Extended Data Fig. 6: The PAIR1-bound IRE1α* dimer interface is partially disrupted through Asp 620 displacement.

(a) Superimposition of the active IRE1α* (gray, PDB: 5HGI) and PAIR1-IRE1α* complex (orange) dimers. (b,c) Zoom-in views of the dimer interface contacts that are similar between active IRE1α* (gray, PDB: 5HGI) and the PAIR1-IRE1α* complex (orange) dimers. (d) 2Fo-Fc electron density maps of Asp620, contoured to 1.0σ shown as gray isomesh, reveal equal distribution of Asp620 between ‘in’ and ‘out’ conformations. (e) Superimposition of dimer interface residue Asp620 from active IRE1α* (gray, PDB: 5HGI) and the PAIR1-IRE1α* complex (orange) shows that Asp620-IN is in a similar conformation as active IRE1α* and forms a salt bridge with the side-chains of Arg594 and Arg627 of the adjacent IRE1α* protomer. In the other conformation, Asp620-OUT, the side-chain of Asp620 is displaced 11.6 Å and can no longer form a salt bridge with Arg594 and Arg627. This inter-dimer salt bridge between Asp620 and Arg594/Arg627 is essential for RNase active dimer formation and although Asp620 is found equally in conformations productive (Asp620-IN) and unproductive (Asp620-OUT) for RNase active dimer formation, the cumulative effect is partial disruption of the RNase active dimer interface. (f) Superimposition of kinase catalytic residues: K599, Glu612, and the DFG-motif (Asp712, Gly713, Phe714) from the PAIR1-IRE1α* complex (orange) and the AMG-16-IRE1α* complex (teal, PDB:4U6R) reveal that residues outside of the helix-αC (K599 and the DFG-motif) are in similar conformations, suggesting that differences in the pharmacology between PAIRs and KIRAs stems mainly from helix-αC movement.