title: AAA+ chaperones and acyldepsipeptides activate the ClpP protease via conformational control.
abstract: The Clp protease complex degrades a multitude of substrates, which are engaged by a AAA+ chaperone such as ClpX and subsequently digested by the dynamic, barrel-shaped ClpP protease. Acyldepsipeptides (ADEPs) are natural product-derived antibiotics that activate ClpP for chaperone-independent protein digestion. Here we show that both protein and small-molecule activators of ClpP allosterically control the ClpP barrel conformation. We dissect the catalytic mechanism with chemical probes and show that ADEP in addition to opening the axial pore directly stimulates ClpP activity through cooperative binding. ClpP activation thus reaches beyond active site accessibility and also involves conformational control of the catalytic residues. Moreover, we demonstrate that substoichiometric amounts of ADEP potently prevent binding of ClpX to ClpP and, at the same time, partially inhibit ClpP through conformational perturbance. Collectively, our results establish the hydrophobic binding pocket as a major conformational regulatory site with implications for both ClpXP proteolysis and ADEP-based anti-bacterial activity.
authors.affiliation: Department of Chemistry and Center for Integrated Protein Science Munich (CIPSM), Technische Universität München, Lichtenbergstraße 4, 85748 Garching, Germany.
authors.forename: Malte
authors.initials: M
authors.lastname: Gersch
authors.affiliation: Institute for Pharmaceutical Biology and Biotechnology, University of Düsseldorf, Universitätsstraße 1, 40225 Düsseldorf, Germany.
authors.forename: Kirsten
authors.initials: K
authors.lastname: Famulla
authors.affiliation: Department of Chemistry and Center for Integrated Protein Science Munich (CIPSM), Technische Universität München, Lichtenbergstraße 4, 85748 Garching, Germany.
authors.forename: Maria
authors.initials: M
authors.lastname: Dahmen
authors.affiliation: 1] Department of Chemistry and Center for Integrated Protein Science Munich (CIPSM), Technische Universität München, Lichtenbergstraße 4, 85748 Garching, Germany [2] Institute of Structural Biology, Helmholtz Zentrum München, 85764 Neuherberg, Germany.
authors.forename: Christoph
authors.initials: C
authors.lastname: Göbl
authors.affiliation: Institute for Pharmaceutical Biology and Biotechnology, University of Düsseldorf, Universitätsstraße 1, 40225 Düsseldorf, Germany.
authors.forename: Imran
authors.initials: I
authors.lastname: Malik
authors.affiliation: Department of Chemistry and Center for Integrated Protein Science Munich (CIPSM), Technische Universität München, Lichtenbergstraße 4, 85748 Garching, Germany.
authors.forename: Klaus
authors.initials: K
authors.lastname: Richter
authors.affiliation: Department of Chemistry and Center for Integrated Protein Science Munich (CIPSM), Technische Universität München, Lichtenbergstraße 4, 85748 Garching, Germany.
authors.forename: Vadim S
authors.initials: VS
authors.lastname: Korotkov
authors.affiliation: Institute for Pharmaceutical Biology and Biotechnology, University of Düsseldorf, Universitätsstraße 1, 40225 Düsseldorf, Germany.
authors.forename: Peter
authors.initials: P
authors.lastname: Sass
authors.affiliation: AiCuris GmbH &Co. KG, Friedrich-Ebert-Straße 475, 42117 Wuppertal, Germany.
authors.forename: Helga
authors.initials: H
authors.lastname: Rübsamen-Schaeff
authors.affiliation: 1] Department of Chemistry and Center for Integrated Protein Science Munich (CIPSM), Technische Universität München, Lichtenbergstraße 4, 85748 Garching, Germany [2] Institute of Structural Biology, Helmholtz Zentrum München, 85764 Neuherberg, Germany [3] Institute of Molecular Biology &Biochemistry, Center of Molecular Medicine, Medical University of Graz, 8010 Graz, Austria.
authors.forename: Tobias
authors.initials: T
authors.lastname: Madl
authors.affiliation: Institute for Pharmaceutical Biology and Biotechnology, University of Düsseldorf, Universitätsstraße 1, 40225 Düsseldorf, Germany.
authors.forename: Heike
authors.initials: H
authors.lastname: Brötz-Oesterhelt
authors.affiliation: Department of Chemistry and Center for Integrated Protein Science Munich (CIPSM), Technische Universität München, Lichtenbergstraße 4, 85748 Garching, Germany.
authors.forename: Stephan A
authors.initials: SA
authors.lastname: Sieber
mesh_terms: D000495:Allosteric Site; D001426:Bacterial Proteins / Q000737:chemistry*; D001665:Binding Sites; D002151:Calorimetry; D002384:Catalysis; D020134:Catalytic Domain; D002845:Chromatography; D047630:Depsipeptides / Q000737:chemistry*; D049071:Endopeptidase Clp / Q000737:chemistry*; D004926:Escherichia coli / Q000737:chemistry; D016650:Fluorescein-5-isothiocyanate; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D008027:Light; D008089:Listeria monocytogenes / Q000737:chemistry; D013058:Mass Spectrometry; D018832:Molecular Chaperones / Q000737:chemistry*; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D013211:Staphylococcus aureus / Q000737:chemistry; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D047630:Depsipeptides; D018832:Molecular Chaperones; D049071:Endopeptidase Clp; D016650:Fluorescein-5-isothiocyanate