Structural basis for c-di-AMP-dependent regulation of the bacterial stringent response by receptor protein DarB

The bacterial second messenger c-di-AMP controls essential cellular processes, including potassium and osmolyte homeo-stasis. This makes synthesizing enzymes and components involved in c-di-AMP signal transduction intriguing as poten-tial targets for drug development. The c-di-AMP receptor protein DarB of Bacillus subtilis binds the Rel protein and triggers the Rel-dependent stringent response to stress condi-tions; however, the structural basis for this trigger is unclear. Here, we report crystal structures of DarB in the ligand-free state and of DarB complexed with c-di-AMP, 3'3'-cGAMP, and AMP. We show that DarB forms a homodimer with a parallel, head-to-head assembly of the monomers. We also confirm the DarB dimer binds two cyclic dinucleotide mole-cules or two AMP molecules; only one adenine of bound c-di -AMP is specifically recognized by DarB, while the second protrudes out of the donut-shaped protein. This enables DarB to bind also 3'3'-cGAMP, as only the adenine fits in the active site. In absence of c-di-AMP, DarB binds to Rel and stimulates (p)ppGpp synthesis, whereas the presence of c-di-AMP abol-ishes this interaction. Furthermore, the DarB crystal structures reveal no conformational changes upon c-di-AMP binding, leading us to conclude the regulatory function of DarB on Rel must be controlled directly by the bound c-di-AMP. We thus derived a structural model of the DarB-Rel complex via in silico docking, which was validated with mass spectrometric analysis of the chemically crosslinked DarB-Rel complex and mutagenesis studies. We suggest, based on the predicted complex structure, a mechanism of stringent response regula-tion by c-di-AMP.

Automated summary

The bacterial second messenger c-di-AMP controls essential cellular processes, and its binding to the protein DarB regulates the stringent response. We determined the crystal structures of DarB in different states and found that DarB forms a homodimer, binding two cyclic dinucleotide molecules or two AMP molecules. Our findings provide insights into the mechanism of stringent response regulation by c-di-AMP and have implications for drug development.