Structural basis of allosteric activation of Mycobacterium tuberculosis isocitrate lyase 2

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.