The hIRE1α cLD forms a stable dimer validated by hydrogen-deuterium exchange experimental data.

(A) The PDB model: structural model of hIRE1α cLD dimer as obtained by Zhou et al.24 (PDB ID 2HZ6) with the addition of missing residues, in particular DR1 (cyan) and DR2 (orange). Backbone in cartoon representation, areas of interest highlighted in different colors. (B) The AF model: AlphaFold 2 Multimer prediction of hIRE1α cLD dimer (P29-P369). Representation style as in Fig. 1A. (C) The deuterated fraction obtained from experimental results (dashed line) published by Amin-Wetzel et al. 25 and the fraction computed from MD simulations (solid lines, blue for TIP3P water and orange for TIP4PD water) for the PDB and AF model at time points 0.5 min and 5 min. Below each absolute value plot, we report the discrepancy, which is defined as the difference between the simulated and experimental deuterated fractions. (D) Visualization of the discrepancy values from Fig. 1C onto the molecular structures of the PDB and AF model simulated in TIP4P-D water (TIP3P shown in Fig. 1 Suppl. 2A). Shades of blue indicate that the simulated structure is more flexible and solvent accessible with respect to what was reported from experiments, while red indicates that the simulated structure is more rigid and less solvent accessible than measured experimentally.

The human IRE1α cLD dimer has a narrower and more occluded central groove than its yeast homolog in equilibrium MD simulations.

(A) Top panel: structure of human IRE1α (hIRE1α) cLD dimer (AlphaFold model). Bottom panel: structure of the S. Cerevisiae IRE1 (yIre1) cLD dimer (PDB ID 2BE1). (B) Close-up on hIRE1α (top panel) and yIre1 (bottom panel) structures on the central groove delimited by the αA-helices. Helices delimiting the groove are depicted in blue cartoon and side chains in licorice representation, while the rest of the dimer is transparent. The arrows indicate the distance between Cα atoms used to compute the groove width. (C) Probability density distribution of the groove width of: hIRE1α cLD dimer computed between Cα of Q105 during simulations in TIP3P and TIP4P-D water (top panel); yIre1 luminal domain dimer computed between Cα of S200 during simulations in TIP3P and TIP4P-D water (bottom panel). Dashed lines indicate the groove width of the dimer model prior to energy minimization.

Unfolded peptides can bind with specificity to the center of the cLD dimer, even if the groove is closed.

(A) AF model of the cLD dimer (gray cartoon) simulated with the unfolded peptide MPZ1-N (violet) positioned parallel to the principal axis of the central groove (t = 0 µs). During the simulation, MPZ1-N rearranges to a vertical position (t = 1 µs). The direction of the principal axis of the central groove is represented by a solid arrow, while the direction of the principal axis of the peptide is indicated by a dashed arrow. (B) AF model of the cLD dimer simulated with unfolded peptide MPZ1N-2X (orange), which stably binds to the center of the dimer. (C) AF model of the cLD dimer simulated with unfolded peptide MPZ1N-2X-RD (red), which unbinds in half of the replicas. (D) Box plot distributions of the minimum groove-peptide distance. Each box plot reports on the results from 3 replicas (in TIP3P or in TIP4PD) of a system containing a dimer and one of the unfolded peptides.

Single point mutation of cLD alters the binding affinity of unfolded peptide.

(A) Analysis of contacts between unfolded peptide and cLD dimer for all MD simulations performed in TIP4P-D water. The contact score reported on the AlphaFold model of the cLD dimer was obtained by computing the contacts from all the simulations, summing them, and normalizing them by the number of frames. Lower values indicate fewer contacts observed. (B) Important areas for peptide binding on the surface of the cLD dimer: the red area identifies a hydrophobic-aromatic binding pocket characterized by residues Y117, F98, and L103; the yellow area identifies a deeper polar-charged binding pocket and is characterized by residues Y161, D181, D79. (C) Side view snapshot after 1 µs of simulation of wild-type hIRE1α cLD dimer (gray) in complex with MPZ1N-2X (orange). Amino acid Y161 on both monomers is represented in green licorice. (D) Time series of the minimum groove-peptide distance for MPZ1N-2X simulated in complex with wild-type hIRE1α cLD dimer in TIP3P (3 replicas) and TIP4P-D (3 replicas) water. (E) Side view snapshot after 1 µs of simulation of Y161R hIRE1α cLD dimer (gray) in complex with MPZ1N-2X (orange). Amino acid R161 on both monomers is represented in blue licorice.(F) Time series of the minimum groove-peptide distance for MPZ1N-2X simulated in complex with Y161R hIRE1α cLD dimer in TIP3P (3 replicas) and TIP4P-D (3 replicas) water.

hIRE1α cLD intermolecular interactions.

(A) Model of two cLD dimers interacting via an unfolded peptide (MPZ1N-2X): the system setup is shown (t = 0 ns) and a snapshot of the simulation (t = 200 ns). (B) BiP-cLD monomer complex as predicted by AlphaFold (BiP in shades of purple, cLD in orange). In the initial model, before the simulation (t = 0 ns), the interface (inset) is constituted by DR2 and SBD, while at the end of the simulation (t = 460 ns), the N-term of BiP rearranges to form a helix and comes in contact with the αB-helix. The two proteins are represented as cartoons, and the side chains of the interface residues at the end of the simulation are shown as sticks. The SBD (residues M1-D408) is colored in dark magenta and the NDB (residues C420-E650) in purple, and the interdomain linker (residues D409-V419) and KDEL motif (residues K651-L654) in light purple.

Schematic illustrating the proposed sequence of events occurring in the early stages of hIRE1α activation within the ER lumen.

Before the onset of ER stress conditions, hIRE1α is prevented from forming assemblies by the chaperone BiP, which occupies the cLD’s oligomeric interface. As ER stress begins, unfolded proteins start to accumulate in the ER lumen, and pre-formed hIRE1α cLD dimers are released from BiP. Now, hIRE1α can interact with unfolded proteins, which brings multiple copies of the protein together to allow the formation of larger assemblies.

(A) Superposition of AF model (light violet) and PDB model (orange) of the hIRE1α cLD dimer before the simulation (RMSD = 3.34 Å). (B) Superposition of cLD dimer models predicted by AlphaFold 2 Multimer (light violet) and AlphaFold 3 (dark violet). (C) PDB model of the hIRE1α cLD dimer after 2 µs of production run from one of the TIP3P replicas. (D) AF model of the hIRE1α cLD dimer after 2 µs of production run from one of the TIP3P replicas. (E) Details of the dimeric interface of the hIREα cLD dimer, consisting of an antiparallel β-sheet with four hydrogen bonds between residues G119-D123. The renders show the dimer interface in the AF model (upper row) and of the PDB model (lower row) prior to energy minimization and production run (t = 0 µs) and after 2 µs of simulation. The backbone atoms of residues G119-D123 are in licorice (C cyan, H white, O red, N blue) and the rest of the structure is rendered as a transparent ribbon. The backbone of the protein is shown as transparent cartoon. (F) Hydrogen bond analysis of all the hIREα cLD dimer simulation replicas. This analysis shows the distance between donor and acceptor atoms (left y-axis, blue line) and the angle between donor-hydrogen-acceptor (right y-axis, orange line) for all replicas of PDB and AF models in TIP3P and TIP4P-D water. The hydrogen bonds analyzed here are those formed at the dimeric interface by residues G119, K121, and N123. The horizontal blue and orange lines indicate, respectively, the ideal distance (3.2 Å) and angle (180°) for a hydrogen bond. Whenever the distance is greater than 3.2 Å or the angle is lower than 120°, the frame is highlighted by a grey or red vertical bar, respectively. Therefore, the vertical bars indicate when the hydrogen bond is lost. The data are plotted every 50th frame for the lines and every frame for the vertical bars. (G) Early PDB model of the hIRE1α cLD dimer after 2 µs of production run. Replica showing a partial dissociation of the dimer. (H) Detail of the side of the dimeric interface of the early PDB model that dissociates during simulations. The side chains of residues L116, Y166, and W125 are highlighted in three different models: the early PDB model (gray), the final PDB model (orange), and the AlphaFold model (light violet). The backbone of the protein is shown as transparent cartoon. (I) The DR1 is folded in the AF model (Fig. 1B). The modified AF model with unfolded DR1 (left, t = 0 µs). The AlphaFold model with unfolded DR1 is stable after 500 ns of simulation (right, t= 500 µs).

(A) Visualization of the discrepancy values from Fig. 1C for TIP3P water onto the molecular structures of the PDB and AF models. Shades of blue indicate that the simulated structure is more flexible and solvent accessible with respect to what was reported from experiments, while red indicates that the simulated structure is more rigid and less solvent accessible than expected. (B) Comparison of deuterated fraction from Amin-Wetzel et al. with deuterated fraction computed from MD simulations (See Method section ‘HDX data comparison’). For each residue, the plot indicates which model gives the better agreement with experimental data (‘best structure’) at each of the time points examined, t = 0.5 min, 5 min or both. The histogram plots the cumulative number of residues that are the closest to experimental data for each model and water. (C) Table comparing the models’ ability to reproduce experimental data. The four variants of the model are compared residue-wise, at both time points, and each model gets a point if it can reproduce the experimental deuterated fraction of a given residue better than the other three models. Points are summed for each model at both time points to get the total. The total score is the value plotted in the histogram in Supplementary Fig.8B. (D) Best structural model of hIRE1α cLD dimer: the colors highlight the best structural representation of each residue at either time point, as plotted in Supplementary Fig.8B.

(A) Probability density distribution of the groove width of: hIRE1α cLD dimer (AlphaFold model) computed between Cα of Q105 during simulations in TIP3P and TIP4P-D water (top panel); hIRE1α cLD dimer (PDB model) computed between Cα of Q105 during simulations in TIP3P and TIP4P-D water (middle panel); yIre1 luminal domain dimer computed between Cα of S200 during simulations in TIP3P and TIP4P-D water (bottom panel). Dashed lines indicate the groove width of the dimer model prior to energy minimization. (B) Representative Q105 side chains arrangements during simulations of the human cLD dimer AlphaFold model (top row) and of the PDB model (bottom row), snapshots are taken from three independent replicas in TIP3P and TIP4P-D.

(A) Additional examples of peptide binding. Here, we report snapshots from simulations at the initial timestep (t= 0 µs) and final timestep (t= 1 µs) of the unfolded peptides not shown in Fig. 3A-C, in order: Valine8, MPZ1-N (perpendicular placement), MPZ1-C, 8ab1, OR-1, V1rb2-1, V1rb2-2. Heterogeneous binding. Peptides that are unstable in the central groove placement tend to rearrange to different parts of IRE1, either to the side of the groove as for V1rb2-1 and MPZ1-C or to the outer part of the dimer in the proximity of the αB-helices, as in the case of MPZ1N-2X-RD. (C) Available binding affinities (K1/2) of the unfolded polypeptides studied to hIRE1α.14,26 (D) Prediction of AlphaFold 3 for hIRE1α cLD dimer in complex with peptide MPZ1N-2X. Colors represent the confidence of the prediction (plDDT). (E) Distributions of the groove width of peptide-bound cLD dimers throughout all simulations performed.

(A) Analysis of contacts between unfolded peptide and cLD dimer for all MD simulations performed in TIP3P water. The contact score reported on the AlphaFold model of the cLD dimer was obtained by computing the contacts from all the simulations, summing them, and normalizing them by the number of frames. Lower values indicate fewer contacts observed. (B) Contact analysis of MPZ1N-2X in complex with wild-type hIRE1α cLD dimer. (C) Contact analysis of MPZ1N-2X in complex with Y161R hIRE1α cLD dimer. The analysis of panels B and C took into consideration all six 1 µs-long replicas in TIP3P and TIP4P-D water for each system.

(A) Prediction of AlphaFold 3 for hIRE1α cLD monomer in complex with ATP-bound BiP. The colors are as in Fig. 5B. (B) Prediction of AlphaFold 3 for hIRE1α cLD monomer in complex with ADP-bound BiP. (C) Prediction of AlphaFold 3 for hIRE1α cLD monomer in complex with BiP not bound to any nucleotide. (D) Structure of hIRE1α cLDBiP- ATP after 2 μs of simulation. (E) Structure of hIRE1α cLD-BiP-ADP after 2 μs ofsimulation. (F) Structure of hIRE1α cLD-BiP after 2 μs of simulation.