Upon accumulation of unfolded proteins at the endoplasmic reticulum (ER), IRE1 activates the unfolded protein response (UPR) to restore protein-folding homeostasis. During ER stress, the ER lumenal domain (LD) of IRE1 drives its clustering on the ER membrane to initiate signaling. How IRE1 LD assembles into high-order oligomers remains largely unknown. By in vitro reconstitution experiments we show that human IRE1 LD forms dynamic biomolecular condensates. IRE1 LD condensates were stabilized when IRE1 LD was tethered to model membranes and upon binding of unfolded polypeptide ligands. Molecular dynamics simulations suggested that weak multivalent interactions are involved in IRE1 LD assemblies. Mutagenesis showed that disordered regions in IRE1 LD control its clustering in vitro and in cells. Importantly, dysregulated clustering led to defects in IRE1 signaling. Our results reveal that membranes and unfolded polypeptides act as scaffolds to assemble dynamic IRE1 condensates into stable, signaling competent clusters.

The Shwachman–Diamond Syndrome (SDS) is an autosomal recessive disease whose majority of patients display mutations in a ribosome assembly protein named Shwachman–Bodian–Diamond Syndrome protein (SBDS). A specific therapy for treating this rare disease is missing, due to the lack of knowledge of the molecular mechanisms responsible for its pathogenesis. Starting from the observation that SBDS single-point mutations, localized in different domains of the proteins, are responsible for an SDS phenotype, we carried out the first comparative Molecular Dynamics simulations on three SBDS mutants, namely R19Q, R126T and I212T. The obtained 450-ns long trajectories were compared with those returned by both the open and closed forms of wild type SBDS and strongly indicated that two distinct conformations (open and closed) are both necessary for the proper SBDS function, in full agreement with recent experimental observations. Our study supports the hypothesis that the SBDS function is governed by an allosteric mechanism involving domains I and III and provides new insights into SDS pathogenesis, thus offering a possible starting point for a specific therapeutic option.

IV.