QM / MM Free Energy Calculations of IRE1 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.