Abstract
Mycobacterium tuberculosis isocitrate lyase 2 (ICL2) is an allosterically regulated enzyme required for growth on non-glycolytic carbon substrates during infection. Although acetyl-CoA and its analogues are known to activate ICL2, the molecular basis of this regulation has remained unclear. Here, we combine protein NMR, crystallography, molecular dynamics, and mutagenesis to show that two structural features unique to ICL2, the C-terminal domain and a helical substructure in the N-terminal catalytic domain, govern its allostery. Acetyl-CoA binding promotes dimerisation of the C-terminal domain and disrupts its contacts with the helical substructure to trigger conformational changes that activate the enzyme. Together, these findings reveal how a non-substrate metabolite drives isocitrate lyase activation, uncovering the allosteric mechanism that controls M. tuberculosis metabolism and informs new therapeutic strategies.
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Data availability
The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.pdb.org (PDB ID 9OBO). The chemical shift assignments for the ICL2 C-terminal domain (ICL2601–766) have been deposited in the BioMagResBank (http://bmrb.io) under the accession numbers 52666 and 52668. Source data underlying the graphs in the main text and Supplementary Figs. are available as Supplementary Data (Supplementary Data 1).
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Acknowledgements
We thank Dr Biswaranjan Mohanty for his assistance and advice with the 3D NMR experiments for the labelled C-terminal domain that was carried out at Sydney Analytical, a core research facility at the University of Sydney. We thank Dr Michael Schmitz from the Nuclear Magnetic Resonance Centre at the University of Auckland for his advice on protein NMR experiments. We acknowledge Dr Richard Hopkinson (University of Leicester) for his helpful comments and discussions on earlier drafts of this paper. We acknowledge the support of the Australian Government in provision of access to ANSTO’s National Deuteration Facility, which is partly funded through the National Collaborative Research Infrastructure Strategy (NCRIS), via NDF proposal 15636 for the generation of the deuterated ICL2 C-terminal domain. We would also like to acknowledge the use of the Magnetic Resonance Facility, Melbourne Protein Characterisation and the Mass Spectrometry and Proteomics Facility at the Bio21 Molecular Biology and Biotechnology Institute for this work. NAMD was developed by the Theoretical and Computational Biophysics Group in the Beckman Institute for Advanced Science and Technology at the University of Illinois at Urbana-Champaign. The authors wish to acknowledge the contribution of NeSI (https://www.nesi.org.nz) high-performance computing facilities to the results of this research. NZ’s national facilities are provided by the NZ eScience Infrastructure and funded jointly by NeSI’s collaborator institutions and through the Ministry of Business, Innovation & Employment’s Research Infrastructure programme. Part of this study was conducted using the MX2 beamline at the Australian Synchrotron, which is part of ANSTO and made use of the ACRF detector. We thank the beamline staff for their enthusiastic and professional support. E.Y.W.H. was supported by the Melbourne Research Scholarship, Rowden White Scholarship, Norma Hilda Scholarship, Dame Margaret Blackwood Soroptimist Scholarship and Dr Albert Shimmins Postgraduate Writing-Up Award from the University of Melbourne. B.X.C.K. was supported by the University of Auckland Doctoral Scholarship. G.B., J.T., W.J. and I.K.H.L. were supported by the Marsden Fund (21-UOA-108) by the Royal Society of New Zealand. G.B. and I.K.H.L. were also supported by the Project Grant from the Maurice & Phyllis Paykel Trust. J.T. was supported by the Ngā Puanga Pūtaiao Fellowship through the Royal Society of New Zealand. M.J.M. was funded by an Australian Research Council (ARC) Future Fellowship (FT180100397). I.K.H.L. and M.J.M. would like to thank the University of Melbourne for support through the Driving Research Momentum (DRM) initiative.
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E.Y.W.H., B.X.C.K. and I.K.H.L. designed research. E.Y.W.H., B.X.C.K., W.J. and M.J.M. performed research. E.Y.W.H., B.X.C.K., W.J., A.S. and M.J.M. analysed data. K.L.W., A.S. and M.J.M. contributed new tools/techniques. E.Y.W.H., B.X.C.K., W.J., G.B., J.T., M.J.M. and I.K.H.L. wrote the paper.
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Huang, E.YW., Kwai, B.X.C., Jiao, W. et al. Structural basis of allosteric activation of Mycobacterium tuberculosis isocitrate lyase 2. Commun Biol (2026). https://doi.org/10.1038/s42003-026-09821-6
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DOI: https://doi.org/10.1038/s42003-026-09821-6
