ISBN 9783843924511

978-3-8439-2451-1, Reihe Biochemie

Maria Dahmen
Structural and biochemical characterization of the two Caseinolytic Protease Complexes (ClpXP) of Listeria monocytogenes

137 Seiten, Dissertation Technische Universität München (2015), Softcover, A5

Zusammenfassung / Abstract

Caseinolytic proteases (ClpPs) are large oligomeric protein complexes that contribute to cell homeostasis. The highly conserved bacterial ClpP is considered a promising target for anti-virulence therapy of bacterial infections. Yet, despite numerous studies about ClpP in general, there is still little research focusing on ClpP derived from the food-borne pathogen Listeria monocytogenes. L. monocytogenes expresses two ClpP isoforms, ClpP1 and ClpP2, whereof ClpP2 forms an active tetradecameric enzyme. ClpP1 is an inactive heptamer and has to form a heterooligomeric complex with ClpP2 to become an active protease. This thesis focuses on the structural and biochemical characterization of the heterocomplex ClpP1/2 as well as its interaction with the associated chaperone ClpX.

After developing an orthogonal affinity-tag strategy to purify the ClpP1/2 heterocomplex, the crystal structure of this complex was solved. The peptidolytic activity of the ClpPs as well as the proteolytic activity of the corresponding ClpXP complexes were probed via in vitro peptidase assays and chaperone-associated proteolytic degradation of ssrA-tagged green fluorescent protein (GFP). Large differences between the activity of the homocomplex ClpP2 and the heterocomplex ClpP1/2 were observed. While the heterocomplex had a decreased peptidase activity, it had in combination with ClpX a 9-fold increased protease activity compared to the corresponding ClpXP2 complex. This elevated catalytic rate may aid the rapid removal of unfolded proteins under stress conditions. Transcriptome analysis of stress-induced changes via real-time PCR revealed a 7-fold up-regulation of ClpP1 and ClpP2 transcript levels upon heat stress. By dissecting the single steps of the whole degradation process it could be demonstrated that the reason for the enhanced proteolytic activity of the heterocomplex is a higher affinity for ClpX. Furthermore, electron microscopy pointed to a unidirectional binding of ClpX to the heterocomplex.

The results help us understand how L. monocytogenes may facilitate its intracellular stress tolerance by the use of two different ClpPs, with the heterocomplex being the enhanced version.