An electrostatic interaction between the proteins ClpC and YjbA mediates their in vivo function in Bacillus subtilis
Date
2022-06-28
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Abstract
Bacillus subtilis sporulation is a developmental process that culminates in the production of an environmentally resistant, metabolically dormant cell type called a spore. However, the molecular mechanisms that drive metabolic dormancy are poorly understood. Genetic and biochemical approaches have led the Camp Lab to a working model in which a previously uncharacterized protein, YjbA, is hypothesized to function as a novel adaptor protein for the AAA+-ATPase chaperone-protease ClpCP. The working model further suggests that the YjbA-ClpC complex targets the degradation of key metabolic enzymes in the forespore, in turn driving metabolic dormancy. One of the many aspects that needs to be tested for this working model is the interaction between YjbA and ClpC. A co-crystal structure of these two proteins generated by collaborators suggested three sites of electrostatic interactions, one of which is the YjbA E116 - ClpC K85 site. My project aims to test the hypothesis that this specific site of interaction is necessary for overall YjbA-ClpC interaction and function in vivo. To do so, I first measured the interactions between mutated versions of YjbA and ClpC lacking E116 and K85, respectively, using an Escherichia coli based two-hybrid assay. This assay revealed that both YjbA E116 and ClpC K85 are necessary for YjbA-ClpC interaction in an E. coli system. I also investigated the importance of both residues for YjbA-ClpC interaction in vegetatively growing B. subtilis by making mutations on each residue and testing for their effects on YjbA-mediated toxicity. My results indicate that interaction at YjbA E116 and ClpC K85 is important for the toxic phenotype in vegetatively growing cells. Overall, my data suggest that the YjbA E116 - ClpC K85 electrostatic site is important for the YjbA-ClpC interaction in vivo.
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Bacillus subtilis, sporulation, site-directed mutagenesis, electrostatic interaction