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

J Biol Chem. 2022 Jul;298(7):102144. doi: 10.1016/j.jbc.2022.102144. Epub 2022 Jun 15.


The bacterial second messenger c-di-AMP controls essential cellular processes, including potassium and osmolyte homeostasis. This makes synthesizing enzymes and components involved in c-di-AMP signal transduction intriguing as potential 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 conditions; 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 molecules 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 abolishes 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 regulation by c-di-AMP.

Keywords: Bacillus subtilis; X-ray crystallography; cyclic dinucleotide; second messenger; stringent response.

MeSH terms

  • Adenine / metabolism
  • Adenosine Monophosphate / metabolism
  • Bacillus subtilis / metabolism
  • Bacterial Proteins* / chemistry
  • Bacterial Proteins* / metabolism
  • Dinucleoside Phosphates* / chemistry
  • Dinucleoside Phosphates* / metabolism


  • Bacterial Proteins
  • Dinucleoside Phosphates
  • cyclic diadenosine phosphate
  • Adenosine Monophosphate
  • Adenine