Characterization of Gain-of-Function Mutant Provides New Insights into ClpP Structure

ATP-dependent
Clp protease (ClpP), a highly conserved serine protease
in vast bacteria, could be converted into a noncontrollable enzyme
capable of degrading mature proteins in the presence of acyldepsipeptides
(ADEPs). Here, we design such a gain-of-function mutant of Staphylococcus aureus ClpP (SaClpP) capable
of triggering the same level of dysfunctional activity that occurs
upon ADEPs treatment. The SaClpPY63A mutant degrades
FtsZ in vivo and inhibits staphylococcal growth.
The crystal structure of SaClpPY63A indicates that
Asn42 would be an important domino to fall for further activation
of ClpP. Indeed, the SaClpPN42AY63A mutant demonstrates
promoted self-activated proteolysis, which is a result of an enlarged
entrance pore as observed in cryo-electron microscopy images. In addition,
the expression of the engineered clpP allele phenocopies
treatment with ADEPs; inhibition of cell division occurs as does showing
sterilizing with rifampicin antibiotics. Collectively, we show that
the gain-of-function SaClpPN42AY63A mutant becomes
a fairly nonspecific protease and kills persisters by degrading over
500 proteins, thus providing new insights into the structure of the
ClpP protease.