6XNV

CRYSTAL STRUCTURE OF LISTERIA MONOCYTOGENES CBPB PROTEIN (LMO1009) IN COMPLEX WITH C-DI-AMP

Summary for 6XNV

• Entry DOI: 10.2210/pdb6xnv/pdb
• Related: 6XNU
• Descriptor: CBS domain-containing protein, (2R,3R,3aS,5R,7aR,9R,10R,10aS,12R,14aR)-2,9-bis(6-amino-9H-purin-9-yl)octahydro-2H,7H-difuro[3,2-d:3',2'-j][1,3,7,9,2,8 ]tetraoxadiphosphacyclododecine-3,5,10,12-tetrol 5,12-dioxide (3 entities in total)
• Functional Keywords: c-di-amp binding protein, cytosolic protein
• Biological source: Listeria monocytogenes
• Total number of polymer chains: 2
• Total formula weight: 38709.47

Authors

Luo, S.,Tong, L. (deposition date: 2020-07-04, release date: 2020-09-09, Last modification date: 2023-10-18)

Primary citation

Peterson, B.N.,Young, M.K.M.,Luo, S.,Wang, J.,Whiteley, A.T.,Woodward, J.J.,Tong, L.,Wang, J.D.,Portnoy, D.A.
(p)ppGpp and c-di-AMP Homeostasis Is Controlled by CbpB in Listeria monocytogenes.
Mbio, 11:-, 2020

PubMed Abstract: The facultative intracellular pathogen , like many related , uses the nucleotide second messenger cyclic di-AMP (c-di-AMP) to adapt to changes in nutrient availability, osmotic stress, and the presence of cell wall-acting antibiotics. In rich medium, c-di-AMP is essential; however, mutations in , the gene encoding c-di-AMP binding protein B, suppress essentiality. In this study, we identified that the reason for -dependent essentiality is through induction of the stringent response by RelA. RelA is a bifunctional RelA/SpoT homolog (RSH) that modulates levels of (p)ppGpp, a secondary messenger that orchestrates the stringent response through multiple allosteric interactions. We performed a forward genetic suppressor screen on bacteria lacking c-di-AMP to identify genomic mutations that rescued growth while was constitutively expressed and identified mutations in the synthetase domain of RelA. The synthetase domain of RelA was also identified as an interacting partner of CbpB in a yeast-2-hybrid screen. Biochemical analyses confirmed that free CbpB activates RelA while c-di-AMP inhibits its activation. We solved the crystal structure of CbpB bound and unbound to c-di-AMP and provide insight into the region important for c-di-AMP binding and RelA activation. The results of this study show that CbpB completes a homeostatic regulatory circuit between c-di-AMP and (p)ppGpp in Bacteria must efficiently maintain homeostasis of essential molecules to survive in the environment. We found that the levels of c-di-AMP and (p)ppGpp, two nucleotide second messengers that are highly conserved throughout the microbial world, coexist in a homeostatic loop in the facultative intracellular pathogen Here, we found that cyclic di-AMP binding protein B (CbpB) acts as a c-di-AMP sensor that promotes the synthesis of (p)ppGpp by binding to RelA when c-di-AMP levels are low. Addition of c-di-AMP prevented RelA activation by binding and sequestering CbpB. Previous studies showed that (p)ppGpp binds and inhibits c-di-AMP phosphodiesterases, resulting in an increase in c-di-AMP. This pathway is controlled via direct enzymatic regulation and indicates an additional mechanism of ribosome-independent stringent activation.

PubMed: 32843560
DOI: 10.1128/mBio.01625-20

Experimental method

X-RAY DIFFRACTION (2.4 Å)