title: Barrel-shaped ClpP Proteases Display Attenuated Cleavage Specificities.
abstract: ClpP is a self-compartmentalizing protease with crucial roles in bacterial and mitochondrial protein quality control. Although the ClpP homocomplex is composed of 14 equivalent active sites, it degrades a multitude of substrates to small peptides, demonstrating its capability to carry out diverse cleavage reactions. Here, we show that ClpP proteases from E. coli, S. aureus, and human mitochondria exhibit preferences for certain amino acids in the P1, P2, and P3 positions using a tailored fluorogenic substrate library. However, this high specificity is not retained during proteolysis of endogenous substrates as shown by mass spectrometric analysis of peptides produced in ClpXP-mediated degradation reactions. Our data suggest a mechanism that implicates the barrel-shaped architecture of ClpP not only in shielding the active sites to prevent uncontrolled proteolysis but also in providing high local substrate concentrations to enable efficient proteolytic processing. Furthermore, we introduce customized fluorogenic substrates with unnatural amino acids that greatly surpass the sensitivity of previously used tools. We used these to profile the activity of cancer-patient- and Perrault-syndrome-derived ClpP mutant proteins.
authors.affiliation: Center for Integrated Protein Science (CIPSM), Technische Universität München , Department of Chemistry, Lichtenbergstraße 4, 85747 Garching, Germany.
authors.forename: Malte
authors.initials: M
authors.lastname: Gersch
authors.affiliation: Center for Integrated Protein Science (CIPSM), Technische Universität München , Department of Chemistry, Lichtenbergstraße 4, 85747 Garching, Germany.
authors.forename: Matthias
authors.initials: M
authors.lastname: Stahl
authors.affiliation: Department of Bioorganic Chemistry, Faculty of Chemistry, Wroclaw University of Technology , Wybrzeże Wyspiańskiego 27, 50-370 Wrocław, Poland.
authors.forename: Marcin
authors.initials: M
authors.lastname: Poreba
authors.affiliation: Center for Integrated Protein Science (CIPSM), Technische Universität München , Department of Chemistry, Lichtenbergstraße 4, 85747 Garching, Germany.
authors.forename: Maria
authors.initials: M
authors.lastname: Dahmen
authors.affiliation: Department of Bioorganic Chemistry, Faculty of Chemistry, Wroclaw University of Technology , Wybrzeże Wyspiańskiego 27, 50-370 Wrocław, Poland.
authors.forename: Anna
authors.initials: A
authors.lastname: Dziedzic
authors.affiliation: Department of Bioorganic Chemistry, Faculty of Chemistry, Wroclaw University of Technology , Wybrzeże Wyspiańskiego 27, 50-370 Wrocław, Poland.
authors.forename: Marcin
authors.initials: M
authors.lastname: Drag
authors.affiliation: Center for Integrated Protein Science (CIPSM), Technische Universität München , Department of Chemistry, Lichtenbergstraße 4, 85747 Garching, Germany.
authors.forename: Stephan A
authors.initials: SA
authors.lastname: Sieber
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism; D020134:Catalytic Domain; D049071:Endopeptidase Clp / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008928:Mitochondria / Q000737:chemistry / Q000201:enzymology / Q000378:metabolism; D008958:Models, Molecular; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D013211:Staphylococcus aureus / Q000737:chemistry / Q000201:enzymology* / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D000596:Amino Acids; D029968:Escherichia coli Proteins; D010455:Peptides; C483572:ClpP protease, E coli; C568646:ClpP protein, human; D049071:Endopeptidase Clp