Engineering Synthetic Adaptors and Substrates for Controlled ClpXP Degradation

Abstract

Facile control of targeted intracellular protein degradation has many potential uses in basic science and biotechnology. One promising approach to this goal is to redesign adaptor proteins, which can regulate proteolytic specificity by tethering substrates to energy-dependent AAA؉ proteases. Using the ClpXP protease, we have probed the minimal biochemical functions required for adaptor function by designing and characterizing variant substrates, adaptors, and ClpX enzymes. We find that substrate tethering mediated by heterologous interaction domains and a small bridging molecule mimics substrate delivery by the wild-type system. These results show that simple tethering is sufficient for synthetic adaptor function. In our engineered system, tethering and proteolysis depend on the presence of the macrolide rapamycin, providing a foundation for engineering highly specific degradation of target proteins in cells. Importantly, this degradation is regulated by a small molecule without the need for new adaptor or enzyme biosynthesis.

A Phosphosignaling Adaptor Primes the AAA+ Protease ClpXP to Drive Cell Cycle-Regulated Proteolysis

“…The scaffolding adaptor SspB adaptor uses the NTD ClpX as a simple tethering site (Dougan et al, 2003; Bolon et al, 2004; Park et al, 2007; Davis et al, 2009). As shown schematically in Figure 3 (box), if CpdR-dependent degradation also uses the NTD ClpX in a similar manner, it should be possible to bypass the need for the NTD ClpX by artificially tethering CpdR to ΔN-ClpX.…”

“…However, if the NTD ClpX serves as more than a passive anchor, simply tethering CpdR would not restore PdeA degradation. We adopted a previously reported tethering system where human FKBP12 protein is fused to E. coli ΔN-ClpX, and F KBP12- r apamycin- b inding (FRB) domain of the rat mTOR protein is fused to E. coli SspB (Davis et al, 2009). Addition of rapamycin induces dimerization of FKBP12 and FRB, tethering SspB to the ΔN-ClpX fusion so adaptor function no longer requires NTD ClpX (Davis et al, 2009).…”

Geobacillus thermodenitrificans YjbH recognizes the C-terminal end of Bacillus subtilis Spx to accelerate Spx proteolysis by ClpXP

“…Hence the weaker K m would result in faster degradation, since the unfolding of native protein is a ratelimiting step for protein degradation by ClpX (Kenniston et al, 2003). The strong interaction of GtYjbH and Spx could increase the effective concentration of Spx to the pore of ClpX, and then the equilibrium would shift towards the generation of degraded products (Davis et al, 2009). Finally, a model mechanism is proposed of how GtYjbH is an adaptor mediating Spx proteolysis by ClpXP.…”

Rapid Depletion of Target Proteins Allows Identification of Coincident Physiological Responses

“…A solution to this problem is to specifically target the encoded gene product for conditional depletion (29). In doing so, the gene remains in its natural context, and the encoded product functions normally at the appropriate dose prior to its removal.Prior studies have demonstrated the utility of this approach for specific cases by reengineering target proteins such that they contain a peptide degradation signal that is recognized by a processive protease (7,15,20,24,29,53). In early examples, a degradation peptide that has a weakened affinity for the protease was selected, and then an adapter protein that increases the effective local concentration of the target near the protease to increase degradation was conditionally expressed (20,29).…”