(75d) Computational and Experimental Study of Palmitate Binding to the IRE1 Protein

IRE1 (inositol-requiring enzyme 1) is an ER (endoplasmic reticulum) transmembrane Ser/Thr protein kinase/endoribonuclease involved in the unfolded protein response (UPR), and is an adaptive intracellular signal transduction pathway induced upon ER stress. Activation of IRE1 by dimerization of the N-terminal luminal domain induces autophosphorylation of its cytosolic kinase domain, thereby activating its C-terminal nuclease domain. Palmitate, a saturated fatty acid, has been recognized to induce ER stress, and as an IRE1 activator will upregulate UPR target genes, i.e. pro-apoptotic genes. Recently, our lab found that palmitate interacts with PKR (protein kinase R) to regulate the PKR kinase activity. Given the similarity in the back-to-back kinase domains of PKR and IRE1, we hypothesize that palmitate also directly binds to the IRE1 protein. We confirmed, using a fluorescence polarization (FP)-based binding assay, that palmitate indeed interacts with the cytosolic domain containing the kinase and nuclease domains, but not the luminal domain of IRE1. To identify potential palmitate-binding sites, we performed molecular dynamics simulations. The computational results suggest that palmitate locates near several sites (i.e. the activation loop and ATP binding site in the kinase domain and the nuclease active site in the nuclease domain) which control IRE1 enzymatic activities. Site-specific mutation experiments further confirmed that the predicted residues are important for palmitate binding and in turn IRE1 enzymatic function. The computationally assisted biochemical studies help confirm the molecular mechanism by which palmitate directly activates the IRE1 functions as well as possibly the mechanism by which palmitate initiates ER stress through the UPR sensor proteins.