Signal peptide chaperoning by SlyD and its role in Tat transport

authored by: Daniel Lindemeier
supervised by: Thomas Brüser
Abstract: Bacterial life depends on a variety of different proteins, some of which facilitate nutrient uptake, while others, known as chaperones, can assist in protein folding. One such chaperone is the widely distributed prokaryotic sensitivity to lysis D (SlyD), a protein of the FK506 binding protein (FKBP) family that has three distinct domains: the FKBP domain, which has peptidylprolyl isomerase functions; the insert-in-flap domain or IF domain, which has chaperone functions; and the unstructured C-terminal domain, which is rich in metal ion-binding residues
such as histidine. Previously, SlyD was found to be able to bind Tat signal peptides of folded proteins translocated by the twin-arginine translocation (Tat) system. In this study, the possible role of SlyD as a Tat chaperone and the previously discovered polar
interactions between peptides were further investigated. An indirect SlyD dependence was
observed during anaerobic DMSO respiration, where DMSO acts as the terminal electron
acceptor. However, DMSO reductase assays revealed that the absence of SlyD did not affect DmsA translocation or conformation, but may affect the electron transport pathway of the DMSO reductase complex. Recently, SlyD has been shown to interact with several proteins involved in iron-sulfur cluster biogenesis, including IscU, which requires peptidyl-prolylisomerization. Here, it is proposed that SlyD may be a potential candidate since iron-sulfur clusters are used for electron transport in DMSO reductases. Site-directed mutagenesis within the hydrophobic binding pocket as well as the FKBP domain disrupted SlyD functionality, further demonstrated the importance of the IF domain for the peptidyl-prolyl activity of SlyD. Previous experiments have shown that SlyD binds to positively charged positions near the hydrophobic h-region of Tat signal peptides, which is similar to the substrate binding of DnaK. Isothermal titration calorimetry (ITC) studies were used to elucidate the importance of key
hydrophobic residues for substrate interaction. Furthermore, two negatively charged residues at the tip of the flexible thumb-like structure (D101) and at the back (E108) of the IF domain were also important for substrate interaction. It was also possible to discover a potential substrate induced fit of the IF domain of SlyD, triggered by the polar interactions between positively charged residues of the substrate and the negatively charged IF domain of SlyD.
Organisation(s): Institute of Microbiology
Type: Doctoral thesis
No. of pages: 199
Publication date: 01.03.2024
Publication status: Published
Electronic version(s): https://doi.org/10.15488/16530 (Access: Embargoed)