Fig. 5: The Core domain of SpoT transduces the allosteric signal from the regulatory CTD and pseudo-SNTH to the enzymatic HD domain.

a, Effects of substitutions at the α6–α7:Core–TGS interface. Interactions stabilizing the α6–α7 motif of the HD-active site with the Core wrapping around α7 and the TGS β-hairpin stabilizing α6. Experimental SAXS curve of SpoT_Ab^E379K/W382K shows it remains in the τ-state. b, Effects of substitutions at the HD–Core–RRM interface with RRM locked in place via the Core and supporting interaction provided by pseudo-SYNTH, indeed the SAXS curve of SpoT_Ab^I637D/R641D is consistent with the dimensions of the τ-state. c, ITC titration of Mn²⁺ into apo-SpoT_Ab. d, HD activity of apo-SpoT_Ab as a function of increasing concentrations of Mn²⁺. e, Structure of the Mn²⁺-free SpoT_Ab^NTD. The HD domain is in purple, and the pseudo-SYNTH is in yellow. The disordered active site is labeled. f, Superposition of the HD domain of SpoT_Ab complexed with ppGpp (in light blue) onto Mn²⁺-free SpoT_Ab (in purple). The key differences in conformation of catalytically crucial active site residues and the structural elements α3, α4 and α8 are highlighted with dashed arrows and shown in bold, respectively. g, Thermal denaturation profile monitored by far UV circular dichroism (CD) spectrum at 222 nm of SpoT_Ab^H54A/H78A, which cannot bind Mn²⁺ in the HD domain. h, Virulence assays in the G. mellonella infection model demonstrate the essentiality of intact allosteric regulation of SpoT_Ab for virulence. G. mellonella larvae were injected with roughly 2 × 10 CFU of A. baumannii (AB5075) strains (10 µl at roughly 2 × 10⁷ CFU per ml), eight larvae were inoculated per strain and incubated at 37 °C in the dark. The viability of the larvae was scored every 24 h. Error bars represent s.d. of three or more independent samples examined over three independent experiments.