Journal: Science Advances

Article Title: The structure of DarB in complex with Rel NTD reveals nonribosomal activation of Rel stringent factors

doi: 10.1126/sciadv.ade4077

Figure Lengend Snippet: ( A ) Binding of c-di-AMP to DarB monitored by ITC. ( B ) Effect of c-di-AMP on DarB and its Rel-dependent activation of pppGpp synthesis. Rel Bs (250 nM) was incubated with ATP and GTP, in the absence or presence of saturating DarB (10-fold excess), c-di-AMP–saturated DarB, or c-di-AMP. ( C ) Binding of Rel to DarB monitored by ITC. ( D ) Analytical size exclusion chromatography of the Rel Bs NTD 2 -DarB 2 complex (in dark blue), Rel NTD (in red), DarB (in green), and the Rel Bs NTD 2 -DarB 2 complex incubated with c-di-AMP (in light blue). The experiment confirms that the presence of c-di-AMP is sufficient to disrupt the complex. ( E ) Titration of DarB into c-di-AMP + Rel monitored by ITC. ( F ) Structure of DarB bound to c-di-AMP. Individual CBS domains of the tandem are labeled. Only one of the two c-di-AMP molecules is shown in the figure for clarity. ( G ) Structural details of the c-di-AMP–binding sites of DarB in the c-di-AMP–bound complex are shown in violet and of apo-DarB in green. Residues involved in the dinucleotide coordination are labeled. ( H ) Heatmaps representing the HDX of DarB (top) and DarB:c-di-AMP complex (center) and the ΔHDX (bottom). Residues involved in the binding to c-di-AMP are outlined by a dashed blue line. RFU, relative fractional uptake; a.u., arbitrary units; A 280nm , absorbance at 280 nm.

Article Snippet: To assess the effect of c-di-AMP on the Rel Bs NTD 2 :DarB 2 complex, 500 μM c-di-AMP (Jena Bioscience) was added to the sample, and the sample was immediately loaded onto the column.

Techniques: Binding Assay, Activation Assay, Incubation, Size-exclusion Chromatography, Titration, Labeling

Journal: Science Advances

Article Title: The structure of DarB in complex with Rel NTD reveals nonribosomal activation of Rel stringent factors

doi: 10.1126/sciadv.ade4077

Figure Lengend Snippet: ( A ) Crystal structure of DarB 2 :Rel Bs NTD 2 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 DarB 2 :Rel Bs NTD 2 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 DarB 2 :Rel Bs NTD 2 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.

Article Snippet: To assess the effect of c-di-AMP on the Rel Bs NTD 2 :DarB 2 complex, 500 μM c-di-AMP (Jena Bioscience) was added to the sample, and the sample was immediately loaded onto the column.

Techniques: Binding Assay, Activity Assay

Journal: Science Advances

Article Title: The structure of DarB in complex with Rel NTD reveals nonribosomal activation of Rel stringent factors

doi: 10.1126/sciadv.ade4077

Figure Lengend Snippet: Effect of the Y279A ( A ) and K290G ( B ) substitutions in Rel Bs NTD to the interaction with DarB, monitored by ITC. Effect of the substitutions to the primary E34R ( C ) or secondary E74G/R75G ( D ) DarB interfaces, to the binding to Rel Bs NTD , monitored by ITC. ( E ) Effect of an E34R substitution on DarB to the activity of DarB monitored as a function of the hydrolase activity of Rel Bs . ( F ) DarB-SYNTH interface color-coded by the conservation score of each amino acid calculated by ConSurf. Residues involved in the primary interface are shown in the conservation bar plots to the right and colored on the basis of their individual conservation profile. The strictly conserved Y of the G loop of Rel is shown in italic. Structural elements of SYNTH ( G ) and DarB ( H ) colored as in (F) underline the strong conservation of the binding interface. The contact regions between both proteins are outlined by dashed black lines with the residues directly involved in the primary binding interface and the PXPGR motif highlighted in (G) and (H) and the location of the c-di-AMP shown as a surface in (H).

Article Snippet: To assess the effect of c-di-AMP on the Rel Bs NTD 2 :DarB 2 complex, 500 μM c-di-AMP (Jena Bioscience) was added to the sample, and the sample was immediately loaded onto the column.

Techniques: Binding Assay, Activity Assay