Towards structure elucidation of the Proline Reductase Complex from Clostridioides difficile

Clostridioides difficile represents the most common cause of antibiotic-induced diarrhea. This work addresses the structural basis of the proline reductase complex. This central enzyme performs important roles in metabolic regulation. Proline reductase consists of three subunits, PrdAβ and PrdAα, which arise from the PrdA protein by selenocysteinolysis, and the selenocysteine-containing PrdB. The complex has a high molecular weight and is membrane-associated. Biophysical and structural biology experiments allowed the proline reductase to be described as a metastable, triacontameric complex with a size of 930 kDa and a high intrinsic flexibility. Limited proteolysis reveals that the N-terminus of PrdAβ is responsible for pentamerization of hexamers. Recombinant isolation methods allowed investigation with cryo-electron microscopy, which may lead to a high-resolution structure of the truncated hexameric subcomplex in the future. Another focus of this work was on two phospholipases, PlaA and PlaB from Legionella pneumophila. These enzymes attack the biomembranes of host cells, altering their integrity and releasing toxic messengers. The bacterium protects itself from toxicity with regulations of phospholipases. The protein structure of PlaA was determined by X-ray crystallography, providing detailed structural information on regulation. Another principle of regulation to protect against self-induced lysis was revealed by characterization of the virulence factor PlaB. The tetrameric structure was determined by an anomalous dispersion experiment. Structure-based mutants were characterized and subcellularly localized. These data suggest that PlaB is inhibited intracellularly by a novel mechanism of NAD(H)-mediated tetramerization. After secretion, PlaB is activated by deoligomerization. The structure revealed why the C-terminus of PlaB is essential for enzymatic activity and how novel structural elements enable membrane association of active PlaB dimers.