(582cv) Functional Role of the Tryptophan Residue On the Transmembrane Domain of the IRE1? Protein

The inositol-requiring 1α protein (IRE1α) is a type I transmembrane protein important in altering gene expression in response to endoplasmic reticulum (ER) stress. IRE1α contains an N-terminal luminal domain that is an ER stress sensor and a C-terminal cytosolic domain that carries protein Ser/Thr kinase and endoribonuclease activities. Under ER stress conditions, the Bip protein dissociates from the luminal domain of IRE1α, promoting self-association of the IRE1α protein. The face-to-face dimerization of the kinase domain facilitates trans-autophosphorylation, subsequently activating the RNase domain. The active form of IRE1α participates in reducing ER stress by upregulating genes involved in enhancing ER protein-folding capacity and degrading unfolded or misfolded proteins. We recently found that the transmembrane (TM) domain of the IRE1α protein dimerizes/oligomerizes in a membrane-mimic environment, and maintains its helical structure. However, functional information on the TM domain of the IRE1α protein is currently unavailable. We performed molecular dynamics (MD) simulations using two model TM peptides inserted in a POPC bilayer. The simulation results revealed that four potential residues (S450, L453, L454, and W457) are located on the dimerization interface of the TM model peptide. TOXCAT and SDS-PAGE analyses showed that alanine mutation of W457 residue dramatically reduces the oligomerization of the TM domain. In addition, mutational studies with MD simulations suggest that W457 serves as a driving force for the TM dimerization process and both the aromatic interaction and hydrogen bond formed by the W457 residue are crucial for stabilizing the dimerization of the TM domain. We further confirmed that the W457A mutation reduces both the kinase and RNase activities of the IRE1α protein in cells. Through the combination of computational and experimental studies, we gained insight into how the TM domain impacts the functions of IRE1α.