Plasticity in AAA+ proteases reveals ATP-dependent substrate specificity principles

Abstract

SummaryIn bacteria, AAA+ proteases such as Lon and ClpXP specifically degrade substrates to promote growth and stress responses. Here, we find that an ATP-binding mutant of ClpX suppresses physiological defects of a Lon-deficient strain by shifting ClpXP protease specificity toward normally Lon-restricted substrates and away from normal ClpXP targets. Reconstitution with purified proteins assigns these effects to changes in direct recognition and processing of substrates. We show that wildtype ClpXP specificity can be similarly altered when ATP hydrolysis is reduced, which unexpectedly accelerates degradation of some substrates. This activation corresponds with changes in ClpX conformation, leading to a model where ClpX cycles between ‘capture’ and ‘processive’ states depending on ATP loading. Limiting ATP binding alters dynamics between states affording better recognition of unorthodox substrates, but worse degradation of proteins specifically bound by the processive state. Thus, AAA+ protease specificities can be directly tuned by differences in ATP hydrolysis rates.HighlightsA mutation in the Walker B region of ClpX induces recognition of new substrates.Proteases are optimized for specific functions but barrier to recognize new substrates is easily overcome.Expanding substrate recognition by a protease comes at the cost of reducing native substrate degradation.Decreasing ATP enhances ClpXP mediated degradation of certain classes of substrates.ClpX adopts distinct conformational states to favor better recognition of some substrates over others.Graphical AbstractIn a wildtype cell, AAA+ proteases Lon and ClpXP promote normal growth by degrading distinct substrates. ClpX*P can compensate for the absence of the Lon protease by tuning ClpXP substrate specificity to better degrade Lon-privileged substrates (such as DnaA, SciP, and misfolded proteins) but this comes at the cost of native ClpXP substrates (such as ssrA-tagged proteins and CtrA). We propose that ClpX alternates between a closed and open conformation and promoting one state over the other leads to alterations in substrate specificity. In the presence of ClpX* or in ATP-limited conditions, the open state is favored, allowing capture and recognition of substrates such as casein. The balance shifts to the closed state under high ATP conditions, allowing degradation of substrates such as GFP-ssrA, which preferentially bind the closed state.