Binding site multiplicity with fatty acid ligands: Implications for the regulation of PKR kinase autophosphorylation with palmitate

Abstract

Saturated long chain-free fatty acids (FFAs), especially palmitate, have been implicated in apoptosis by inhibiting the activity of PKR (double-stranded RNA-dependent protein kinase). We recently found evidence that palmitate interacts directly with the kinase domain of PKR, subsequently inhibiting the autophosphorylation of PKR. To investigate the interactions of palmitate with PKR and its effects on PKR autophosphorylation, we performed extensive unbiased MD simulations combined with biochemical and biophysical experiments. The simulations predict multiple putative binding sites of palmitate on both the phosphorylated and unphosphorylated PKR with similar binding affinities. Ligand-protein interactions involving a large variety of different binding modes challenge the conventional view of highly specific, single binding sites. Key interactions of palmitate involve the αC-helix of PKR, especially near residue R307. Experimental mutation of R307 was found to affect palmitate binding and reduce its inhibitory effect. Based on this study a new allosteric mechanism is proposed where palmitate binding to the αC-helix prevents the inactive-to-active transition of PKR and subsequently reduces its ability to autophosphorylate.

A tale of two proteins: PACT and PKR and their roles in inflammation

“…It is unclear how PKR is activated in this condition, as no requirement for dsRNA or PACT has been demonstrated. However, it was shown that PKR could be activated by free fatty acids (FFA); at high concentrations, the long unsaturated FFA palmitate can interact with the kinase domain of PKR to inhibit kinase activity [224]. It was also shown that PKR activation is dependent on interaction with phosphorylated TRBP, and shows that inhibition of TRBP can lead to increased glucose sensitivity [225].…”

Identification of the differentially expressed proteins in nasopharyngeal carcinoma by proteomics

“…Thus, defects in eIF2 are potentially fatal ( 17 , 18 ). These are currently known to be associated with a wide range of disorders, such as delayed sensitivity, contact dermatitis, impaired glucose tolerance, insulin resistance, Alzheimer's disease, myelodysplastic syndromes, and different types of cancers (breast cancer, leukemia, melanoma, hepatocellular carcinoma, and colon cancer) ( 19 , 20 ).…”