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

Antonio Carlesso; Paolo Conflitti; Sayyed Jalil Mahdizadeh; Giuseppe Deganutti; Leif A Eriksson; Vittorio Limongelli

doi:10.1002/jcc.70288

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

Antonio Carlesso
, Paolo Conflitti
, Sayyed Jalil Mahdizadeh
, Giuseppe Deganutti
, Leif A Eriksson
, Vittorio Limongelli

Research output: Contribution to journal › Article › peer-review

1 Downloads (Pure)

Abstract

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.

Original language	English
Article number	e70288
Number of pages	11
Journal	Journal of Computational Chemistry
Volume	46
Issue number	31
Early online date	4 Dec 2025
DOIs	https://doi.org/10.1002/jcc.70288
Publication status	Published - 5 Dec 2025

Bibliographical note

© 2025 The Author(s). Journal of Computational Chemistry published by Wiley Periodicals LLC.

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Funding

This work was supported by Cancerfonden, 211447-Pj, Elisabeth and Alfred Ahlqvist's Foundation, Postdoctoral funding, The Foundation Blanceflor, and H2020 European Research Council, 101001784. This research was funded by Elisabeth and Alfred Ahlqvists Foundation (AC), The Foundation Blanceflor (AC), the Faculty of Science at the University of Gothenburg and the Swedish Cancer Foundation (grant number 211447-Pj) (LAE). The authors thank the National Academic Infrastructure for Supercomputing in Sweden (NAISS) for generous allocations of computing time at supercomputing centers C3SE and PDC, in part funded by the Swedish Research Council through grant agreement no. 2022-06725. Vittorio Limongelli acknowledges funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (“CoMMBi” ERC grant agreement No. 101001784), the Swiss National Science Foundation (SNSF) (grant number IC00I0-231546), and the Swiss National Supercomputing Centre (CSCS) under project ID lp40 and u8. The authors gratefully acknowledge Filippo Lessio for his help in designing the IRE1 schematic representation. This research was funded by Elisabeth and Alfred Ahlqvists Foundation (AC), The Foundation Blanceflor (AC), the Faculty of Science at the University of Gothenburg and the Swedish Cancer Foundation (grant number 211447‐Pj) (LAE). The authors thank the National Academic Infrastructure for Supercomputing in Sweden (NAISS) for generous allocations of computing time at supercomputing centers C3SE and PDC, in part funded by the Swedish Research Council through grant agreement no. 2022‐06725. Vittorio Limongelli acknowledges funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (“CoMMBi” ERC grant agreement No. 101001784), the Swiss National Science Foundation (SNSF) (grant number IC00I0‐231546), and the Swiss National Supercomputing Centre (CSCS) under project ID lp40 and u8. The authors gratefully acknowledge Filippo Lessio for his help in designing the IRE1 schematic representation. This work was supported by Cancerfonden, 211447‐Pj, Elisabeth and Alfred Ahlqvist's Foundation, Postdoctoral funding, The Foundation Blanceflor, and H2020 European Research Council, 101001784.

Funders	Funder number
Foundation Blanceflor
Horizon Europe
University of Gothenburg
European Research Council
Elisabeth och Alfred Ahlqvists foundation
Centro Svizzero di Calcolo Scientifico	lp40
Vetenskapsrådet	2022‐06725
Horizon Europe	101001784
Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung	IC00I0‐231546
Swedish Cancer Foundation	211447‐Pj

Keywords

Protein Serine-Threonine Kinases/chemistry
Protons
Quantum Theory
Lysine/chemistry
Molecular Dynamics Simulation
Thermodynamics
Endoribonucleases/chemistry
Humans

Access to Document

10.1002/jcc.70288Licence: CC BY

Deganutti2025VoRFinal published version, 1.87 MBLicence: CC BY

Cite this

@article{3c508659cc2d453f9bb8c5db1174d6e5,

title = "QM/MM Free Energy Calculations of IRE1 Reveal a Unique Protonation State of the Catalytic Lys599",

abstract = "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.",

keywords = "Protein Serine-Threonine Kinases/chemistry, Protons, Quantum Theory, Lysine/chemistry, Molecular Dynamics Simulation, Thermodynamics, Endoribonucleases/chemistry, Humans",

author = "Antonio Carlesso and Paolo Conflitti and Mahdizadeh, \{Sayyed Jalil\} and Giuseppe Deganutti and Eriksson, \{Leif A\} and Vittorio Limongelli",

note = "{\textcopyright} 2025 The Author(s). Journal of Computational Chemistry published by Wiley Periodicals LLC. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.",

year = "2025",

month = dec,

day = "5",

doi = "10.1002/jcc.70288",

language = "English",

volume = "46",

journal = "Journal of Computational Chemistry",

issn = "0192-8651",

publisher = "John Wiley \& Sons Inc.",

number = "31",

}

TY - JOUR

T1 - QM/MM Free Energy Calculations of IRE1 Reveal a Unique Protonation State of the Catalytic Lys599

AU - Carlesso, Antonio

AU - Conflitti, Paolo

AU - Mahdizadeh, Sayyed Jalil

AU - Deganutti, Giuseppe

AU - Eriksson, Leif A

AU - Limongelli, Vittorio

N1 - © 2025 The Author(s). Journal of Computational Chemistry published by Wiley Periodicals LLC. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

PY - 2025/12/5

Y1 - 2025/12/5

N2 - 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.

AB - 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.

KW - Protein Serine-Threonine Kinases/chemistry

KW - Protons

KW - Quantum Theory

KW - Lysine/chemistry

KW - Molecular Dynamics Simulation

KW - Thermodynamics

KW - Endoribonucleases/chemistry

KW - Humans

UR - https://www.scopus.com/pages/publications/105023912787

U2 - 10.1002/jcc.70288

DO - 10.1002/jcc.70288

M3 - Article

C2 - 41346185

SN - 0192-8651

VL - 46

JO - Journal of Computational Chemistry

JF - Journal of Computational Chemistry

IS - 31

M1 - e70288

ER -

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

Abstract

Bibliographical note

Funding

Keywords

Access to Document

Other files and links

Fingerprint

Cite this