Mechanisms of protease-substrate interactions in the E. coli cytosol

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The folding state of proteins within cells is controlled by an energy-dependent quality control system, consisting of ATP-dependent proteases and molecular chaperones. Misfolded or aggregated proteins can be either refolded to the native structure or degraded. The functional interplay between proteases and chaperones in this process is poorly understood. While substrate specificities and substrate populations of major chaperones of the E. coli cytosol are known, equivalent data for proteases are missing. We plan to identify natural substrates of major cytosolic proteases (Lon, ClpAP, ClpXP, HslUV) and to determine their substrate binding motifs and the kinetic features of the protease-substrate interactions. We will also determine the relationship between proteases and chaperones in their activity on misfolded soluble and aggregated proteins as well as on reporter proteins with engineered protease and/or chaperone binding sequences. Finally, we will have a major focus on the mechanistic analysis of a novel adaptor protein for the ClpAP protease machine, ClpS, which we discovered recently. ClpS changes the substrate specificity of the ClpAP proteolytic machine by inhibiting the degradation of SsrA-tagged substrates, while stimulating the removal of aggregated proteins. The structural basis of the ClpA-ClpS interaction and the resulting consequences on ClpA substrate interaction and the chaperone mechanism of ClpA will be investigated.