Engineering controllable protein degradation.
Complex biological networks are regulated via alterations in protein expression, degradation, and function. Synthetic control of these processes allows dissection of natural systems and the design of new networks. In E. coli, the adaptor SspB tethers ssrA-tagged substrates to the ClpXP protease, causing a modest increase in their rate of degradation. To engineer controlled degradation, we have designed a series of modified ssrA tags that have weakened interactions with ClpXP. When SspB is present, ClpXP degrades purified substrates bearing these engineered peptide tags 100-fold more efficiently. Importantly, substrates bearing these tags are stable in the absence of SspB in vivo but are rapidly degraded upon SspB induction. Our studies supply a conceptual foundation and working components for controllable degradation, improve mechanistic understanding of adaptor-mediated proteolysis, and demonstrate that the relative importance of adaptor proteins in degradation is correlated with the strength of protease-substrate contacts.
McGinness
Kathleen E
KE
Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Baker
Tania A
TA
Sauer
Robert T
RT
D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D049071:Endopeptidase Clp / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D008954:Models, Biological; D008957:Models, Genetic; D008969:Molecular Sequence Data; D015202:Protein Engineering / Q000379:methods*; D013379:Substrate Specificity
D002352:Carrier Proteins; D029968:Escherichia coli Proteins; C477700:sspB protein, E coli; C483573:ClpXP protease, E coli; D049071:Endopeptidase Clp