title: 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.
authors.affiliation: Laboratory of Chemical Biology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences , Shanghai 201203, China.|University of Chinese Academy of Sciences , Beijing 100049, China.
authors.forename: Tengfeng
authors.initials: T
authors.lastname: Ni
authors.affiliation: College of Life Sciences, Zhejiang Sci-Tech University , Hangzhou 310018, China.|Drug Design and Discovery Center, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences , Shanghai 201203, China.
authors.forename: Fei
authors.initials: F
authors.lastname: Ye
authors.affiliation: CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences , Shanghai 201203, China.
authors.forename: Xing
authors.initials: X
authors.lastname: Liu
authors.affiliation: Laboratory of Chemical Biology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences , Shanghai 201203, China.
authors.forename: Jie
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Laboratory of Chemical Biology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences , Shanghai 201203, China.
authors.forename: Hongchuan
authors.initials: H
authors.lastname: Liu
authors.affiliation: Laboratory of Chemical Biology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences , Shanghai 201203, China.|University of Chinese Academy of Sciences , Beijing 100049, China.
authors.forename: Jiahui
authors.initials: J
authors.lastname: Li
authors.affiliation: National Center for Protein Science Shanghai, Institute of Biochemistry and Cell Biology, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences , Shanghai 201210, China.
authors.forename: Yingyi
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Experiment Center for Science and Technology, Shanghai University of Traditional Chinese Medicine , Shanghai 201203, China.
authors.forename: Yinqiang
authors.initials: Y
authors.lastname: Sun
authors.affiliation: CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences , Shanghai 201203, China.
authors.forename: Meining
authors.initials: M
authors.lastname: Wang
authors.affiliation: Drug Design and Discovery Center, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences , Shanghai 201203, China.
authors.forename: Cheng
authors.initials: C
authors.lastname: Luo
authors.affiliation: University of Chinese Academy of Sciences , Beijing 100049, China.|Drug Design and Discovery Center, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences , Shanghai 201203, China.
authors.forename: Hualiang
authors.initials: H
authors.lastname: Jiang
authors.affiliation: Laboratory of Chemical Biology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences , Shanghai 201203, China.|University of Chinese Academy of Sciences , Beijing 100049, China.
authors.forename: Lefu
authors.initials: L
authors.lastname: Lan
authors.affiliation: School of Life Sciences, Fudan University , Shanghai 200433, China.
authors.forename: Jianhua
authors.initials: J
authors.lastname: Gan
authors.affiliation: University of Chinese Academy of Sciences , Beijing 100049, China.|CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences , Shanghai 201203, China.
authors.forename: Ao
authors.initials: A
authors.lastname: Zhang
authors.affiliation: University of Chinese Academy of Sciences , Beijing 100049, China.|CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences , Shanghai 201203, China.
authors.forename: Hu
authors.initials: H
authors.lastname: Zhou
authors.affiliation: Laboratory of Chemical Biology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences , Shanghai 201203, China.|University of Chinese Academy of Sciences , Beijing 100049, China.
authors.forename: Cai-Guang
authors.initials: CG
authors.lastname: Yang
mesh_terms: D000483:Alleles; D018360:Crystallography, X-Ray; D049071:Endopeptidase Clp / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008854:Microscopy, Electron; D009154:Mutation; D011487:Protein Conformation; D013211:Staphylococcus aureus / Q000254:growth & development
chemical_list: D049071:Endopeptidase Clp