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Projekt Druckansicht

Structure-function analysis of DegP

Fachliche Zuordnung Stoffwechselphysiologie, Biochemie und Genetik der Mikroorganismen
Förderung Förderung von 2008 bis 2015
Projektkennung Deutsche Forschungsgemeinschaft (DFG) - Projektnummer 73451336
 
Erstellungsjahr 2016

Zusammenfassung der Projektergebnisse

A novel biological principle was uncovered i.e. that cooperative enzymes can be activated by substoichiometic binding of an inhibitor that targets the active site. This was done by using the HtrA protease DegP as a model. DegP is an unusual protease that switches from an inactive hexamer to higher order oligomers of up to 24mer representing the active state. Each of these oligomeric particles are build of trimeric units. Each trimeric building block displays an up to 6 fold positive cooperativity comprising 3 ligand binding sites in the active sites of the protease domains and three additional ligand binding sites in its PDZ1 domains. Cooperativity is mediated by loops of the protease domain that sense ligand binding to PDZ1 domains. in addition, some loops are shared between protomers. The latter architectural feature mediates communication between the protease domains. These results have wide implications. For example, if an inhibitor that targets a cooperative enzyme is not equally distributed across all tissues (the well known problem of bioavailability) the inhibitor will work in tissues where bioavailability is good but will activate the target protein in tissues where bioavailability is poor causing the opposite of the desired effect. This effect might be important for basic research involving e.g. animal models as well as for therapeutic approaches, i.e. drug treatments. To obtain further insights into the precise events during the transition between the resting and active states, native mass spectrometry was used to demonstrate that binding of multiple peptide ligands to the DegP resting state occurs prior to the transition into the active higher oligomeric conformation. The transition from the resting 6mer to the 9meric and 12meric active states of DegP occurred at an occupancy of 40% for each peptide ligand. We observed substrate-specific 9-mer formation with a maximum load of 9 peptides, whereas other substrates led to 12-mers accommodating 24 peptides. In additional studies, another PDZ protease of E. coli, Tsp (tail specific protease) that is also involved in protein quality control was biochemically studied. In contrast to DegP, Tsp is a monomer and its PDZ domain is not at the C-terminus but internal. In this work, we have identified synthetic substrates, boronic acid-derived inhibitors, several of which had sub-µM IC50s, and allosteric activators. Interestingly, the antimicrobial peptide Drosocin produced by fruit flies serves as an unexpected activator of Tsp but not of DegP. These data suggest that at least for some types of antibiotics, specific protein quality control proteases such as Tsp or stress response systems are activated to counteract the potentially lethal effects of naturally occurring antibiotics. Moreover, an inhibitor of Chlamydia DegP that was rather specific and worked even in cell based assays (because Chlamydia is an obligate intracellular pathogen) was characterised.

Projektbezogene Publikationen (Auswahl)

  • 2011. HtrA proteases: Regulated proteolysis in protein quality control. Nat Rev Mol Cell Biol 12:152-162
    Clausen, T., Kaiser, M., Huber, R. and M. Ehrmann
  • 2011. Protein quality control in the bacterial periplasm. Annu Rev Microbiol 65:149-168
    Merdanovic, M., Clausen, T., Kaiser, M., Huber, R. and M. Ehrmann
  • 2012. Chemical biology approaches reveal conserved features of a C-terminal processing PDZ protease. ChemBioChem 13:402-408
    Weski, J., Meltzer, M., Spaan, L., Mönig, T., Oeljeklaus, J., Hauske, P., Vouilleme, L., Volkmer, R., Boisguerin, P., Boyd, D., Huber, R., Kaiser, M. and M. Ehrmann
    (Siehe online unter https://doi.org/10.1002/cbic.201100643)
  • 2013 Identification of a serine protease inhibitor which causes inclusion vacuole reduction and is lethal to Chlamydia trachomatis. Mol Microbiol 89:676-689
    Gloeckl, S; Ong, VA; Patel, P; Tyndall, JDA; Timms, P; Beagley, KW; Allan, JA; Armitage, CW; Turnbull, L; Whitchurch, CB; Merdanovic, M; Ehrmann, M; Verdoes, M; Bogyo, M; and WM Huston
    (Siehe online unter https://doi.org/10.1111/mmi.12306)
  • 2013. The diversity of allosteric regulation in proteases. ACS Chem Biol 8:19-26
    Merdanovic, M, Mönig, T, Ehrmann, M and M Kaiser
    (Siehe online unter https://doi.org/10.1021/cb3005935)
  • 2014. Substrate Occupancy at the Onset of Oligomeric Transitions of DegP. Structure 22: 281–290
    Thompson, NJ, Merdanovic, M, Ehrmann, M, van Duijn, E, and Heck, AJR
    (Siehe online unter https://doi.org/10.1016/j.str.2013.11.010)
 
 

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