Fig. 2. Structure of the Rel_Bs^NTD₂:DarB heterotetrameric complex.

(A) Crystal structure of DarB₂:Rel_Bs^NTD₂ heterotetrameric complex with the disc-shaped DarB dimer located at the center of the complex (colored in pink and lilac) and the two Rel_Bs^NTD bound at both sides of the DarB dimer. For each Rel_Bs^NTD molecule of the complex, the HD domain is colored in light blue, and the SYNTH domain is in yellow. In the nonsymmetrical hetero-complex, the Rel_Bs^NTD in the SYNTH-primed state (left, outlined with a black dashed line) is observed in a more open and less structured conformation than the resting Rel_Bs^NTD molecule (right, outlined with a light gray dashed line). The relative HD-to-SYNTH distance in each Rel_Bs^NTD monomer is indicated. (B) Details of Rel_Bs^NTD in the SYNTH-primed state highlighting key structural elements. (C) Heatmaps showing the HDX signal kinetics of Rel_Bs^NTD (top) and Rel_Bs^NTD as part of the DarB₂:Rel_Bs^NTD₂ complex (center) and the ΔHDX (bottom). Both catalytic domain of Rel_Bs and all the secondary structural elements of the NTD are shown in the figure. Residues involved in the binding interface with DarB are outlined by a dashed blue line, and the regions with increased deuterium uptake, which include the G loop that becomes exposed upon binding to DarB and the alarmone allosteric site, are shaded in red. (D) Topology representation of Rel_Bs^NTD colored as a function of the ΔHDX. (E) Heatmaps representing the HDX of DarB (top) and DarB as part of the DarB₂:Rel_Bs^NTD₂ complex (center) and the ΔHDX (bottom). Residues involved in the binding interface with Rel_Bs^NTD are indicated by a dashed blue line, and those involved in the binding to c-di-AMP are shaded in green. (F) Effect of DarB on the SYNTH activity of Rel_Bs in the presence or absence of “starved” ribosomes.