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Peer-reviewedCC BYCell Reports

Activation mechanism and structural assembly of the Mycobacterium tuberculosis ClpP1P2 protease and its associated ATPases

  • Katharina Weinhäupl1,2
  • Tatos N. Akopian3,4
  • Dmitry A. Semchonok5,4
  • Olga Krandor3,6
  • Rocío Arranz7,8
  • M. Teresa Bueno-Carrasco9
  • Marcos Gragera10,8
  • Maelenn Chevreuil11,12
  • Bertrand D. E. Raynal11,12
  • Samuel Zinga3,13
  • Yuxin Liu14,13
  • Jack Hung-Sen Lai14,13
6 more authors
  • Wengen Wu14,13
  • William W. Bachovchin14,15
  • Diego F. Gauto16,4
  • Alfred Lewis Goldberg17,18
  • Eric J. Rubin19
  • Hugo Fraga20
20 affiliations
  1. 1i3S, Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal
  2. 2Universidade do Porto Instituto de Investigacao e Inovacao em Saude
  3. 3Harvard Chan School of Public Health, Department of Immunology and Infectious Diseases, Harvard University, Cambridge, MA, USA
  4. 4Harvard School of Public Health
  5. 5Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Lisbon, Portugal
  6. 6Instituto de Tecnologia Quimica e Biologica
  7. 7Cryo-EM Facility, Department of Macromolecular Structures, Centro Nacional de Biotecnología-CSIC, Madrid, Spain; Department of Macromolecular Structure, National Center of Biotechnology (CNB-CSIC), Madrid 28049, Spain
  8. 8Centro Nacional de Biotecnologia
  9. 9Cryo-EM Facility, Department of Macromolecular Structures, Centro Nacional de Biotecnología-CSIC, Madrid, Spain; Department of Macromolecular Structure, National Institute of Biotechnology (CNB-CSIC), Madrid 28049, Spain; Institute for Chemical Research (IIQ), Scientific Research Center Isla de la Cartuja (cicCartuja), University of Seville-CSIC, Seville, Spain
  10. 10Cryo-EM Facility, Department of Macromolecular Structures, Centro Nacional de Biotecnología-CSIC, Madrid, Spain; Department of Macromolecular Structure, National Institute of Biotechnology (CNB-CSIC), Madrid 28049, Spain
  11. 11Plateforme de biophysique moléculaire, C2RT, Institut Pasteur, Université Paris Cité, 75015 Paris, France
  12. 12Institut Pasteur
  13. 13Tufts University School of Medicine
  14. 14Department of Developmental, Molecular and Chemical Biology, Tufts, Medford, MA, USA
  15. 15Harvard Medical School
  16. 16Institut de Chimie des Substances Naturelles (ICSN), Centre national de la recherche scientifique (CNRS), Gif-Sur-Yvette, Paris, France
  17. 17Department of Cell Biology, Harvard Medical School, Boston, MA, USA
  18. 18Faculdade de Medicina da Universidade do Porto
  19. 19Harvard Chan School of Public Health, Department of Immunology and Infectious Diseases, Harvard University, Cambridge, MA, USA. Electronic address: [email protected]
  20. 20i3S, Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal; Departamento de Biomedicina, Faculdade de Medicina da Universidade do Porto, Porto, Portugal. Electronic address: [email protected]
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Abstract

Supramolecular assemblies are integral to cellular biochemical processes, relying on their dynamic nature to fulfill essential functions. The protease ClpP1P2, paired with ATPase partners ClpC1 or ClpX, is vital for the survival of Mycobacterium tuberculosis ( Mtb ). While the ClpP1P2 complex requires activation by specific N-blocked dipeptides (e.g., Z-Leu-Leu) to exhibit proteolytic activity in vitro, the mechanism of in vivo activation remains unclear. In this study, we identified novel activators that enabled the structural determination of the ClpC1P1P2 complex, providing insights into its assembly. Furthermore, we discovered that trehalose - a key metabolite and molecular crowding agent in Mtb , significantly enhances the activity of both ClpC1P1P2 and ClpXP1P2 complexes without the need for activating peptides. Analytical ultracentrifugation revealed that trehalose promotes the formation of these active complexes, mimicking intracellular conditions. These findings propose a new model of Clp system activation in vivo , offering promising avenues for therapeutic targeting in tuberculosis treatment. Significance Statement The proteolytic complex formed by the essential proteins ClpP1 and ClpP2, along with their specific ATP-dependent activators ClpX and ClpC1, has emerged as a highly attractive target for anti-tuberculosis drug development. While previous studies have shown that ClpP1P2 can be activated in vitro by small peptide activators, its in vivo activation mechanism remains unclear. In this study, we identify novel activators and demonstrate that trehalose, a key metabolite in Mycobacterium tuberculosis , enhances ClpC1P1P2 and ClpXP1P2 activity without the need for activating peptides. These findings propose a new model for Clp system activation in Mycobacterium tuberculosis , advancing our understanding of its regulation and potential as a therapeutic target.

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Cite

BibTeXRIS
BibTeX
@article{weinhaupl2026activation,
  title = {{Activation mechanism and structural assembly of the Mycobacterium tuberculosis ClpP1P2 protease and its associated ATPases}},
  author = {Katharina Weinhäupl and Tatos N. Akopian and Dmitry A. Semchonok and Olga Krandor and Rocío Arranz and M. Teresa Bueno-Carrasco and Marcos Gragera and Maelenn Chevreuil and Bertrand D. E. Raynal and Samuel Zinga and Yuxin Liu and Jack Hung-Sen Lai and Wengen Wu and William W. Bachovchin and Diego F. Gauto and Alfred Lewis Goldberg and Eric J. Rubin and Hugo Fraga},
  journal = {Cell Reports},
  year = {2026},
  month = may,
  doi = {10.1016/j.celrep.2026.117400},
  url = {https://doi.org/10.1016/j.celrep.2026.117400},
  note = {CC BY},
}
RIS
TY  - JOUR
TI  - Activation mechanism and structural assembly of the Mycobacterium tuberculosis ClpP1P2 protease and its associated ATPases
AU  - Katharina Weinhäupl
AU  - Tatos N. Akopian
AU  - Dmitry A. Semchonok
AU  - Olga Krandor
AU  - Rocío Arranz
AU  - M. Teresa Bueno-Carrasco
AU  - Marcos Gragera
AU  - Maelenn Chevreuil
AU  - Bertrand D. E. Raynal
AU  - Samuel Zinga
AU  - Yuxin Liu
AU  - Jack Hung-Sen Lai
AU  - Wengen Wu
AU  - William W. Bachovchin
AU  - Diego F. Gauto
AU  - Alfred Lewis Goldberg
AU  - Eric J. Rubin
AU  - Hugo Fraga
PY  - 2026
DA  - 2026/05/30
JO  - Cell Reports
DO  - 10.1016/j.celrep.2026.117400
UR  - https://doi.org/10.1016/j.celrep.2026.117400
AB  - Supramolecular assemblies are integral to cellular biochemical processes, relying on their dynamic nature to fulfill essential functions. The protease ClpP1P2, paired with ATPase partners ClpC1 or ClpX, is vital for the survival of Mycobacterium tuberculosis ( Mtb ). While the ClpP1P2 complex requires activation by specific N-blocked dipeptides (e.g., Z-Leu-Leu) to exhibit proteolytic activity in vitro, the mechanism of in vivo activation remains unclear. In this study, we identified novel activators that enabled the structural determination of the ClpC1P1P2 complex, providing insights into its assembly. Furthermore, we discovered that trehalose - a key metabolite and molecular crowding agent in Mtb , significantly enhances the activity of both ClpC1P1P2 and ClpXP1P2 complexes without the need for activating peptides. Analytical ultracentrifugation revealed that trehalose promotes the formation of these active complexes, mimicking intracellular conditions. These findings propose a new model of Clp system activation in vivo , offering promising avenues for therapeutic targeting in tuberculosis treatment. Significance Statement The proteolytic complex formed by the essential proteins ClpP1 and ClpP2, along with their specific ATP-dependent activators ClpX and ClpC1, has emerged as a highly attractive target for anti-tuberculosis drug development. While previous studies have shown that ClpP1P2 can be activated in vitro by small peptide activators, its in vivo activation mechanism remains unclear. In this study, we identify novel activators and demonstrate that trehalose, a key metabolite in Mycobacterium tuberculosis , enhances ClpC1P1P2 and ClpXP1P2 activity without the need for activating peptides. These findings propose a new model for Clp system activation in Mycobacterium tuberculosis , advancing our understanding of its regulation and potential as a therapeutic target.
SN  - 2211-1247
SN  - 2639-1856
ER  -