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

Abstract

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.

Selective Activation of Human Caseinolytic Protease P (ClpP)

“…[6] Different conformational intermediates are also observed for bacterial ClpPs, which either exist in an active extended, inactive compact, or inactive compressed conformation. [10,11] Furthermore,a lso small molecules were shown to exert conformational control. Binding of ClpX to the hydrophobic pockets results in conformational selection of the active,extended complex;however,the exact interplay between chaperone binding and the catalytic hotspots remains to be elucidated.…”

“…However,switching the two fluorine substituents to a3 ,5 position (4), for example,a so bserved in ADEP2, decreased activity further. An unsubstituted benzyl group (6)a sw ell as ab enzyl group bearing electron-donating methoxy (12,13), methylenedioxy (10)a nd methyl (11) groups showed no activation effect. An unsubstituted benzyl group (6)a sw ell as ab enzyl group bearing electron-donating methoxy (12,13), methylenedioxy (10)a nd methyl (11) groups showed no activation effect.…”

“…[11] We cloned and purified the respective hClpP Y118A mutant which showed enhanced peptidase activity in absence of D9 and even higher activity in presence of D9 suggesting that this activation mutation can be transferred to the human orthologue (Supporting Information, Figure S7). However,arecently reported gain-of-function mutation in SaClpP (Y63A) attracted our attention owing to ah igher tetradecamer stability and corresponding chance for forming crystals.…”

Selektive Aktivierung der humanen caseinolytischen Protease P (ClpP)

“…[11] Wirk lonierten und reinigten die entsprechende humane Mutante hClpP Y118A, die eine verstärkte Peptidase-Aktivitätauch ohne D9 zeigte. Eine Kokristallisation der Wildtyp-hClpP mit D9 war zunächst nicht mçglich.…”

“…[4,5] Das Chaperon ist verantwortlich fürdie Erkennung des Substrats sowie dessen Entfaltung und Tr anslokation. [10,11] Daneben kçnnen auch niedermolekulare Verbindungen konformationelle Kontrolle ausüben. [6] Verschiedene konformationelle Intermediate kçnnen auch fürb akterielle ClpPs beobachtet werden, die entweder in einer aktiven gestreckten oder in inaktiven kompakten oder komprimierten Formen vorkommen.…”