<scp>QM</scp> / <scp>MM</scp> Free Energy Calculations of <scp>IRE1</scp> Reveal a Unique Protonation State of the Catalytic Lys599

Inositol Requiring Enzyme 1 (IRE1) is a bifunctional serine/threonine kinase and endoribonuclease identified as therapeutic target in multiple diseases. Inspired by the recent work on the assessment of lysine and cysteine reactivities, we present a simple and intuitive protocol for the assessment of reactive lysine, while characterizing a unique protonation state of Lys599 located in the kinase domain. Using Quantum Mechanics/Molecular Mechanics (QM/MM) calculations, QM/MM well-tempered metadynamics simulations (QM/MM WT-MetaD), and classical Molecular Dynamics (MD), we have investigated inhibitor binding in three different states of the IRE1 kinase: (i) DFG-in/αC-in (DICI) conformation; (ii) the DFG-out/αC-out (DOCO) conformation, and (iii) the DFG-in/αC-out (DICO) conformation. Our findings reveal a unique proton transfer from the sidechain of the β3-strand Lys599 to Glu612 of the αC-helix. Our results allow for accurately defining the geometry of the hydrogen bonds occurring in the IRE1 kinase active state and distinguishing structurally closely related inactive states by analyzing the formation/disruption of crucial hydrogen bonds in the Lys599-Glu612-Asp711 triad. Our work prompts further studies in IRE1 and other kinases to characterize possibly conserved drug binding mechanisms that might lead to a novel structural paradigm in kinase drug discovery.