The cryo-electron microscopy supramolecular structure of the bacterial stressosome unveils its mechanism of activation

Abstract

The stressosome is the epicenter of the stress response in bacteria, and one of the largest bacterial nanomachines. How the stressosome integrates and transmits stress signals from the environment has remained elusive. The stressosome consists of multiple copies of three proteins RsbR, RsbS and RsbT a kinase that is important for its activation. Here using cryo-electron microscopy, we determined the atomic organization of the Listeria monocytogenes stressosome at 3.38Å resolution. The structure shows that RsbR and RsbS are organized in a 60 protomers truncated icosahedron. Two phosphorylation sites on RsbR (T175, T209) and one on RsbS (S56) are arranged on a horizontal row that is interrupted by a 13 amino acid flexible loop in RsbR. RsbR T175 and RsbS S56 are accessible on the surface and are phosphorylated under normal stress conditions. Access to T209 is partially hidden by the RsbR flexible loop, whose “open” or “closed” position, could modulate stressosome activation. Modification of the flexible loop or of residues involved in RsbR and RsbS interaction, results in a dominant negative phenotype. In addition, we showed that the interaction between three glutamic acids in the N terminal domain of RsbR and the membrane bound mini-protein Prli42 is essential for Listeria survival to stress. Taken together, our data provide the first atomic model of the stressosome core assembly, and highlight a loop that is important for stressosome activation, paving the way towards elucidating the structural basis of stressosome function in bacteria