The activation mechanism of the Bacillus subtilis stressosome signalling hub

The survival of all organisms is dependent on their ability to adapt to changes in their environment and the stress response of Bacillus subtilis, and a number of its close Gram-positive relatives, provides these organisms with a protective mechanism against a wide range of physical and chemical stresses. This research proposal concerns the mechanism by which B. subtilis is able to respond to environmental stimuli. At the top of the stress-response pathway is a structure resembling a small virus that acts as a signalling hub, and has been named the stressosome. This stressosome is composed of a number of proteins that sense stress-inducing stimuli that are related to a protein called RsbR and another small protein, RsbS, that together act to sequester a protein kinase, RsbT, required to kick-start a cascade of interactions that ultimately leads to the activation of over two hundred genes whose products provide the cell with resistance to stress. The structure of the stressosome that we have recently determined, displays an unusual arrangement of protein components providing tantalising clues as to the mechanism of action in response to stress. We aim to use a blue-light responsive signalling protein to generate a stressosome that we can use in structural studies to determine any gross-structural changes in the complex that lead to the release of the protein kinase and thus the mechanism by which the stress response is initiated. Furthermore, we will investigate the role of the RsbS protein, the absence of which causes bacteria to develop as small and sickly cells, in stress-response by creating stressosomes that consist only of an RsbR-like protein. It is also apparent that the proteins related to RsbR may function as signalling modules. A full understanding of how these proteins work is dependent on the determination of their structures by X-ray crystallography, which provides a molecular model of the protein that can be used to assess potential activating signals that are recognised by their specific shapes. Finally, we will investigate the role of a number of other proteins that are known to mediate the length of the stress signal, as the requirement to turn off the stress response in a timely fashion is almost as important as the ability to respond to stress in the first place.

Macromolecular assemblies are increasingly being seen to play key roles in many cellular processes and a mechanistic understanding of the function of these is vital to our basic scientific knowledge. We aim to capitalise on our previous studies and determine the structural nature of stressosome activation. This complex is an extremely elegant solution to the problem of integrating diverse inputs to a single signalling outcome and we are in a position to elucidate the full activation mechanism. From the sensing of a specific signal to conformational changes concomitant with this activation and the changes that occur in order to facilitate the phosphorylation of the structure by the RsbT kinase and its subsequent release to begin the cascade that leads to the activation of sigmaB and the ultimate response of gene expression. The most significant of our objectives is to determine the cryo-EM structure of an activated stressosome, using a blue-light activated RsbR paralogue to simulate activation in vitro. This structure will highlight molecular changes that occur on the receipt of a stress signal. We will complement this with a structure of an activated stressosome bound to a catalytically inactive form of the kinase; this will show the structure poised in the intermediate state between the resting and fully phosphorylated 'active' state. Concurrent with this objective we will pursue crystallisation and X-ray diffraction studies of the sensor domains from the RsbR paralogues to investigate their structure and elucidate their function as the sequences of these proteins give little clue as to their overall fold and function. Finally, we will investigate the role of other proteins in the mediation of the stress response. We will use standard biochemical techniques to determine if proteins that have been identified by yeast-two hybrid methods bind to stressosomes and using crystallographic and cryo-EM methods elucidate the structural basis for their role.