to PKR
PKR (double-stranded RNA-activated protein kinase) is an important component of the innate immunity, antiviral and apoptotic pathways. Recently, our group found that palmitic acid (PA), a saturated fatty acid, is involved in apoptosis by reducing the autophosphorylation of PKR at the Thr451 residue in HepG2 cells, however, the molecular mechanism by which palmitate reduces PKR autophosphorylation is not known. In the present study, we investigate how palmitic acid modulates the activity of PKR using both experimental and computational techniques. To determine the binding affinity of palmitate to PKR proteins at the physiological level of unbounded FFA concentration of 10 nM, we developed a fluorescence polarization (FP)-based palmitate interaction assay using fluorescently labeled palmitate molecule, Bodipy-PA. Unphosphorylated PKR-WT and phosphorylated PKR-pWT had KD values of 23.22±1.28 nM and 24.90±1.00 nM, respectively, suggesting that both have similar binding affinities to Bodipy-PA. However, the FPmax for PKR-pWT (FPmax=0.20) was higher than for PKR-WT (FPmax=0.16), indicating that PKR-pWT has a statistically higher capacity to bind to Bodpy-PA. Computational docking experiments suggested that PA may dock onto the ATP-binding site on PKR, near Lys296 or Asp432. Since the ATP-binding site is highly conserved, we confirmed computationally that palmitate could also locate onto the ATP-binding site of other kinases, i.e. Akt1, CDK4, and MAKAPK3. We further experimentally demonstrated through ATP competition assays that PA binds to the ATP-binding site on the PKR protein. Furthermore, in-vitro experiments showed that PA binding onto the ATP-binding site blocks PKR autophosphorylation at Thr446 and Thr451, which we also observed in HepG2 cells. These results suggest that PA directly interacts with the PKR protein to alter its autophosphorylation, thereby regulating its activity. Based on the computational docking results, we further performed a mutation binding study to search the specific PA-binding residues on PKR. The mutation results further support that PA locates in the ATP-binding site on the PKR protein. Thus, a better understanding of how palmitate interacts with the PKR protein, as well as other kinases, could shed light into possible mechanisms by which palmitate mediates kinase signaling pathways, that could have implications on the efficacy of current drug therapies that target kinases.