Fig. 4. G-1749 modifies the conformation of the IRE1 kinase activation loop.

a Model of activation loop and αEF-αF loop segments (yellow cartoon) of the IRE1 kinase for which electron density was missing in the G-1749/IRE1 co-crystal structure, built using the crystal structure model (Chain A) as template, using the Homology Model algorithm in the Molecular Operating Environment (MOE) software, overlaid with IRE1 apo 0P crystal structure (blue cartoon). The model shown here was the one reported as optimized among ten determined by the program (see Supplementary Fig. 4). b Hydrogen–deuterium exchange (HX-MS) results for peptide R687-L695 in indicated samples, shown as number of deuterons over time. Samples were incubated in deuterated buffer from 30 s up to 8 h and then subjected to protease digestion after quenching. Peptides were separated by HPLC, and mass spectrometry was utilized to measure deuteration levels of peptides at the indicated timepoints. The peptide from the sample with IRE1-0P in the presence of G-1749 showed a much faster deuteration than IRE1-0P apo and in complex with AMG-18. Source data are provided as a Source Data file. Data are presented as the mean for measurements from two independent experiments (n = 2). c Data from panel b were used to calculate protection factors, pF (see Methods and SI), for indicated samples, compared to IRE1-0P. pF above a value of 1 indicate protection/stabilization, while pF between 0 and 1 indicate deprotection/destabilization of the region. Source data are provided as a Source Data file. Data are presented as the mean for measurements from two independent experiments (n = 2). d pF for all detected peptides are reported on the corresponding structures as cartoon putty, with thicker cartoon corresponding to larger values, and vice versa. Stabilization (pF > 1) is shown as blue, destabilization (0 < pF < 1) is shown as red. No change is shown as gray. Source data are provided as a Source Data file.